WO1997027276A2 - Nucleic acids encoding polypeptides having absidia lipase activity - Google Patents

Nucleic acids encoding polypeptides having absidia lipase activity Download PDF

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Publication number
WO1997027276A2
WO1997027276A2 PCT/US1997/000598 US9700598W WO9727276A2 WO 1997027276 A2 WO1997027276 A2 WO 1997027276A2 US 9700598 W US9700598 W US 9700598W WO 9727276 A2 WO9727276 A2 WO 9727276A2
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Prior art keywords
acid sequence
nucleic acid
seq
set forth
absidia
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PCT/US1997/000598
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French (fr)
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WO1997027276A3 (en
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Randy M. Berka
Karuppan C. Boominathan
Thomas Sandal
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Novo Nordisk Biotech, Inc.
Novo Nordisk A/S
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Priority to AU17477/97A priority Critical patent/AU1747797A/en
Priority to EP97904764A priority patent/EP0876489A2/en
Priority to JP52690697A priority patent/JP3333521B2/en
Publication of WO1997027276A2 publication Critical patent/WO1997027276A2/en
Publication of WO1997027276A3 publication Critical patent/WO1997027276A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • the present invention relates to isolated nucleic acid sequences encoding polypeptides having Absidia lipase activity.
  • the invention also relates to nucleic acid constructs, vectors, and host cells compnsing the nucleic acid sequences as well as recombinant methods for producing the polypeptides.
  • Detergents formulated with lipolytic enzymes are known to have improved properties for removing fatty stains.
  • LIPOLASETM NovartisTM
  • Thermomyces lanuginosus also called Humicola lanugtnosa
  • U.S. Patent No. 3,634,195 desc ⁇ bes the production of lipase from Absidia cylindrospora var rhizomorpha NRRL 2815 and Absidia blakesleeana NRRL 1305.
  • Ko ⁇ tala et al. (1987, Journal of the American Oil Chemists Society 64: 509-513) disclose that soybean oil was partially hydrolyzed when incubated with Absidia coerula NRRL 5926 and Absidia ramosa NRRL 1309.
  • Satyanarayana (1981, Current Science 50: 680-682) discloses the secretion of lipase by a strain of Absidia corymbifera Aisaka et al. (1979, Agricultural Biological Chemistry 43: 2125-2129) describe the formation of a lipoprotein lipase from Absidia hyalospora strain KY 303 (now classified as Absidia blakesleeana)
  • the present invention relates to isolated nucleic acid sequences encoding polypeptides having lipase activity selected from the group consisting of:
  • nucleic acid sequence which encodes a polypeptide endogenous to an Absidia strain with an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10;
  • nucleic acid sequence endogenous to an Absidia strain which is capable of hybridizing under medium stringency conditions with (i) the nucleic acid sequence set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or (ii) any of their complementary strands;
  • nucleic acid sequence which is capable of hybridizing under medium stringency conditions with (l) the nucleic acid sequence set forth in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or (ii) any of their complementary strands;
  • SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10;
  • the present invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.
  • Figure 1 shows an agarose gel purification of hpase-specific PCR products from Absidia griseola and Absidia griseola var. iguchii genomic DNA.
  • Lane 1 Hi ' ndlll-digested lambda DNA and
  • Absidia griseola var. iguchii PCR product Both PCR products appear to be approximately 870 bp in size.
  • Figure 2 shows an autoradiogram from Southern hybridization analysis of genomic DNA digests from several Absidia species probed with a radiolabeled lipase gene segment from Absidia griseola.
  • the sizes of HmdIII-digested lambda DNA and H ⁇ elll-digested ⁇ X17RF-DNA size standards are indicated on the right side of the autoradiogram.
  • Lanes 1-3 Absidia sporophora- variabihs DNA (EcoRI plus Aspllil, Aspl ⁇ % ⁇ and £coRI, respectively); lanes 4-6: Absidia cor ⁇ mbifera DNA (EcoRI plus AspllSl, ⁇ sp718I and EcoRI, respectively); lanes 7-9: Absidia blakesleeana DNA (EcoRI plus AspllSl, Aspll ⁇ l and £coRI, respectively); lanes 10-12: Absidia griseola var.
  • Figure 3 shows the DNA sequence and deduced amino acid sequence of Absidia griseola var iguchii lipase (S ⁇ Q ID NOS. 1 and 2, respectively) Introns are marked by a solid hne Regions corresponding to previously determined peptide sequences are underlined ( — )
  • Figure 4 shows the DNA sequence and deduced amino acid sequence of Absidia blakesleeana lipase (S ⁇ Q ID NOS. 3 and 4, respectively) Introns are marked by a solid line Regions corresponding to previously determined peptide sequences are underlined ( — )
  • Figure 5 shows the DNA sequence and deduced ammo acid sequence of Absidia corymbifera lipase (S ⁇ Q ID NOS 5 and 6, respectively) Introns are marked by a solid line. Regions corresponding to previously determined peptide sequences are underlined ( — ).
  • Figure 6 shows the DNA sequence and deduced ammo acid sequence oi Absidia sporophora- vanabi s lipase (S ⁇ Q ID NOS. 7 and 8, respectively). Introns are marked by a solid line
  • Figure 7 shows the DNA sequence and deduced ammo acid sequence of Absidia reflexa lipase (S ⁇ Q ID NOS 9 and 10, respectively).
  • Figure 8 shows a comparison of the ammo acid sequence homology among Absidia lipases compared to Rhizomucor miehei lipase (S ⁇ Q ID NO: 15) and Humicola lanuginosa (S ⁇ Q ID NO: 16) Identical residues are boxed.
  • Figure 9 shows a restriction map of pBANe ⁇ .
  • Figure 10 shows a restriction map of pKB2.
  • Figure 1 1 shows a restriction map of pRamB19.
  • the present invention relates to isolated nucleic acid sequences which encode polypeptides having lipase activity with an amino acid sequence set forth in S ⁇ Q ID NO.2, S ⁇ Q ID NO:4, S ⁇ Q ID NO:6, S ⁇ Q ID NO:8, or S ⁇ Q ID NO: 10.
  • the nucleic acid sequences are set forth in S ⁇ Q ID NO:l, S ⁇ Q ID NO:3, S ⁇ Q ID NO:5, S ⁇ Q ID NO 7, and S ⁇ Q ID NO:9.
  • nucleic acid sequences of the present invention also encompass nucleic acid sequences which encode a polypeptide having the am o acid sequence set forth m S ⁇ Q ID NO.2, S ⁇ Q ID NO:4, S ⁇ Q ID NO:6, S ⁇ Q ID NO:8, or S ⁇ Q ID NO: 10, but differ from S ⁇ Q ID NO: l , S ⁇ Q ID NO:3.
  • the present invention also relates to subsequences of S ⁇ Q ID NO:l , S ⁇ Q ID NO 3, S ⁇ Q LD NO:5, S ⁇ Q ID NO:7, or S ⁇ Q ID NO:9, which encode a fragment of S ⁇ Q ID NO:2, S ⁇ Q ID NO-4, S ⁇ Q ID N0 6, S ⁇ Q ID NO:8, or S ⁇ Q ID NO: 10, respectively, and retain lipase activity
  • the nucleic acid sequences of the present invention are the nucleic acid sequences contained m plasmids pZL-NLl, pZL-NL61, pZL-NL95, and pZL-NL124, which are contained in Escherichia coli NRRL B-21520, NRRL B-21521 , NRRL B-21522, and NRRL B-21523, respectively.
  • isolated nucleic acid sequence refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences
  • lipase is defined herein as a lipolytic enzyme classified under the Enzyme Classification number E.C. 3.1.1 - (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB) Lipolytic enzymes thus exhibit hydrolytic activity towards at least one of the types of ester bonds mentioned m the context of E.C.
  • the lipolytic enzymes of the present invention may have activity towards triglycerides (lipase activity, E C 3 1 1.3), e g , 1,3 -positionally specific lipase activity
  • the present invention relates to isolated nucleic acid sequences encoding polypeptides with lipase activity which are capable of hybridizing under high, medium, or low stringency conditions with an oligonucleotide probe which hybridizes under the same conditions with the nucleic acid sequence set forth in SEQ ID NO.l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO 7, or SEQ LD NO:9, its complementary strand or a subsequence thereof (J.
  • hybridization indicates that the analogous nucleic acid sequence hybridizes to the oligonucleotide probe corresponding to the polypeptide encoding part of the nucleic acid sequence shown in SEQ ID NO:l, SEQ ID NO.3, SEQ ID NO:5, SEQ ID NO.7, or SEQ ID NO 9, under low to high stringency conditions (for example, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA, and either 50, 35 or 25% formamide for high, medium and low stringencies, respectively), following standard Southern blotting procedures
  • the nucleic acid sequences are capable of hybridizing under medium stringency conditions and most preferably under high stringency conditions
  • the nucleic acid sequences are capable of hybridizing with the
  • SEQ ID NO:l SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO.7, or SEQ ID NO"9, as well as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10, or subsequences thereof may be used to design an oligonucleotide probe to isolate homologous genes encoding lipases from other strains of different genera or species according to methods well known in the art.
  • a genomic or cDNA library prepared from such other organisms may be screened for DNA which hybridizes with such probes followmg standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 40 nucleotides in length. Longer probes, preferably no more than 1200 nucleotides in length, can also be used. Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, biotin, or avidin)
  • a PCR reaction using the degenerate probes mentioned herein and genomic DNA or first-strand cDNA from an Absidia strain can also yield an Absidia lipase-specific product which can then be used as a probe to clone the corresponding genomic or cDNA
  • Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable earner material In order to identify clones or DNA which are homologous with SEQ ID NO: l, SEQ ID NO:3.
  • the carrier material is used in a Southern blot m which the carrier material is finally washed three times for 30 minutes each using 2X SSC, 0 2% SDS at preferably not higher than 40 °C, more preferably not higher than 45 °C, more preferably not higher than 50°C, more preferably not higher than 55 °C, even more preferably not higher than 60 °C, especially not higher than 65 °C.
  • Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using X-ray film.
  • the present invention also relates to isolated nucleic acid sequences which have a degree of identity to the nucleic acid sequence set forth m SEQ ID NO:l, SEQ ID NO.3, SEQ ID NO:5, SEQ ID NO 7, or SEQ ID NO:9 of at least about 65%, preferably about 70%, preferably about 75%, preferably about 80%, more preferably about 85%, even more preferably about 90%, most preferably about 95%, and even most preferably about 97%, which encode an active polypeptide
  • the degree of identity between two nucleic acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48: 443-453)
  • the Clustal method (DNASTAR, Inc., Madison, WI) is used with an identity table, a gap penalty of 10, and a gap length of 10
  • the present invention relates to isolated nucleic acid sequences encoding polypeptides with lipase activity which have an amino acid sequence with a degree of identity to the amino acid sequence set forth m SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, or SEQ ID NO: 10 of at least about 65%, preferably about 70%, preferably about 75%, preferably about 80%, more preferably about 85%, even more preferably about 90%, most preferably about 95%, and even most preferably about 97%, which qualitatively retain the activity of the polypeptides (hereinafter "homologous polypeptides").
  • the homologous polypeptides have an amino acid sequence which differs by five ammo acids, preferably by four ammo acids, more preferably by three ammo acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO-4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10
  • the degree of identity between two or more amino acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48- 443-453).
  • amino acid sequences of the homologous polypeptides encoded by the nucleic acid sequences of the present invention may differ from the ammo acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10 by an insertion or deletion of one or more ammo acid residues and/or the substitution of one or more ammo acid residues by different amino acid residues
  • am o acid changes are of a minor nature, that is conservative ammo acid substitutions which do not significantly affect the folding and/or activity of the protein: small deletions, typically of one to about 30 amino acids, small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about
  • the isolated nucleic acid sequences of the present invention which are capable of hybridizing with an oligonucleotide probe which hybridizes with the nucleic acid sequence set forth in SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO:5, SEQ ID NO 7, or SEQ ID NO:9, its complementary strand or a subsequence thereof, may be obtained from microorganisms of any genus, for example, from a bactenal or fungal source, but preferably from a fungal cell, and more preferably from a filamentous fungal cell or a yeast cell.
  • the term "obtained from” (or endogenous to) as used herein in connection with a given source shall mean that the polypeptide is produced by the source or by a cell in which a gene from the source has been inserted
  • Preferred sources for homologous genes are strains of the genus Absidia and species thereof available in public depositones
  • homologous genes may be identified and obtained from other sources includedmg microorganisms isolated from nature (e g , soil, composts, water, etc ) usmg the above- mentioned probes Techniques for isolating microorganisms from natural habitats are well known in the art.
  • the nucleic acid sequence may then be derived by similarly screening a cDNA library of another microorganism
  • Particularly preferred strains are filamentous fungus strains, such as an Acremomum, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe.
  • Mucor Mycehophthora, Neocalhmastix, Neurospora, Paecilomyces, Penicilhum, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma strain or yeast strains, such as a Candida, Kluyveromyces , Pichia, Saccharomyces , Schizosaccharomyces, or Yarrowia strain.
  • a nucleic acid sequence of the present invention is obtained from a strain of the genus Absidia, as described in M.A.A. Schipper, Persoonia, Vol 14, Part 2, pp 133- 148 (1990), such as a strain of Absidia griseola, Absidia sporophora-variabihs, Absidia griseola var iguchii, Absidia corymbifera, or Absidia blakesleeana.
  • the nucleic acid sequence is obtained from Absidia blakesleeana NN100826 (NRRL 1304), e g , the nucleic acid sequence set forth in SEQ ID NO.3; Absidia corymbifera NNI 00062 (IFO 8084), e g , the nucleic acid sequence set forth in SEQ ID N0 5; Absidia griseola NN000987 (ATCC 20430), Absidia griseola var iguchii NN000591 (ATCC 20431), e g , the nucleic acid sequence set forth in SEQ ID NO.l: Absidia sporophora-variabihs NN102427 (ATCC 36019), e g , the nucleic acid sequence set forth in SEQ ID NO:7; and Absidia reflexa NNI 02427 (ATCC 44896), e g , the nucleic acid sequence set forth m SEQ ID NO:9.
  • a corymbifera A NNI 00060 CBS 100 31, IFO corymbifera 4009, NRRL 2982
  • strains are readily accessible to the public in a number of culture collections, such as the Amencan Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
  • ATCC Amencan Type Culture Collection
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the sequence may be isolated or cloned by utilizing techniques which are well known to those of ordinary skill in the art (see, e g., Sambrook et ai, supra)
  • the known techniques used to isolate or clone a nucleic acid sequence include isolation from genomic DNA, preparation from cDNA, or a combination thereof.
  • the cloning of the nucleic sequences of the present invention from such genomic DNA can be effected, e , by using the well known polymerase chain reaction (PCR).
  • nucleic acid sequence may be cloned from a strain of Absidia producing the polypeptide, or another or related organism and thus, for example, may be an allelic or species vanant of the polypeptide encoding region ofthe nucleic acid sequence
  • Modification of the nucleic acid sequence encoding the polypeptide may be necessary for the synthesis of polypeptides substantially similar to the polypeptide
  • the term "substantially similar" to the polypeptide refers to non-naturally occurnng forms of the polypeptide.
  • These polypeptides may differ m some engineered way from the polypeptide isolated from its native source For example, it may be of interest to synthesize vanants of the polypeptide where the variants differ in specific activity, thermostability, oxidative stability, pH optimum, or the like using, for example, site-directed mutagenesis.
  • the analogous sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding region of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO 5 , SEQ ID NO:7, or SEQ ID NO:9, a sub-sequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production oi ' the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence
  • nucleotide substitution see, e.g , Ford et ai, 1991 , Protein Expression and Purification 2: 95-107.
  • amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for lipase activity to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of substrate- enzyme interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et ai, 1992, Science 255: 306-312; Smith et ai, 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et ai, 1992, FEBS Letters 309: 59-64).
  • Polypeptides encoded by the nucleic acid sequences of the present invention also include fused polypeptides in which another polypeptide is fused at the N-terminus or the C-termmus of the polypeptide or fragment thereof.
  • a fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention.
  • Techniques for producing fusion polypeptides are known in the art, and include, hgatmg the coding sequences encoding the polypeptides so that they are in frame and that expression ofthe fused polypeptide is under control ofthe same promoter(s) and terminator.
  • polypeptides having lipolytic activity encoded by the nucleic acid sequences of this invention are characterized by having a high activity at alkaline pH (about pH 9-10), even in the absence of free Ca".
  • these polypeptides have optimum lipolytic activity at about pH 9 or higher (have a higher activity at pH 9 than at pH 8) when tested in the absence of free Ca" .
  • Certam preferred nucleic acid sequences encode polypeptides having lipase activity when tested at pH 9 without free Ca ++ .
  • lipolytic enzymes can be obtained from strains of Absidia subgenus Mycocladus, e.g., the species and strains listed above.
  • nucleic acid sequences encode polypeptides having a higher lipolytic activity at pH 10 than pH 9 in the absence of Ca ++ .
  • Such a nucleic acid sequence can be obtained from Absidia reflexa NNI 02427 (ATCC 44896).
  • a further group of preferred nucleic acid sequences encodes polypeptides retaining more than 90% residual activity after 30 minutes incubation at pH 10, 45 °C.
  • Such a sequence can be obtained from a strain of Absidia sporophora-variabihs, e.g , Absidia sporophora-variabihs NN102427 (ATCC 36019).
  • the present invention also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences capable of directing the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • Nucleic acid construct is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurnng gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.
  • the term nucleic acid construct may be synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention.
  • coding sequence as defined herein is a sequence which is transcribed into mRNA and translated into a polypeptide of the present invention when placed under the control of the approp ⁇ ate control sequences.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • An isolated nucleic acid sequence encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide Manipulation of the nucleic acid sequence encoding a polypeptide prior to its insertion mto a vector may be desirable or necessary depending on the expression vector.
  • the techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art.
  • control sequences is defined herein to include all components which are necessary or advantageous for expression of the coding sequence of the nucleic acid sequence.
  • Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide
  • control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator
  • the control sequences include a promoter, and transcriptional and translational stop signals
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide
  • the control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell for expression of the nucleic acid sequence.
  • the promoter sequence contains transcription control sequences which mediate the expression of the polypeptide.
  • the promoter may be any nucleic acid sequence which shows transc ⁇ ptional activity in the host cell of choice and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the nucleic acid constructs of the present invention are the promoters obtained from the E coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicilhnase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et ai, 1978, Proceedings ofthe National Academy of Sciences USA 75: 3727-3731), as well as the tae promote
  • promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus ntger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae t ⁇ ose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described m U.S.
  • Patent No. 4,288,627 which is incorporated herein by reference
  • Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tp ⁇ (a hybrid of the promoters from the genes encodmg Aspergillus niger neutral a-amylase and Aspergillus oryzae tnose phosphate isomerase), and glaA promoters.
  • useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokmase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3 -phosphogly cerate kinase gene.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactokmase gene
  • ADH2/GAP Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes
  • Saccharomyces cerevisiae 3 -phosphogly cerate kinase gene Other useful promoters for yeast host cells are descnbed by Romanos et
  • useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bov e papilloma virus (BPV).
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription The terminator sequence is operably hnked to the 3 ' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used m the present invention.
  • Preferred terminators for filamentous fungal host cells are obtained from the genes encoding
  • Aspergillus oryzae TAKA amylase Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-hke protease.
  • Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYCl), or
  • Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase Other useful terminators for yeast host cells are described by Romanos et ai, 1992, supra Terminator sequences are well known m the art for mammalian host cells.
  • the control sequence may also be a suitable leader sequence, a nontranslated region of a mRNA which is important for translation by the host cell.
  • the leader sequence is operably linked to the 5 ' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence which is functional in the host cell of choice may be used in the present invention.
  • Preferred leaders for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase and Aspergillus oryzae t ⁇ ose phosphate isomerase.
  • Suitable leaders for yeast host cells are obtained from the Saccharomyces cerevisiae enolase
  • ENO-1 gene the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene, the Saccharomyces cerevisiae alpha-factor, and the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3- phosphate dehydrogenase genes (ADH2/GAP).
  • the control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
  • Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes encodmg Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-amylase
  • polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990. Polyadenylation sequences are well known in the art for mammalian host cells.
  • the control sequence may also be a signal peptide coding region, which codes for an ammo acid sequence hnked to the ammo terminus of the polypeptide which can direct the expressed polypeptide mto the cell's secretory pathway.
  • the 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked m translation reading frame with the segment of the coding region which encodes the secreted polypeptide
  • the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to that portion of the coding sequence which encodes the secreted polypeptide
  • the foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the lipase relative to the natural signal peptide coding region normally associated with the coding sequence
  • An effective signal peptide coding region for filamentous fungal host cells is the signal peptide coding region obtained from Aspergillus oryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, the Rhizomucor miehei aspartic proteinase gene, the Humicola lanuginosa cellulase gene, or the Rhizomucor miehei lipase gene
  • Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase Other useful signal peptide coding regions are described by Romanos et al , 1992, supra
  • the control sequence may also be a propeptide coding region, which codes for an ammo acid sequence positioned at the amino terminus of a polypeptide
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases)
  • a propolypeptide is generally inactive and can be converted to mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide
  • the propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factor gene, or the Myce ophthora thermophilum laccase gene (WO 95/33836)
  • the nucleic acid constructs of the present invention may also compnse one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide,
  • An activator is a protein which activates transcription of a nucleic acid sequence encoding a polypeptide (Kudla et al , 1990, EMBO Journal 9 1355-1364; Jarai and Buxton, 1994, Current Genetics 26: 2238-244, Verdier, 1990, Yeast 6: 271-297).
  • the nucleic acid sequence encoding an activator may be obtained from the genes encoding Bacillus stearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activator protein 1 (hapl), Saccharomyces cerevisiae galactose metabolizing protein 4 (gal4), and Aspergillus nidulans ammonia regulation protem (areA)
  • nprA Bacillus stearothermophilus NprA
  • hapl Saccharomyces cerevisiae heme activator protein 1
  • gal4 Saccharomyces cerevisiae galactose metabolizing protein 4
  • Aspergillus nidulans ammonia regulation protem are also examples.
  • a chaperone is a protem which assists another polypeptide folding properly (Hartl et al ,
  • the nucleic acid sequence encoding a chaperone may be obtained from the genes encoding Bacillus subtilis GroE proteins, Aspergillus oryzae protein disulphide isomerase, Saccharomyces cerevisiae calnexm, Saccharomyces cerevisiae B ⁇ P/GRP78, and Saccharomyces cerevisiae Hsp70 For further examples, see Gething and Sambrook, 1992, supra, and Hartl et ai, 1994, supra
  • a processing protease is a protease that cleaves a propeptide to generate a mature biochemically active polypeptide (Enderlm and Ogrydziak, 1994, Yeast 10: 67-79; Fuller et al , 1989, Proceedings of the National Academy of Sciences USA 86: 1434-1438; Julius et ai, 1984, Cell 37 1075-1089; Julius et ai, 1983, Cell 32: 839-852).
  • the nucleic acid sequence encoding a processing protease may be obtained from the genes Saccharomyces cerevisiae dipeptidylammopeptidase, Saccharomyces cerevisiae Kex2, and Yarrowia hpolytica dibasic processing endoprotease (xpr ⁇ ). It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound Regulatory systems in prokaryotic systems would include the lac, tae, and trp operator systems.
  • the ADH2 system or GAL1 system may be used.
  • the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences
  • Other examples of regulatory sequences are those which allow for gene amplification In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals In these cases, the nucleic acid sequence encoding the polypeptide would be placed in tandem w th the regulatory sequence
  • the present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals
  • the various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites
  • the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropnate vector for expression
  • the coding sequence is located m the vector so that the coding sequence is operably linked with the appropnate control sequences for expression, and possibly secretion
  • the recombinant expression vector may be any vector (e g , a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence
  • T e vectors may be linear or closed circular plasmids.
  • the vector may be an autonomously replicating vector, i e , a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e g , a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assunng self-replication
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated
  • the vector system may be a smgle vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon
  • the vectors of the present invention may be integrated into the host cell genome when introduced into a host cell
  • the vector may rely on the nucleic acid sequence encodmg the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination
  • the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell.
  • the additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise locat ⁇ on(s) in the chromosome(s).
  • the mtegrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination
  • the mtegrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell
  • the mtegrational elements may be non-encoding or encoding nucleic acid sequences
  • the vector may be integrated mto the genome of the host cell by non-homologous recombination
  • These nucleic acid sequences may be any sequence that is homologous with a target sequence in the genome of the host cell, and, furthermore, may be non-encoding or en
  • the vector may further compnse an origin of replication enabling the vector to replicate autonomously in the host cell in question
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication m E coli, and pUBl 10, pE194, pTA1060, and pAMBl permitting replication in Bacillus
  • origin of replications for use m a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARSl
  • the origin of replication may be one having a mutation which makes its functioning temperature- sensitive in the host cell (see, e g , Ehrhch, 1978, Proceedings of the National Academy of Sciences USA 75 1433)
  • the expression vector may be pZL-NLl, pZL-NL61 , pZI - NL95, or pZL-NL124
  • the vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like
  • Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphe col or tetracycline resistance
  • a frequently used mammalian marker is the dihydrofolate reductase gene Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRPl, and URA3
  • a selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithme carbamoyltransferase), bar (phosphinoth ⁇ cin acetyltrans
  • the present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides.
  • host cell encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur dunng replication
  • the cell is preferably transformed with a vector comprising a nucleic acid sequence of the invention followed by integration of the vector into the host chromosome.
  • Transformation means introducing a vector comprising a nucleic acid sequence of the present invention into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector mto the host chromosome occurs homologous or non-homologous recombination as described above
  • the choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the host cell may be a unicellular microorganism, e g , a prokaryote, or a non-unicellular microorganism, e.g , a eukaryote.
  • Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g , Bacillus alkalophilus, Bacillus amyloliquefacien , Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus.
  • a Bacillus cell e.g , Bacillus alkalophilus, Bacillus amyloliquefacien , Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus.
  • the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell
  • the transformation of a bacterial host cell may, for instance, be effected by protoplast transformation (see, e g , Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e g , Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971 , Journal of Molecular Biology 56: 209-221), by electroporation (see,
  • the host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell.
  • a mammalian cell such as a mammalian cell, an insect cell, a plant cell or a fungal cell.
  • Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.
  • the host cell is a fungal cell.
  • "Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et ai, In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al, 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al, 1995, supra).
  • Examples of Basidiomycota include mushrooms, rusts, and smuts.
  • Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi.
  • Representative groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicillium, Candida, and Alternaria.
  • Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.
  • the fungal host cell is a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).
  • the ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces).
  • Schizosaccharomycoideae e.g., genus Schizosaccharomyces
  • Nadsonioideae e.g., Lipomycoideae
  • Saccharomycoideae e.g., genera Pichia
  • the basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus , Filobasidium, and Filobasidiella.
  • Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980.
  • yeast host cell is a cell of a species of Candida
  • yeast host cell is a Saccharomyces carlsbergensis
  • Saccharomyces cerevisiae Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
  • the yeast host cell is a Kluyveromyces lactis cell.
  • the yeast host cell is a Yarrowia lipolytica cell.
  • the fungal host cell is a filamentous fungal cell.
  • filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al, 1995, supra).
  • the filamentous fungi are characterized by a vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obhgately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma.
  • the filamentous fungal host cell is an Aspergillus cell.
  • the filamentous fungal host cell is an Acremonium cell.
  • the filamentous fungal host cell is a Fusarium cell.
  • the filamentous fungal host cell is a Humicola cell.
  • the filamentous fungal host cell is a Mucor cell. In another even more preferred embodiment, the filamentous fungal host cell is a Myceliophthora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Neurospora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Penicillium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Thielavia cell. In another even more preferred embodiment, the filamentous fungal host cell is a Tolypocladium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Trichoderma cell.
  • the filamentous fungal host cell is an Aspergillus awamon, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell.
  • the filamentous fungal host cell is a Fusarium cerealis, Fusarium crookwellense, Fusarium graminearum, Fusarium oxysporum, Fusarium sambucinum or Fusarium sulphureum cell.
  • the filamentous fungal host cell is a Humicola insolens or Humicola lanuginosa cell.
  • the filamentous fungal host cell is a Mucor miehei cell. In another most preferred embodiment, the filamentous fungal host cell is a Myceliophthora thermophilum cell. In another most preferred embodiment, the filamentous fungal host cell is a Neurospora crassa cell. In another most preferred embodiment, the filamentous fungal host cell is a Penicillium purpurogenum cell. In another most preferred embodiment, the filamentous fungal host cell is a Thielavia terrestris cell. In another most preferred embodiment, the Trichoderma cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma Iongibrachiatum, Trichoderma reesei or Trichoderma viride cell.
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et ai, 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. A suitable method of transforming Fusarium species is described by Malardier et ai, 1989, Gene 78: 147-156 or in copending US Serial No. 08/269,449, inco ⁇ orated herein by reference. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
  • Mammalian cells may be transformed by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52: 546).
  • the present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive to expression of the polypeptide; and (b) recovering the polypeptide.
  • the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art.
  • the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).
  • suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it is recovered from cell lysates.
  • the polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.
  • the production of lipase activity can be determined by any method known in the art. In one method, one Lipase Unit (LU) is the amount of enzyme which liberates 1 0 ⁇ mol of titratable fatty acid per minute with tnbuty ⁇ n as substrate and gum arabic as an emulsifier at 30°C, pH 7 0 (phosphate buffer) Lipolytic enzyme activity in the absence of free Ca" in the range pH 7-10 is tested with a substrate emulsion of olive oil.
  • LU Lipase Unit
  • the resulting polypeptide may be recovered by methods known in the art
  • the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • the recovered polypeptide may then be further purified by a variety of chromatographic procedures, e g . ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like
  • polypeptides of the present invention may be purified by a variety of procedures known m the art including, but not limited to, chromatography ( , ion exchange, affinity, hydrophobic, chromatofocusmg, and size exclusion), electrophoretic procedures (e.g , preparative lsoelectnc focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e , Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • chromatography , ion exchange, affinity, hydrophobic, chromatofocusmg, and size exclusion
  • electrophoretic procedures e.g , preparative lsoelectnc focusing (IEF)
  • differential solubility e.g., ammonium sulfate precipitation
  • extraction see, e , Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
  • the recombinant polypeptides enoded by the nucleic acid sequences of the invention may be used in conventional applications of lipolytic enzyme, particularly at a high pH, e.g., m laundry and dishwashing detergents, institutional and industrial cleaning and leather processing
  • the lipolytic enzymes of the invention can also be used for lntereste ⁇ fication, for total hydrolysis of fats and oils, and in optical isomer resolution process.
  • Absidia griseola NN000987 (ATCC 20430), Absidia sporophora-variabihs N I 02427
  • the frozen myce a preparations were ground to a fine powder m an electnc coffee grinder, and the powders were each added to a disposable plastic centrifuge tube containing 20 ml of TE buffer and 5 ml of 20% w/v sodium dodecylsulfate (SDS).
  • SDS sodium dodecylsulfate
  • the mixtures were gently inverted several times to ensure mixing, and extracted twice with an equal volume of phenol:chloroform: ⁇ soamyl alcohol (25:24:1 v/v/v).
  • Sodium acetate (3 M solution) was added to the extracted samples to a final concentration of 0.3 M followed by 2.5 volumes of ice cold ethanol to precipitate the DNA.
  • the tubes were centnfuged at 15,000 x g for 30 minutes to pellet the DNA
  • the DNA pellets were allowed to air-dry for 30 minutes before resuspension m 0.5 ml of TE buffer.
  • DNase-free ⁇ bonuclease A was added to the resuspended DNA pellets to a concentration of 100 ⁇ g/ml and the mixtures were then incubated at 37°C for 30 min Proteinase K (200 ⁇ g/ml) was added and each tube was incubated an additional one hour at 37°C
  • each sample was extracted twice with phenol:chloroform: ⁇ soamyl alcohol (25:24: 1 v/v/v) before precipitating the DNA with sodium acetate and ethanol.
  • the DNA pellets were d ⁇ ed under vacuum, resuspended TE buffer, and stored at 4°C.
  • Example 2 PCR Amplification of Absidia griseola NN000987 and Absidia griseola var. iguchii NN000591 Lipase Gene Segments Based on the ammo acid sequences of the Absidia griseola NN000987 lipase and the Absidia griseola var.
  • iguchii NN000591 lipase as disclosed by Gormsen et ai in Patent Application DK 95/00424 (the contents of which are inco ⁇ orated herein by reference), the oligonucleotide primers shown below were synthesized with an Applied Biosystems Model 394 DNA RNA Synthesizer, according to the manufacturer's instructions, to PCR amplify lipase gene fragments from Absidia griseola NN000987 and Absidia griseola var.
  • Oligonucleotide (- strand): 5'-ARRAANCCRAAIGCNCCIGGNGG-3' (SEQ ID NO: 14)
  • Amplification reactions (100 ml) were prepared using approximately 1 ⁇ g of Absidia griseola NN000987 or Absidia griseola var. iguchii NN000591 genomic DNA as the template. Each reaction contained the following components: 1 ⁇ g of genomic DNA, 40 pmol of forward primer, 40 pmol of reverse primer, 200 mM each of dATP, dCTP, dGTP, and dTTP, 1 x Taq polymerase buffer (Perkm-Elmer Co ⁇ ., Branchburg, NJ), and 5 Units of Taq polymerase (Perkin-Elmer Co ⁇ , Branchburg, NJ).
  • Lipase gene segments of approximately 870 bp were amplified from Absidia griseola NN000987 and Absidia griseola var. iguchii NN000591 as shown in Figure 1 with the lipase-specific PCR primers described above. DNA sequence analysis showed that the amplified gene segments encode portions of the corresponding Absidia lipase genes. In addition, the DNA sequence data confirmed that these two gene products are probably identical and share regions of homology with the Rhizomucor miehei lipase.
  • Example 3 Hybridization Analysis of Genomic DNA
  • Total cellular DNA samples prepared from the five Absidia strains desc ⁇ bed in Example 1 were analyzed by Southern hybridization (Maniatis et ai, 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York). Approximately 2-5 ⁇ g of each DNA sample was digested with EcoRI, Aspl 1%1 or EcoRI plus Asp7 ⁇ Sl and fractionated on a 1% agarose gel.
  • the gel was photographed under short wavelength UV light and soaked for 15 minutes in 0.5 M NaOH- 1.5 M NaCl followed by 15 minutes in 1.5 M NaCl- 1 M Tris-HCl pH 8 DNA in the gel was transferred onto Nytran ⁇ hybridization membrane (Schleicher & Schuell, Keene, NH) by capillary blotting in 20 X SSP ⁇ (3 M sodium chlo ⁇ de-0.2 M sodium dibasic phosphate-0.02 M disodium ⁇ DTA) according to Davis et al (1980, Advanced Bacterial Genetics, A Manual for Genetic Engineering, Cold Spnng Harbor Press, Cold Spnng Harbor, New York).
  • the DNA was cross-linked onto the membrane using a UV Stratahnker (Stratagene, La Jolla, CA), and the membrane was soaked for 2 hours in the following hybridization buffer at 45 °C with gentle agitation 5 X SSP ⁇ , 35% formamide (v/v), 0.3% SDS, and 200 mg/ml denatured and sheared salmon testes DNA.
  • the lipase-specific probe fragment isolated from the Absidia griseola NN000987 PCR-clone described in Example 2 was radiolabeled by nick translation (Maniatis et al , 1982, supra) with [ 2 P]dCTP (Amersham, Arlington Heights, IL), denatured by adding NaOH to a final concentration of 0.1 M, and added to the hybridization buffer at an activity of approximately 1 x 10 6 cpm per ml of buffer. The mixture was incubated overnight at 45 °C in a shaking water bath.
  • the membranes were washed once in 0.2 X SSPE with 0.1% SDS at 45 °C followed by two washes in 0.2 X SSPE (no SDS) at the same temperature.
  • the membranes were allowed to dry on paper towels for 15 minutes, then wrapped in Saran-WrapTM and exposed to X-ray film overnight at -70°C with intensifying screens (Kodak, Rochester, NY).
  • Enriched genomic DNA libraries were constructed m the bacteriophage cloning vector lZipLox (Life Technologies, Gaithersburg, MD). First, total cellular DNA was digested with EcoRI and size-fractionated on 1% agarose gels. DNA fragments migrating m the size range corresponding to hybridization signals previously observed on Southern blots described m Example 3 were excised and eluted from the gel using Prep-a-Gene reagents (BioRad Laboratories, Hercules, CA). The approximate sizes of the DNA fragments in these fractions were as follows: 1.9-2.5 kb (Absidia griseola var.
  • iguchii NN000591 1.9-2.5 kb (Absidia blakesleeana NN100826), 1.9-2.5 kb (Absidia corymbifera NN100062) and 2.5-4.3 kb (Absidia sporophora-variabihs NN102427).
  • the eluted DNA fragments were ligated with EcoRI-cleaved and dephosphorylated ⁇ ZipLox vector arms (Life Technologies, Gaithersburg, MD), and the ligation mixtures were packaged using commercial packaging extracts (Stratagene, La Jolla, CA) The packaged DNA libraries were plated and amplified in Escherichia coli Y1090ZL cells (Life Technologies, Gaithersburg, MD).
  • the titers of recombinant phage in each library ranged from 1.3-5.4 X 10 5 pfu/ml (background titers with no DNA were 1.7-2.0 X 10 4 pfu ml).
  • Approximately 15,000-30,000 plaques from each unamplified library were screened by plaque-hybridization using the lipase-specific PCR fragment from Absidia griseola NN000987 as the probe (Davis et ai, 1980, supra). Plaques, which gave strong hybridization signals with the probe, were purified twice in E.
  • coli Y1090ZL cells and the lipase genes were subsequently excised from the ⁇ ZipLox vector as pZL 1 -derivatives (D'Alessio et al , 1992, Focus® 14: 76).
  • the recombinant DNA segments were inserted within the phagemid pZLl portion of the vector, and the phagemid harboring the cloned insert was recovered in the autonomously replicating pZLl using in vivo excision by infection of E coli DHlOBzip cells (Life Technologies, Gaithersburg, MD)
  • the lipase clones isolated in this manner were prepared for DNA sequence analysis using a Wizard 373 DNA pu ⁇ fication kit (Promega, Madison, WI)
  • DNA sequencing of the lipase clones descnbed in Example 4 was performed with an Applied Biosystems Model 373A Automated DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) on both strands using the pnmer walking technique with dye-terminator chemistry (Giesecke et al , 1992, Journal of Virol Methods 38 47-60) Oligonucleotide sequencing pn ers were synthesized on an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions
  • Absidia reflexa lipase In order to isolate a gene encoding the Absidia reflexa lipase, a strain of Absidia reflexa N I 02427 (ATCC 44896) was grown on optimal medium with jojoba oil as an induction component A cDNA library was prepared from this strain, and, using the Absidia corymbifera gene descnbed above as a probe, a cDNA clone was identified by colony hybndization The sequence of the Absidia reflexa gene is provided m Figure 7 (SEQ ID NO 9) This lipase is approximately 99% identical with the Absidia sporophora-variabihs lipase
  • the intron-exon organization among Absidia lipase genes is very similar in that all are composed of seven exons and six mtrons as shown in Table 1
  • the size of the exons 2 through 6 are stnetly conserved
  • the first exon shows slight vanabi ty in size due to some vanation in the region encoding the signal peptide and propeptide portions of the enzyme
  • the nucleotide sequence homology between corresponding exons is also very high with 85-97% identity
  • the lengths of the introns va ⁇ ed, and with the exception of the Absidia corymbifera N I 00062 and Absidia sporophora-variabihs NNI 02427 lipase genes there is little sequence homology between corresponding introns
  • the 5 ' and 3' flanking sequences of the lipase genes also shows sequence divergence
  • Absidia lipases share extensive amino acid sequence homology between each other with 87-96% identity, and limited homology to Rhizomucor miehei lipase (SEQ ID NO 15) with 53-55% identity and Humicola lanuginosa lipase (SEQ ID NO 16) with 22-24% identity as shown in Table 3 and Figure 8 Table 3 Amino acid sequence similarity among lipases from Absidia species, R miehei, and H lanuginosa
  • Absidia lipases are most closely related to the lipases from other members of the Zygomycete class of fungi Yeast, bacterial, and mammalian lipases as well as fungal cutinases appear to be very distantly related if at all.
  • the clone and the nucleotide sequences of the Absidia corymbifera lipase gene described above were used for subcloning of the gene and expression in an Aspergillus host. PCR was used to subclone the lipase gene (without its own promoter) from the isolated genomic clone NL95A using primers designed from the nucleotide sequences.
  • pBANe ⁇ Figure 9
  • Swal and Pad restriction enzyme sites were introduced.
  • the vector pBANE6 contained the TAKA promoter, NA2-tp ⁇ leader, and AMG terminator as regulatory sequences.
  • the plasmid also contained the Aspergillus nidulans amdS gene as a selectable marker for fungal transformations
  • the following primers were used for PCR amplification: Forward Primer: 5'-CCCATTTAAATATGCGTTTTTATTCAGTAGTATCAT-3' (SEQ ID NO: 17)
  • Reverse primer 5'-CTCGGCTTAATTAAAATGGGTTATAAGCAGAGACCAGTG-3' (SEQ ID NO: 18) PCR was performed using Pwo polymerase (Boehringer Mannheim, Indianapolis, IN) according to manufacturer's specifications. The PCR amplified product was gel isolated, cut with Swal and Pad enzymes, and gel purified. The purified fragment was ligated to the pBANe ⁇ vector (already cut with Swal and Pad) to yield the plasmid pKB2 ( Figure 10) in which transcription of the lipase gene was under the control of the TAKA promoter. The plasmid pKB2 was transformed mto E coli DH5 cells E coli transformants containing the pKB2 plasmid were isolated and plasmid DNA was prepared for transformation and expression in Aspergillus.
  • Protoplasts were prepared from Aspergillus oryzae strain BANe3 in which the amdS gene of the host strain was deleted. Protoplast preparation and transformation were done as previously described (Ch ⁇ stensen et ai, supra). Aspergillus oryzae transformants expressing acetamidase were selected based on their ability to utilize acetamide as the sole nitrogen source. A total of 42 transformants were generated and spore purified twice on selective plates The spore purified transformants were used for further analysis
  • the transformants were screened for lipase expression by cultivation in shake flasks (25 ml medium in 125 ml flasks) using a medium containing 50 g of maltose, 2.0 g of MgS0 4 -7H 2 0, 10 g of KH,P0 4 , 2 0 g of K,S0 4 , 2.0 g of citric acid, 10 g of yeast extract, 0 5 ml of trace metals solution, and 2 0 g of urea per liter
  • the trace metals solution was comprised of 14.3 g of ZnS0 4 -7H 2 0, 2.5 g of CuS0 4 -5H 2 0, 0.5 g of N ⁇ Cl,-6H 2 0), 13.8 g of FeS0 4 -7H 2 0), 8.5 g of MnSOschreib-H 2 0, and 3.0 g of citric acid per liter
  • the pH of the medium was adjusted to 6.5 before sterilization by autoclaving.
  • Flasks were inoculated with freshly harvested spores and incubated in an incubator at 34 °C and 200 ⁇ m Cultures were assayed for lipase activity daily after 48 hours of cultivation Since nothing was known about the suitable substrate for this lipase, enzyme activity was assayed by three methods: I) lipase plate assay using olive oil as the substrate, n) colo ⁇ met ⁇ cally using p-mtrophenylbutyrate as the substrate and in) titration using t ⁇ butyrm (tnglyce ⁇ de of butyric acid) A lipase plate assay was performed using a plate medium that contained the following. 0.1 M
  • Lipase activity was also measured colonmet ⁇ cally using p-nitrophenylbutyrate as a substrate.
  • p-Nitrophenylbutyrate was prepared by adding 10 ml of this compound to 1.0 ml dimethylsulfoxide (DMSO) and 4.0 ml of 0.1 M Tns pH 9.0 buffer One hundred microliters of suitably diluted culture broth were added to each well The reaction was started by adding 100 ml of the p-nitrophenylbutyrate substrate and the absorbance was measured at 405 nm for 3-5 min. The enzyme activity was calculated from a curve made with a known amount of Humicola lanuginosa lipase as the standard. The untransformed strain produced little or no activity while different transformants produced lipase after 48 hours of cultivation.
  • Lipase activity was further determined by titration based on the hydrolysis of t ⁇ buty ⁇ n catalyzed by the lipase. The liberation of butync acid was followed by alkaline titration in a pH-stat. The assay was performed on culture broths from selected transformants as well as from the untransformed control strain. The results showed that the untransformed control strain produced no lipase activity, while the transformants produced detectable lipase activity.
  • Example 8 Expression of the Absidia sporophora-variabihs Lipase Gene in Aspergillus oryzae.
  • the coding region of the A. sporophora-variabihs lipase was amplified using the original genomic clone as the template for Pwo polymerase (Boehringer-Mannheim Biochemicals, Indainapohs, IN) in a PCR reaction which contained the following: 61 ml sterile water, 10 ml of diluted template DNA (ca. 5 ng/ml), 1 ml pnmer 1 (ca. 30 pmol. dATGATGCATTCTCATTTTGTAGTCTTATTG, SEQ ID NO:19), 1 ml primer 2 (ca.
  • Amplification conditions were as follows: First cycle at 95 °C for 5 minutes, 45 °C for 2 minutes, and 67 °C for 5 minutes; cycles 2 through 30 at 95°C for 2 minutes, 45°C for 1 minute, and 67°C for 2 minutes; and a soak cycle at 4°C.
  • the amplified lipase gene segment was digested with Pad and isolated by preparative agarose gel electrophoresis, excised and purified using Prep-a-Gene reagents (BioRad Laboratories, Hercules, CA). The purified fragment was ligated with pBANe6 which had been cleaved with Swal and Pad to generate the lipase expression vector pRaMB19 ( Figure 11). pRaMB19 was subsequently used to transform an alkaline protease-deficient Aspergillus oryzae host JaL142 using standard methods (Ch ⁇ stensen et al. 1988.
  • transformants After a 48 hour incubation at 30 °C, 80 of 84 transformants showed distinct clea ⁇ ng zones on the tributynn agar plates indicating production of extracellular lipase activity. Ten of these transformants were further tested in shake flask cultures of MY50 medium containing 50 g of maltodextrin, 2 g of MgS0 4 -7H 2 0, 10 g of KH 2 P0 4J 2 g of citric acid, 2 g of K 2 S0 4 , 0.5 ml of trace metals solution, 10 g of yeast extract, 2 g of urea pH 6.0 per liter.
  • E. coli DH 1 OB (pZL-NL61 ) - Absidia corymbifera NRRL B-21521
  • E. coli DH10B (pZL-NL95) - Absidia griseola-iguchn NRRL B-21522
  • E. coli DH 1 OB (pZL-NL124) - Absidia sporophora-variablihs NRRL B-21523

Abstract

The present invention relates to isolated nucleic acid sequences encoding polypeptides having Absidia lipase activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides. The invention further relates to compositions comprising the polypeptides.

Description

NUCLEIC ACIDS ENCODING POLYPEPTIDES HAVING ABSD3IA LIPASE ACTIVITY
Background of the Invention Field ofthe Invention
The present invention relates to isolated nucleic acid sequences encoding polypeptides having Absidia lipase activity. The invention also relates to nucleic acid constructs, vectors, and host cells compnsing the nucleic acid sequences as well as recombinant methods for producing the polypeptides.
Description of the Related Art Detergents formulated with lipolytic enzymes are known to have improved properties for removing fatty stains. For example, LIPOLASE™ (Novo Nordisk A/S, Bagsvaerd, Denmark), a microbial lipase obtained from the fungus Thermomyces lanuginosus (also called Humicola lanugtnosa), has been introduced into many commercial brands of detergent.
Other microbial lipases from Pseudomonas cepacta (U.S. Patent No 4,876,024), Streptomycetes (WO 94/14940), and Gongronella butleri strain NRJRL 3521 (U.S. Patent No. 3,634,195, the strain was previously named Absidia butleri, see K.H. Domsch et al, Compendium of Soil Fungi, Academic Press 1980, p. 381) have also been suggested for use m detergents.
U.S. Patent No. 3,634,195 descπbes the production of lipase from Absidia cylindrospora var rhizomorpha NRRL 2815 and Absidia blakesleeana NRRL 1305. Koπtala et al. (1987, Journal of the American Oil Chemists Society 64: 509-513) disclose that soybean oil was partially hydrolyzed when incubated with Absidia coerula NRRL 5926 and Absidia ramosa NRRL 1309. Satyanarayana (1981, Current Science 50: 680-682) discloses the secretion of lipase by a strain of Absidia corymbifera Aisaka et al. (1979, Agricultural Biological Chemistry 43: 2125-2129) describe the formation of a lipoprotein lipase from Absidia hyalospora strain KY 303 (now classified as Absidia blakesleeana)
Many detergents are alkaline m solution (e g , around pH 10) and contain a builder to bind Ca** ions. There is a need for new lipolytic enzymes with high activity at high pH in the absence of Ca"" Lipases of the genus Absidia possess these characteristics, and therefore, are highly desirable for use in detergent compositions. Heretofore, however, there has been no means of producing these enzymes recombinantly.
It is an object of the present invention to provide for recombinant production of these valuable enzymes Summary of the Invention
The present invention relates to isolated nucleic acid sequences encoding polypeptides having lipase activity selected from the group consisting of:
(a) a nucleic acid sequence which encodes a polypeptide endogenous to an Absidia strain with an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10;
(b) a nucleic acid sequence endogenous to an Absidia strain which is capable of hybridizing under medium stringency conditions with (i) the nucleic acid sequence set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or (ii) any of their complementary strands;
(c) a nucleic acid sequence which is capable of hybridizing under medium stringency conditions with (l) the nucleic acid sequence set forth in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or (ii) any of their complementary strands;
(d) a nucleic acid sequence encodmg a polypeptide having hpase activity with an ammo acid sequence which has at least 65% identity with the ammo acid sequence set forth m SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10;
(e) an allelic form of (a), (b), (c), or (d); and
(f) a fragment of (a), (b), (c), (d), or (e).
The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.
Brief Description of the Figures
Figure 1 shows an agarose gel purification of hpase-specific PCR products from Absidia griseola and Absidia griseola var. iguchii genomic DNA. Lane 1 : Hi'ndlll-digested lambda DNA and
Hαelll-digested øX17RF-DNA size standards; lane 2: Absidia griseola PCR product; and lane 3:
Absidia griseola var. iguchii PCR product. Both PCR products appear to be approximately 870 bp in size.
Figure 2 shows an autoradiogram from Southern hybridization analysis of genomic DNA digests from several Absidia species probed with a radiolabeled lipase gene segment from Absidia griseola. The sizes of HmdIII-digested lambda DNA and Hαelll-digested øX17RF-DNA size standards are indicated on the right side of the autoradiogram. Lanes 1-3: Absidia sporophora- variabihs DNA (EcoRI plus Aspllil, Aspl\%\ and £coRI, respectively); lanes 4-6: Absidia corγmbifera DNA (EcoRI plus AspllSl, Λsp718I and EcoRI, respectively); lanes 7-9: Absidia blakesleeana DNA (EcoRI plus AspllSl, Aspllϋl and £coRI, respectively); lanes 10-12: Absidia griseola var. iguchii DNA (EcoRI plus Asp7\ il, Asp7\ 8l and EcoRI, respectively); and lanes 13-15 Absidia griseola DNA (EcoRI plus Asp7\ 81, Asp71&\ and EcoRI, respectively)
Figure 3 shows the DNA sequence and deduced amino acid sequence of Absidia griseola var iguchii lipase (SΕQ ID NOS. 1 and 2, respectively) Introns are marked by a solid hne Regions corresponding to previously determined peptide sequences are underlined ( — )
Figure 4 shows the DNA sequence and deduced amino acid sequence of Absidia blakesleeana lipase (SΕQ ID NOS. 3 and 4, respectively) Introns are marked by a solid line Regions corresponding to previously determined peptide sequences are underlined ( — )
Figure 5 shows the DNA sequence and deduced ammo acid sequence of Absidia corymbifera lipase (SΕQ ID NOS 5 and 6, respectively) Introns are marked by a solid line. Regions corresponding to previously determined peptide sequences are underlined ( — ).
Figure 6 shows the DNA sequence and deduced ammo acid sequence oi Absidia sporophora- vanabi s lipase (SΕQ ID NOS. 7 and 8, respectively). Introns are marked by a solid line
Figure 7 shows the DNA sequence and deduced ammo acid sequence of Absidia reflexa lipase (SΕQ ID NOS 9 and 10, respectively).
Figure 8 shows a comparison of the ammo acid sequence homology among Absidia lipases compared to Rhizomucor miehei lipase (SΕQ ID NO: 15) and Humicola lanuginosa (SΕQ ID NO: 16) Identical residues are boxed.
Figure 9 shows a restriction map of pBANeό. Figure 10 shows a restriction map of pKB2.
Figure 1 1 shows a restriction map of pRamB19.
Detailed Description of the Invention
Nucleic Acid Sequences
In a first embodiment, the present invention relates to isolated nucleic acid sequences which encode polypeptides having lipase activity with an amino acid sequence set forth in SΕQ ID NO.2, SΕQ ID NO:4, SΕQ ID NO:6, SΕQ ID NO:8, or SΕQ ID NO: 10. In a specific embodiment, the nucleic acid sequences are set forth in SΕQ ID NO:l, SΕQ ID NO:3, SΕQ ID NO:5, SΕQ ID NO 7, and SΕQ ID NO:9. The nucleic acid sequences of the present invention also encompass nucleic acid sequences which encode a polypeptide having the am o acid sequence set forth m SΕQ ID NO.2, SΕQ ID NO:4, SΕQ ID NO:6, SΕQ ID NO:8, or SΕQ ID NO: 10, but differ from SΕQ ID NO: l , SΕQ ID NO:3. SΕQ ID NO:5, SΕQ ID NO:7, or SΕQ ID NO:9, respectively, by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SΕQ ID NO:l , SΕQ ID NO 3, SΕQ LD NO:5, SΕQ ID NO:7, or SΕQ ID NO:9, which encode a fragment of SΕQ ID NO:2, SΕQ ID NO-4, SΕQ ID N0 6, SΕQ ID NO:8, or SΕQ ID NO: 10, respectively, and retain lipase activity In a preferred embodiment, the nucleic acid sequences of the present invention are the nucleic acid sequences contained m plasmids pZL-NLl, pZL-NL61, pZL-NL95, and pZL-NL124, which are contained in Escherichia coli NRRL B-21520, NRRL B-21521 , NRRL B-21522, and NRRL B-21523, respectively The term "isolated nucleic acid sequence" as used herein refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used m genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incoφoration of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof
The term "lipase" is defined herein as a lipolytic enzyme classified under the Enzyme Classification number E.C. 3.1.1 - (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB) Lipolytic enzymes thus exhibit hydrolytic activity towards at least one of the types of ester bonds mentioned m the context of E.C. 3.1.1, for example, ester bonds present in mono-, di- and triglycerides, phospholipids (all classes), thioesters, cholesterol esters, wax-esters, cutin, subeπn, synthetic esters, etc As an example, the lipolytic enzymes of the present invention may have activity towards triglycerides (lipase activity, E C 3 1 1.3), e g , 1,3 -positionally specific lipase activity In a second embodiment, the present invention relates to isolated nucleic acid sequences encoding polypeptides with lipase activity which are capable of hybridizing under high, medium, or low stringency conditions with an oligonucleotide probe which hybridizes under the same conditions with the nucleic acid sequence set forth in SEQ ID NO.l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO 7, or SEQ LD NO:9, its complementary strand or a subsequence thereof (J. Sambrook, E.F Fπtsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). Hybridization indicates that the analogous nucleic acid sequence hybridizes to the oligonucleotide probe corresponding to the polypeptide encoding part of the nucleic acid sequence shown in SEQ ID NO:l, SEQ ID NO.3, SEQ ID NO:5, SEQ ID NO.7, or SEQ ID NO 9, under low to high stringency conditions (for example, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 50, 35 or 25% formamide for high, medium and low stringencies, respectively), following standard Southern blotting procedures In a preferred embodiment, the nucleic acid sequences are capable of hybridizing under medium stringency conditions and most preferably under high stringency conditions In another preferred embodiment, the nucleic acid sequences are capable of hybridizing with the nucleic acid sequence set forth in SEQ ID NO: l, SEQ ID NO:3, SEQ ID N0 5, SEQ ID NO 7, or SEQ ID NO:9, or its complementary strand.
SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO.7, or SEQ ID NO"9, as well as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10, or subsequences thereof may be used to design an oligonucleotide probe to isolate homologous genes encoding lipases from other strains of different genera or species according to methods well known in the art. Thus, a genomic or cDNA library prepared from such other organisms may be screened for DNA which hybridizes with such probes followmg standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 40 nucleotides in length. Longer probes, preferably no more than 1200 nucleotides in length, can also be used. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, biotin, or avidin) A PCR reaction using the degenerate probes mentioned herein and genomic DNA or first-strand cDNA from an Absidia strain can also yield an Absidia lipase-specific product which can then be used as a probe to clone the corresponding genomic or cDNA Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable earner material In order to identify clones or DNA which are homologous with SEQ ID NO: l, SEQ ID NO:3. SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, the carrier material is used in a Southern blot m which the carrier material is finally washed three times for 30 minutes each using 2X SSC, 0 2% SDS at preferably not higher than 40 °C, more preferably not higher than 45 °C, more preferably not higher than 50°C, more preferably not higher than 55 °C, even more preferably not higher than 60 °C, especially not higher than 65 °C. Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using X-ray film. The present invention also relates to isolated nucleic acid sequences which have a degree of identity to the nucleic acid sequence set forth m SEQ ID NO:l, SEQ ID NO.3, SEQ ID NO:5, SEQ ID NO 7, or SEQ ID NO:9 of at least about 65%, preferably about 70%, preferably about 75%, preferably about 80%, more preferably about 85%, even more preferably about 90%, most preferably about 95%, and even most preferably about 97%, which encode an active polypeptide The degree of identity between two nucleic acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48: 443-453) For purposes of determining the degree of identity between two nucleic acid sequences for the present invention, the Clustal method (DNASTAR, Inc., Madison, WI) is used with an identity table, a gap penalty of 10, and a gap length of 10
In a third embodiment, the present invention relates to isolated nucleic acid sequences encoding polypeptides with lipase activity which have an amino acid sequence with a degree of identity to the amino acid sequence set forth m SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, or SEQ ID NO: 10 of at least about 65%, preferably about 70%, preferably about 75%, preferably about 80%, more preferably about 85%, even more preferably about 90%, most preferably about 95%, and even most preferably about 97%, which qualitatively retain the activity of the polypeptides (hereinafter "homologous polypeptides"). In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five ammo acids, preferably by four ammo acids, more preferably by three ammo acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO-4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10 The degree of identity between two or more amino acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48- 443-453). For purposes of determining the degree of identity between two ammo acid sequences for the present invention, the Clustal method (DNASTAR, Inc., Madison, WI) is used with an identity table, a gap penalty of 10, and a gap length of 10. The amino acid sequences of the homologous polypeptides encoded by the nucleic acid sequences of the present invention may differ from the ammo acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10 by an insertion or deletion of one or more ammo acid residues and/or the substitution of one or more ammo acid residues by different amino acid residues Preferably, am o acid changes are of a minor nature, that is conservative ammo acid substitutions which do not significantly affect the folding and/or activity of the protein: small deletions, typically of one to about 30 amino acids, small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates punfication by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain Examples of conservative substitutions are withm the group of basic ammo acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic ammo acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalamne, tryptophan and tyrosine), and small ammo acids (such as glycine, alanine, serine, threonine and methionine) Ammo acid substitutions which do not generally alter the specific activity are known in the art and are described, e g , by H Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York The most commonly occurnng exchanges are: Ala Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu Val, Ala/Glu, Asp/Gly as well as these in reverse
The isolated nucleic acid sequences of the present invention which are capable of hybridizing with an oligonucleotide probe which hybridizes with the nucleic acid sequence set forth in SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO:5, SEQ ID NO 7, or SEQ ID NO:9, its complementary strand or a subsequence thereof, may be obtained from microorganisms of any genus, for example, from a bactenal or fungal source, but preferably from a fungal cell, and more preferably from a filamentous fungal cell or a yeast cell. For purposes of the present invention, the term "obtained from" (or endogenous to) as used herein in connection with a given source shall mean that the polypeptide is produced by the source or by a cell in which a gene from the source has been inserted Preferred sources for homologous genes are strains of the genus Absidia and species thereof available in public depositones Furthermore, homologous genes may be identified and obtained from other sources includmg microorganisms isolated from nature (e g , soil, composts, water, etc ) usmg the above- mentioned probes Techniques for isolating microorganisms from natural habitats are well known in the art. The nucleic acid sequence may then be derived by similarly screening a cDNA library of another microorganism Particularly preferred strains are filamentous fungus strains, such as an Acremomum, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe. Mucor, Mycehophthora, Neocalhmastix, Neurospora, Paecilomyces, Penicilhum, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma strain or yeast strains, such as a Candida, Kluyveromyces , Pichia, Saccharomyces , Schizosaccharomyces, or Yarrowia strain.
In a preferred embodiment, a nucleic acid sequence of the present invention is obtained from a strain of the genus Absidia, as described in M.A.A. Schipper, Persoonia, Vol 14, Part 2, pp 133- 148 (1990), such as a strain of Absidia griseola, Absidia sporophora-variabihs, Absidia griseola var iguchii, Absidia corymbifera, or Absidia blakesleeana. In an even more preferred embodiment, the nucleic acid sequence is obtained from Absidia blakesleeana NN100826 (NRRL 1304), e g , the nucleic acid sequence set forth in SEQ ID NO.3; Absidia corymbifera NNI 00062 (IFO 8084), e g , the nucleic acid sequence set forth in SEQ ID N0 5; Absidia griseola NN000987 (ATCC 20430), Absidia griseola var iguchii NN000591 (ATCC 20431), e g , the nucleic acid sequence set forth in SEQ ID NO.l: Absidia sporophora-variabihs NN102427 (ATCC 36019), e g , the nucleic acid sequence set forth in SEQ ID NO:7; and Absidia reflexa NNI 02427 (ATCC 44896), e g , the nucleic acid sequence set forth m SEQ ID NO:9.
Withm the genus Absidia, the following subgenera, groups, species and strains are preferred Variants and mutants thereof capable of producing lipolytic enzyme are also encompassed It is noted that a number of previously recognized species names were reclassified by Schipper, Op cit , and for convenience the previously used names of some strains are also listed below where multiple numbers in the same box indicate multiple deposits of the same strain
Subgenus, Species name Previous Inventors' Deposit number(s) group species name strain No
Subgenus A blakesleeana NRRL 1304, Mvcocladus A NN100826 ATCC 10148a, blakesleeana CBS 100.28, CMI 111736
A NN 102406 CBS 100.36 blakesleeana
A NNI 02407 CBS 102.36, blakesleeana NRRL 2696
A NNI 02408 CBA 420 70 blakesleeana
A NN102413 NRRL 1305 blakesleeana
A griseola NN000987 ATCC 20430
A griseola NNI 02403 CBS 519 71, ATCC 22618, IFO 9472
A griseola NN000591 ATCC 20431 var. iguchii
A hyalospora NN102432 CBS 173 67, NRRL 2916
A blakesleeana A atrospora NNI 02423 CBS 518.71, var. atrospora ATCC 22617, IFO 9471
A corymbifera A NNI 00060 CBS 100 31, IFO corymbifera 4009, NRRL 2982
A NNI 00062 IFO 8084 corymbifera
A NNI 02404 CBS 102.48 corymbifera
A NN102405 CBS 582.65, corymbifera ATCC 22574, NRRL 1309
A hesseltinu NNI 02426 CBS 958.68, ATCC 24263 Subenus Absidia, A. cylindrospora NNI 02422 CBS 154.63, Group B var. rhizomorpha NRRL 2815 pseudocyhndrospo ra-
NN 102434 ATCC
24169, CBS
100 62, NRRL
2770
- A. reflexa - NNI 02424 ATCC 44896, IFO 5874
- A. sporophora- - NNI 02427 ATCC 36019 variabilis
The above-mentioned strains are readily accessible to the public in a number of culture collections, such as the Amencan Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
Once a nucleic acid sequence has been detected with the probe(s) described above, the sequence may be isolated or cloned by utilizing techniques which are well known to those of ordinary skill in the art (see, e g., Sambrook et ai, supra) The known techniques used to isolate or clone a nucleic acid sequence include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic sequences of the present invention from such genomic DNA can be effected, e , by using the well known polymerase chain reaction (PCR). See, for example, Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York The nucleic acid sequence may be cloned from a strain of Absidia producing the polypeptide, or another or related organism and thus, for example, may be an allelic or species vanant of the polypeptide encoding region ofthe nucleic acid sequence
Modification of the nucleic acid sequence encoding the polypeptide may be necessary for the synthesis of polypeptides substantially similar to the polypeptide The term "substantially similar" to the polypeptide refers to non-naturally occurnng forms of the polypeptide. These polypeptides may differ m some engineered way from the polypeptide isolated from its native source For example, it may be of interest to synthesize vanants of the polypeptide where the variants differ in specific activity, thermostability, oxidative stability, pH optimum, or the like using, for example, site-directed mutagenesis. The analogous sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding region of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO 5, SEQ ID NO:7, or SEQ ID NO:9, a sub-sequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production oi' the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence For a general description of nucleotide substitution, see, e.g , Ford et ai, 1991 , Protein Expression and Purification 2: 95-107.
It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Ammo acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for lipase activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate- enzyme interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et ai, 1992, Science 255: 306-312; Smith et ai, 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et ai, 1992, FEBS Letters 309: 59-64).
Polypeptides encoded by the nucleic acid sequences of the present invention also include fused polypeptides in which another polypeptide is fused at the N-terminus or the C-termmus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include, hgatmg the coding sequences encoding the polypeptides so that they are in frame and that expression ofthe fused polypeptide is under control ofthe same promoter(s) and terminator.
The polypeptides having lipolytic activity encoded by the nucleic acid sequences of this invention are characterized by having a high activity at alkaline pH (about pH 9-10), even in the absence of free Ca".
More specifically, these polypeptides have optimum lipolytic activity at about pH 9 or higher (have a higher activity at pH 9 than at pH 8) when tested in the absence of free Ca" .
Certam preferred nucleic acid sequences encode polypeptides having lipase activity when tested at pH 9 without free Ca++. Such lipolytic enzymes can be obtained from strains of Absidia subgenus Mycocladus, e.g., the species and strains listed above.
Another group of preferred nucleic acid sequences encode polypeptides having a higher lipolytic activity at pH 10 than pH 9 in the absence of Ca++. Such a nucleic acid sequence can be obtained from Absidia reflexa NNI 02427 (ATCC 44896). A further group of preferred nucleic acid sequences encodes polypeptides retaining more than 90% residual activity after 30 minutes incubation at pH 10, 45 °C. Such a sequence can be obtained from a strain of Absidia sporophora-variabihs, e.g , Absidia sporophora-variabihs NN102427 (ATCC 36019).
Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences capable of directing the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
"Nucleic acid construct" is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurnng gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct may be synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term "coding sequence" as defined herein is a sequence which is transcribed into mRNA and translated into a polypeptide of the present invention when placed under the control of the appropπate control sequences. The boundaries of the coding sequence are generally determined by a translation start codon ATG at the 5 '-terminus and a translation stop codon at the 3 '-terminus. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
An isolated nucleic acid sequence encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide Manipulation of the nucleic acid sequence encoding a polypeptide prior to its insertion mto a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art.
The term "control sequences" is defined herein to include all components which are necessary or advantageous for expression of the coding sequence of the nucleic acid sequence. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcription control sequences which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcπptional activity in the host cell of choice and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicilhnase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et ai, 1978, Proceedings ofthe National Academy of Sciences USA 75: 3727-3731), as well as the tae promoter (DeBoer et ai, 1983, Proceedings ofthe National Academy of Sciences USA 80: 21-25). Further promoters are descπbed in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook et ai, 1989, supra.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus ntger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae tπose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described m U.S. Patent No. 4,288,627, which is incorporated herein by reference), and hybrids thereof. Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpι (a hybrid of the promoters from the genes encodmg Aspergillus niger neutral a-amylase and Aspergillus oryzae tnose phosphate isomerase), and glaA promoters.
In a yeast host, useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokmase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3 -phosphogly cerate kinase gene. Other useful promoters for yeast host cells are descnbed by Romanos et ai, 1992, Yeast 8: 423-488. In a mammalian host cell, useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bov e papilloma virus (BPV). The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription The terminator sequence is operably hnked to the 3 ' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used m the present invention. Preferred terminators for filamentous fungal host cells are obtained from the genes encoding
Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-hke protease.
Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYCl), or
Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et ai, 1992, supra Terminator sequences are well known m the art for mammalian host cells.
The control sequence may also be a suitable leader sequence, a nontranslated region of a mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5 ' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence which is functional in the host cell of choice may be used in the present invention.
Preferred leaders for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase and Aspergillus oryzae tπose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the Saccharomyces cerevisiae enolase
(ENO-1) gene, the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene, the Saccharomyces cerevisiae alpha-factor, and the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3- phosphate dehydrogenase genes (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes encodmg Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-amylase
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990. Polyadenylation sequences are well known in the art for mammalian host cells.
The control sequence may also be a signal peptide coding region, which codes for an ammo acid sequence hnked to the ammo terminus of the polypeptide which can direct the expressed polypeptide mto the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked m translation reading frame with the segment of the coding region which encodes the secreted polypeptide Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to that portion of the coding sequence which encodes the secreted polypeptide The foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the lipase relative to the natural signal peptide coding region normally associated with the coding sequence The signal peptide coding region may be obtained from a glucoamylase or an amylase gene from an Aspergillus species, a lipase or proteinase gene from a Rhizomucor species, the gene for the alpha- factor from Saccharomyces cerevisiae, an amylase or a protease gene from a Bacillus species, or the calf preprochymosin gene However, any signal peptide coding region capable of directing the expressed lipase into the secretory pathway of a host cell of choice may be used in the present invention An effective signal peptide coding region for bacterial host cells is the signal peptide coding region obtained from the altogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subti sin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilis prsA gene Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57 109-137
An effective signal peptide coding region for filamentous fungal host cells is the signal peptide coding region obtained from Aspergillus oryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, the Rhizomucor miehei aspartic proteinase gene, the Humicola lanuginosa cellulase gene, or the Rhizomucor miehei lipase gene Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase Other useful signal peptide coding regions are described by Romanos et al , 1992, supra
The control sequence may also be a propeptide coding region, which codes for an ammo acid sequence positioned at the amino terminus of a polypeptide The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases) A propolypeptide is generally inactive and can be converted to mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide The propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factor gene, or the Myce ophthora thermophilum laccase gene (WO 95/33836) The nucleic acid constructs of the present invention may also compnse one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e , an activator (e g , a trans-act g factor), a chaperone, and a processing protease Any factor that is functional in the host cell of choice may be used in the present invention T e nucleic acids encodmg one or more of these factors are not necessanly in tandem with the nucleic acid sequence encoding the polypeptide
An activator is a protein which activates transcription of a nucleic acid sequence encoding a polypeptide (Kudla et al , 1990, EMBO Journal 9 1355-1364; Jarai and Buxton, 1994, Current Genetics 26: 2238-244, Verdier, 1990, Yeast 6: 271-297). The nucleic acid sequence encoding an activator may be obtained from the genes encoding Bacillus stearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activator protein 1 (hapl), Saccharomyces cerevisiae galactose metabolizing protein 4 (gal4), and Aspergillus nidulans ammonia regulation protem (areA) For further examples, see Verdier, 1990, supra and MacKenzie et al , 1993, Journal of General Microbiology 139: 2295-2307. A chaperone is a protem which assists another polypeptide folding properly (Hartl et al ,
1994, TIBS 19: 20-25; Bergeron et al., 1994, TIBS 19: 124-128; Demolder et ai, 1994, Journal of Biotechnology 32- 179-189; Craig, 1993, Science 260: 1902-1903; Gething and Sambrook, 1992, Nature 355: 33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry 269. 7764-7771; Wang and Tsou, 1993, The FASEB Journal 7: 1515-1 1157; Robinson et ai, 1994, Bio/Technology 1 : 381- 384) The nucleic acid sequence encoding a chaperone may be obtained from the genes encoding Bacillus subtilis GroE proteins, Aspergillus oryzae protein disulphide isomerase, Saccharomyces cerevisiae calnexm, Saccharomyces cerevisiae BιP/GRP78, and Saccharomyces cerevisiae Hsp70 For further examples, see Gething and Sambrook, 1992, supra, and Hartl et ai, 1994, supra
A processing protease is a protease that cleaves a propeptide to generate a mature biochemically active polypeptide (Enderlm and Ogrydziak, 1994, Yeast 10: 67-79; Fuller et al , 1989, Proceedings of the National Academy of Sciences USA 86: 1434-1438; Julius et ai, 1984, Cell 37 1075-1089; Julius et ai, 1983, Cell 32: 839-852). The nucleic acid sequence encoding a processing protease may be obtained from the genes Saccharomyces cerevisiae dipeptidylammopeptidase, Saccharomyces cerevisiae Kex2, and Yarrowia hpolytica dibasic processing endoprotease (xprό). It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound Regulatory systems in prokaryotic systems would include the lac, tae, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences Other examples of regulatory sequences are those which allow for gene amplification In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals In these cases, the nucleic acid sequence encoding the polypeptide would be placed in tandem w th the regulatory sequence
Expression Vectors
The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropnate vector for expression In creating the expression vector, the coding sequence is located m the vector so that the coding sequence is operably linked with the appropnate control sequences for expression, and possibly secretion
The recombinant expression vector may be any vector (e g , a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced T e vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i e , a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e g , a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assunng self-replication Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated The vector system may be a smgle vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon The vectors of the present invention may be integrated into the host cell genome when introduced into a host cell For integration, the vector may rely on the nucleic acid sequence encodmg the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise locatιon(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the mtegrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination The mtegrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell Furthermore, the mtegrational elements may be non-encoding or encoding nucleic acid sequences On the other hand, the vector may be integrated mto the genome of the host cell by non-homologous recombination These nucleic acid sequences may be any sequence that is homologous with a target sequence in the genome of the host cell, and, furthermore, may be non-encoding or encoding sequences
For autonomous replication, the vector may further compnse an origin of replication enabling the vector to replicate autonomously in the host cell in question Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication m E coli, and pUBl 10, pE194, pTA1060, and pAMBl permitting replication in Bacillus Examples of origin of replications for use m a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARSl The origin of replication may be one having a mutation which makes its functioning temperature- sensitive in the host cell (see, e g , Ehrhch, 1978, Proceedings of the National Academy of Sciences USA 75 1433) In a specific embodiment, the expression vector may be pZL-NLl, pZL-NL61 , pZI - NL95, or pZL-NL124
The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphe col or tetracycline resistance A frequently used mammalian marker is the dihydrofolate reductase gene Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRPl, and URA3 A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithme carbamoyltransferase), bar (phosphinothπcin acetyltransferase), hygB (hygromycin phosphotransferase), maD (nitrate reductase), pyrG (orotιdme-5 ' -phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate resistance markers, as well as equivalents from other species Preferred for use m an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus Furthermore, selection may be accomplished by co-transformation, e g , as described in WO 91/17243, where the selectable marker is on a separate vector More than one copy of a nucleic acid sequence encoding a polypeptide of the present invention may be inserted mto the host cell to amplify expression of the nucleic acid sequence Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.
The procedures used to ligate the elements descnbed above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e g . Sambrook et al , 1989, supra).
Host Cells
The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. The term "host cell" encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur dunng replication The cell is preferably transformed with a vector comprising a nucleic acid sequence of the invention followed by integration of the vector into the host chromosome. "Transformation" means introducing a vector comprising a nucleic acid sequence of the present invention into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector mto the host chromosome occurs homologous or non-homologous recombination as described above
The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e g , a prokaryote, or a non-unicellular microorganism, e.g , a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g , Bacillus alkalophilus, Bacillus amyloliquefacien , Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus. Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thurtngiensis; or a Streptomyces cell, e g , Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E coli and Pseudomonas sp In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell The transformation of a bacterial host cell may, for instance, be effected by protoplast transformation (see, e g , Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e g , Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971 , Journal of Molecular Biology 56: 209-221), by electroporation (see, e g , Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-5278).
The host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell. Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.
In a preferred embodiment, the host cell is a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et ai, In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al, 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al, 1995, supra). Representative groups of Ascomycota include, e.g., Neurospora, Eupenicillium (=Penicillium), Emericella (= Aspergillus), Eurotium (= Aspergillus), and the true yeasts listed above. Examples of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.
In a preferred embodiment, the fungal host cell is a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus , Filobasidium, and Filobasidiella. Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biology of yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J., and Stopani, A.O.M., editors, 2nd edition, 1987; The Yeasts, Rose, A.H., and Harrison, J.S., editors, 2nd edition, 1987; and 77ιe Molecular Biology of the Yeast Saccharomyces, Strathern et al, editors, 1981). In a more preferred embodiment, the yeast host cell is a cell of a species of Candida,
Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, or Yarrowia. In a most preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis,
Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.
In a preferred embodiment, the fungal host cell is a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al, 1995, supra). The filamentous fungi are characterized by a vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obhgately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative. In a more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma. In an even more preferred embodiment, the filamentous fungal host cell is an Aspergillus cell. In another even more preferred embodiment, the filamentous fungal host cell is an Acremonium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Fusarium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Humicola cell. In another even more preferred embodiment, the filamentous fungal host cell is a Mucor cell. In another even more preferred embodiment, the filamentous fungal host cell is a Myceliophthora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Neurospora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Penicillium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Thielavia cell. In another even more preferred embodiment, the filamentous fungal host cell is a Tolypocladium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Trichoderma cell.
In a most preferred embodiment, the filamentous fungal host cell is an Aspergillus awamon, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal host cell is a Fusarium cerealis, Fusarium crookwellense, Fusarium graminearum, Fusarium oxysporum, Fusarium sambucinum or Fusarium sulphureum cell. In another most preferred embodiment, the filamentous fungal host cell is a Humicola insolens or Humicola lanuginosa cell. In another most preferred embodiment, the filamentous fungal host cell is a Mucor miehei cell. In another most preferred embodiment, the filamentous fungal host cell is a Myceliophthora thermophilum cell. In another most preferred embodiment, the filamentous fungal host cell is a Neurospora crassa cell. In another most preferred embodiment, the filamentous fungal host cell is a Penicillium purpurogenum cell. In another most preferred embodiment, the filamentous fungal host cell is a Thielavia terrestris cell. In another most preferred embodiment, the Trichoderma cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma Iongibrachiatum, Trichoderma reesei or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et ai, 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. A suitable method of transforming Fusarium species is described by Malardier et ai, 1989, Gene 78: 147-156 or in copending US Serial No. 08/269,449, incoφorated herein by reference. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al, 1983, Journal of Bacteriology 153: 163; and Hinnen et al, 1978, Proceedings of the National Academy of Sciences USA 75: 1920. Mammalian cells may be transformed by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52: 546).
Methods of Production
The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive to expression of the polypeptide; and (b) recovering the polypeptide. In these methods, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it is recovered from cell lysates.
The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide. The production of lipase activity can be determined by any method known in the art. In one method, one Lipase Unit (LU) is the amount of enzyme which liberates 1 0 μmol of titratable fatty acid per minute with tnbutyπn as substrate and gum arabic as an emulsifier at 30°C, pH 7 0 (phosphate buffer) Lipolytic enzyme activity in the absence of free Ca" in the range pH 7-10 is tested with a substrate emulsion of olive oil. 2% PVA solution (1 :3) at 40°C for 10 minutes, at a specified pH At the end of the reaction, the reaction mixture is extracted by chloroform: methanol (1:1) at acidic conditions, and the fatty acid released during the reaction is measured by TLC-FID analysis (Iatroscan). One OPID unit (OPIDU) is taken as the release of 1 0 μmole of fatty acid per minute In each test, 10 mM EDTA is used together with 200 mM of buffer (Tris-HCl buffer at pH 7 and 8, diethanol amme buffer at pH 8, 9 and 10)
The resulting polypeptide may be recovered by methods known in the art For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The recovered polypeptide may then be further purified by a variety of chromatographic procedures, e g . ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like
The polypeptides of the present invention may be purified by a variety of procedures known m the art including, but not limited to, chromatography ( , ion exchange, affinity, hydrophobic, chromatofocusmg, and size exclusion), electrophoretic procedures (e.g , preparative lsoelectnc focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e , Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
Uses
The recombinant polypeptides enoded by the nucleic acid sequences of the invention may be used in conventional applications of lipolytic enzyme, particularly at a high pH, e.g., m laundry and dishwashing detergents, institutional and industrial cleaning and leather processing
The lipolytic enzymes of the invention can also be used for lnteresteπfication, for total hydrolysis of fats and oils, and in optical isomer resolution process.
The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.
Examples
Example 1 : Genomic DNA Extraction
Absidia griseola NN000987 (ATCC 20430), Absidia sporophora-variabihs N I 02427
(ATCC 36019), Absidia griseola var. iguchii NN000591 (ATCC 20431), Absidia corymbifera NN100062 (IFO 8084), and Absidia blakesleeana NN100826 (NRRL 1304) were each grown in 25 ml of 0.5% yeast extract-2% glucose (YEG) medium for 24 hours at 32°C and 250 rpm. Myceha from each culture were then collected by filtration through Miracloth (Calbiochem, La Jolla, CA) and washed once with 25 ml of 10 mM Tπs-0.1 M EDTA (TE) buffer. Excess buffer was drained from the myceha preparations which were subsequently frozen in liquid nitrogen. The frozen myce a preparations were ground to a fine powder m an electnc coffee grinder, and the powders were each added to a disposable plastic centrifuge tube containing 20 ml of TE buffer and 5 ml of 20% w/v sodium dodecylsulfate (SDS). The mixtures were gently inverted several times to ensure mixing, and extracted twice with an equal volume of phenol:chloroform:ιsoamyl alcohol (25:24:1 v/v/v). Sodium acetate (3 M solution) was added to the extracted samples to a final concentration of 0.3 M followed by 2.5 volumes of ice cold ethanol to precipitate the DNA. The tubes were centnfuged at 15,000 x g for 30 minutes to pellet the DNA The DNA pellets were allowed to air-dry for 30 minutes before resuspension m 0.5 ml of TE buffer. DNase-free πbonuclease A was added to the resuspended DNA pellets to a concentration of 100 μg/ml and the mixtures were then incubated at 37°C for 30 min Proteinase K (200 μg/ml) was added and each tube was incubated an additional one hour at 37°C Finally, each sample was extracted twice with phenol:chloroform:ιsoamyl alcohol (25:24: 1 v/v/v) before precipitating the DNA with sodium acetate and ethanol. The DNA pellets were dπed under vacuum, resuspended TE buffer, and stored at 4°C.
Example 2: PCR Amplification of Absidia griseola NN000987 and Absidia griseola var. iguchii NN000591 Lipase Gene Segments Based on the ammo acid sequences of the Absidia griseola NN000987 lipase and the Absidia griseola var. iguchii NN000591 lipase as disclosed by Gormsen et ai in Patent Application DK 95/00424 (the contents of which are incoφorated herein by reference), the oligonucleotide primers shown below were synthesized with an Applied Biosystems Model 394 DNA RNA Synthesizer, according to the manufacturer's instructions, to PCR amplify lipase gene fragments from Absidia griseola NN000987 and Absidia griseola var. iguchii NN000591 (Note: R = A or G, I = inosine, Y = T or C, H = A or T or C, and N = A or T or C or G)- 1. Forward pnmer
Amino acid sequence: GluThrGluIleGlnAlaHisThrPhe (SEQ ID NO: 11) Oligonucleotide (+ strand): 5'-GARACIGARATHCARGCICAYACITT-3' (SEQ ID NO: 12) 2. Reverse primer
Ammo acid sequence: ProProGlyAlaPheGlyPheLeu (SEQ ID NO: 13)
Oligonucleotide (- strand): 5'-ARRAANCCRAAIGCNCCIGGNGG-3' (SEQ ID NO: 14)
Amplification reactions (100 ml) were prepared using approximately 1 μg of Absidia griseola NN000987 or Absidia griseola var. iguchii NN000591 genomic DNA as the template. Each reaction contained the following components: 1 μg of genomic DNA, 40 pmol of forward primer, 40 pmol of reverse primer, 200 mM each of dATP, dCTP, dGTP, and dTTP, 1 x Taq polymerase buffer (Perkm-Elmer Coφ., Branchburg, NJ), and 5 Units of Taq polymerase (Perkin-Elmer Coφ , Branchburg, NJ). Sterile mineral oil (100 μl) was layered on top of each reaction mixture, and the reactions were incubated in a Perkin-Elmer Model 480 Thermal Cycler programmed as follows Cycle 1 - 95°C for 5 minutes, 45°C for 2 minutes, and 67°C for 5 minutes; Cycle 2-30 - 95°C for 2 minutes; 45 °C for 2 minutes, and 67 °C for 2 minutes; and Soak cycle at 4°C. The reaction products were isolated on a 1% low melting point agarose gel (Sigma Chemical Co., St. Louis, MO) and the major PCR product band from each reaction was excised from the gel and purified using β-agarase (New England Biolabs, Beverly, MA) according to the manufacturer's instructions. The purified PCR products were subsequently cloned into a pCRII vector (Invitrogen, San Diego, CA) and the DNA sequences were determined using lac forward and reverse primers (New England BioLabs, Beverly, MA).
Lipase gene segments of approximately 870 bp were amplified from Absidia griseola NN000987 and Absidia griseola var. iguchii NN000591 as shown in Figure 1 with the lipase-specific PCR primers described above. DNA sequence analysis showed that the amplified gene segments encode portions of the corresponding Absidia lipase genes. In addition, the DNA sequence data confirmed that these two gene products are probably identical and share regions of homology with the Rhizomucor miehei lipase.
Example 3: Hybridization Analysis of Genomic DNA Total cellular DNA samples prepared from the five Absidia strains descπbed in Example 1 were analyzed by Southern hybridization (Maniatis et ai, 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York). Approximately 2-5 μg of each DNA sample was digested with EcoRI, Aspl 1%1 or EcoRI plus Asp7\Sl and fractionated on a 1% agarose gel. The gel was photographed under short wavelength UV light and soaked for 15 minutes in 0.5 M NaOH- 1.5 M NaCl followed by 15 minutes in 1.5 M NaCl- 1 M Tris-HCl pH 8 DNA in the gel was transferred onto Nytranό hybridization membrane (Schleicher & Schuell, Keene, NH) by capillary blotting in 20 X SSPΕ (3 M sodium chloπde-0.2 M sodium dibasic phosphate-0.02 M disodium ΕDTA) according to Davis et al (1980, Advanced Bacterial Genetics, A Manual for Genetic Engineering, Cold Spnng Harbor Press, Cold Spnng Harbor, New York). The DNA was cross-linked onto the membrane using a UV Stratahnker (Stratagene, La Jolla, CA), and the membrane was soaked for 2 hours in the following hybridization buffer at 45 °C with gentle agitation 5 X SSPΕ, 35% formamide (v/v), 0.3% SDS, and 200 mg/ml denatured and sheared salmon testes DNA. The lipase- specific probe fragment isolated from the Absidia griseola NN000987 PCR-clone described in Example 2 was radiolabeled by nick translation (Maniatis et al , 1982, supra) with [ 2P]dCTP (Amersham, Arlington Heights, IL), denatured by adding NaOH to a final concentration of 0.1 M, and added to the hybridization buffer at an activity of approximately 1 x 106 cpm per ml of buffer. The mixture was incubated overnight at 45 °C in a shaking water bath. Following incubation, the membranes were washed once in 0.2 X SSPE with 0.1% SDS at 45 °C followed by two washes in 0.2 X SSPE (no SDS) at the same temperature. The membranes were allowed to dry on paper towels for 15 minutes, then wrapped in Saran-Wrap™ and exposed to X-ray film overnight at -70°C with intensifying screens (Kodak, Rochester, NY).
Analysis of the total cellular DNA samples from each of the Absidia species by Southern blotting under conditions of moderate stringency using the PCR-deπved lipase gene segment probe from Absidia griseola var. iguchii NN000591 demonstrated that the lipase genes of all the Absidia species tested cross-hybridized to the probe (Figure 2). All the species tested also showed a single hybridization signal m the EcoRI digest of approximately 2.1 kb with the exception of Absidia sporophora-variabihs NN102427 which gave a hybridization signal with a 4 kb band. Furthermore, it appeared that all of the species tested contain a single copy of the correspondmg lipase gene in their genomes
Example 4: DNA Libraries and Identification of Lipase Clones
Enriched genomic DNA libraries were constructed m the bacteriophage cloning vector lZipLox (Life Technologies, Gaithersburg, MD). First, total cellular DNA was digested with EcoRI and size-fractionated on 1% agarose gels. DNA fragments migrating m the size range corresponding to hybridization signals previously observed on Southern blots described m Example 3 were excised and eluted from the gel using Prep-a-Gene reagents (BioRad Laboratories, Hercules, CA). The approximate sizes of the DNA fragments in these fractions were as follows: 1.9-2.5 kb (Absidia griseola var. iguchii NN000591), 1.9-2.5 kb (Absidia blakesleeana NN100826), 1.9-2.5 kb (Absidia corymbifera NN100062) and 2.5-4.3 kb (Absidia sporophora-variabihs NN102427). The eluted DNA fragments were ligated with EcoRI-cleaved and dephosphorylated λZipLox vector arms (Life Technologies, Gaithersburg, MD), and the ligation mixtures were packaged using commercial packaging extracts (Stratagene, La Jolla, CA) The packaged DNA libraries were plated and amplified in Escherichia coli Y1090ZL cells (Life Technologies, Gaithersburg, MD). The titers of recombinant phage in each library ranged from 1.3-5.4 X 105 pfu/ml (background titers with no DNA were 1.7-2.0 X 104 pfu ml). Approximately 15,000-30,000 plaques from each unamplified library were screened by plaque-hybridization using the lipase-specific PCR fragment from Absidia griseola NN000987 as the probe (Davis et ai, 1980, supra). Plaques, which gave strong hybridization signals with the probe, were purified twice in E. coli Y1090ZL cells and the lipase genes were subsequently excised from the λZipLox vector as pZL 1 -derivatives (D'Alessio et al , 1992, Focus® 14: 76). The recombinant DNA segments were inserted within the phagemid pZLl portion of the vector, and the phagemid harboring the cloned insert was recovered in the autonomously replicating pZLl using in vivo excision by infection of E coli DHlOBzip cells (Life Technologies, Gaithersburg, MD) The lipase clones isolated in this manner were prepared for DNA sequence analysis using a Wizard 373 DNA puπfication kit (Promega, Madison, WI)
Example 5: DNA Sequence Analysis of Lipase Genes
DNA sequencing of the lipase clones descnbed in Example 4 was performed with an Applied Biosystems Model 373A Automated DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) on both strands using the pnmer walking technique with dye-terminator chemistry (Giesecke et al , 1992, Journal of Virol Methods 38 47-60) Oligonucleotide sequencing pn ers were synthesized on an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions
The nucleotide sequences of the genes encoding the Absidia lipases are shown in Figures 3- 6 (SEQ ID NOS 1 , 3, 5, and 7) The assignment of mtrons within each gene was based on (a) known amino acid sequence data denved from peptide fragments (Boel et al , 1988, Lipids 23 701- 706), (b) alignment to the deduced amino acid sequence of Rhizomucor miehei lipase (SEQ ID NO 15) (Gurr et al , In Kinghorn, J R [ed], Gene Structure in Eukaryotic Microbes, pp 93-139, IRL Press, Oxford), and (c) known consensus sequences for introns of filamentous fungi (von Heijne, 1984, Journal of Molecular Biology 173 243-251 )
In order to isolate a gene encoding the Absidia reflexa lipase, a strain of Absidia reflexa N I 02427 (ATCC 44896) was grown on optimal medium with jojoba oil as an induction component A cDNA library was prepared from this strain, and, using the Absidia corymbifera gene descnbed above as a probe, a cDNA clone was identified by colony hybndization The sequence of the Absidia reflexa gene is provided m Figure 7 (SEQ ID NO 9) This lipase is approximately 99% identical with the Absidia sporophora-variabihs lipase
Example 6: Comparison of the Lipase Genes from Absidia Species
The intron-exon organization among Absidia lipase genes is very similar in that all are composed of seven exons and six mtrons as shown in Table 1 The size of the exons 2 through 6 are stnetly conserved The first exon shows slight vanabi ty in size due to some vanation in the region encoding the signal peptide and propeptide portions of the enzyme In addition, the nucleotide sequence homology between corresponding exons is also very high with 85-97% identity In contrast, the lengths of the introns vaπed, and with the exception of the Absidia corymbifera N I 00062 and Absidia sporophora-variabihs NNI 02427 lipase genes, there is little sequence homology between corresponding introns The 5 ' and 3' flanking sequences of the lipase genes also shows sequence divergence
Table 1 Intron-exon organization of the lipase genes from several Absidia species
Species Exon (length in bp) Intron (len gth in bp)
1 2 3 4 5 6 7 1 2 3 4 5 6
A griseola var iguchii 489 58 97 173 81 79 31 82 64 56 61 64 66
A blakesleeana 492 58 97 173 81 79 31 75 52 57 60 64 67
A corymbifera 495 58 97 173 81 79 31 61 62 54 58 53 59
A sporophora- 495 58 97 173 81 79 31 61 62 54 57 53 59 variabihs
Based on the deduced amino acid sequences (SEQ ID NOS 2, 4, 6, 8, and 10), a comparison of the biochemical and biophysical properties of the Absidia lipases is presented in Table 2 All four lipases are very similar in that all are synthesized as preproenzymes of 337-338 amino acids, comprising signal peptides of 17- 21 ammo acids, propeptides of 52-56 ammo acids, and mature enzymes of 264-265 amino acids with molecular weights of approximately 29,000 for the non-glycosylated proteins The calculated lsoelectric points vary from 6 10 to 6 94 with the most alkaline being the lipase from Absidia sporophora-variabihs These calculated isoelectric values are much lower than those observed experimentally on IEF gels (see Gormsen et al in Patent Application DK 95/00424) probably as a result that not all charged residues are on the exposed three- dimensional surface of the protein
Table 2 Predicted biochemical and biophysical properties of from several Absidia species
Species Preprofor Predicte Predicte Mature Molecul Calculat Molar 1 A280 m d signal d lipase ar ed extinctio unit peptide propepti weight isoelectri n (mg/ml) de c point coeficien t
A griseola var 337 aa 18 aa 54 aa 264 aa 29,028 6 45 27,580 1 05 iguchii
A blakesleeana 337 aa 18 aa 55 aa 264 aa 28,959 6 64 24,900 1 16
A corymbifera 338 aa 21 aa 52 aa 265 aa 29,057 6 10 27,460 1 06
A sporophora- 338 aa 17 aa 56 aa 265 aa 28,952 6 94 27,460 1 05 variabihs
The Absidia lipases share extensive amino acid sequence homology between each other with 87-96% identity, and limited homology to Rhizomucor miehei lipase (SEQ ID NO 15) with 53-55% identity and Humicola lanuginosa lipase (SEQ ID NO 16) with 22-24% identity as shown in Table 3 and Figure 8 Table 3 Amino acid sequence similarity among lipases from Absidia species, R miehei, and H lanuginosa
Percent Similarity
1 2 3 4 5 6
Percent 1 - 87. 87. 88. 53. 22. 1 I blakesleeana
Divergence 2 5 1 7 3
2 11. - 86. 87. 54. 22. 2 A. griseola var.
9 3 2 8 7 iguchii
3 11. 11. - 96. 53. 23. 3 A. corymbifera
6 9 4 3 7
4 11. 11. 3.6 - 53. 24. 4 A. sporophora-
0 0 3 1 variabihs
5 41. 40. 41. 41. - 23. 5 R. miehei
6 5 9 9 7
6 70. 70. 70. 70. 68. - 6 Humicola
6 9 0 0 8 lanuginosa
1 2 3 4 5 6
From a phylogenetic standpoint, the Absidia lipases are most closely related to the lipases from other members of the Zygomycete class of fungi Yeast, bacterial, and mammalian lipases as well as fungal cutinases appear to be very distantly related if at all.
Example 7: Expression of the Absidia corymbifera lipase gene
The clone and the nucleotide sequences of the Absidia corymbifera lipase gene described above were used for subcloning of the gene and expression in an Aspergillus host. PCR was used to subclone the lipase gene (without its own promoter) from the isolated genomic clone NL95A using primers designed from the nucleotide sequences. In order to facilitate the subcloning of the gene fragment into an expression vector designated pBANeδ (Figure 9), Swal and Pad restriction enzyme sites, respectively, at the 5' and 3' end of the gene, were introduced. The vector pBANE6 contained the TAKA promoter, NA2-tpι leader, and AMG terminator as regulatory sequences. The plasmid also contained the Aspergillus nidulans amdS gene as a selectable marker for fungal transformations The following primers were used for PCR amplification: Forward Primer: 5'-CCCATTTAAATATGCGTTTTTATTCAGTAGTATCAT-3' (SEQ ID NO: 17)
Reverse primer: 5'-CTCGGCTTAATTAAAATGGGTTATAAGCAGAGACCAGTG-3' (SEQ ID NO: 18) PCR was performed using Pwo polymerase (Boehringer Mannheim, Indianapolis, IN) according to manufacturer's specifications. The PCR amplified product was gel isolated, cut with Swal and Pad enzymes, and gel purified. The purified fragment was ligated to the pBANeό vector (already cut with Swal and Pad) to yield the plasmid pKB2 (Figure 10) in which transcription of the lipase gene was under the control of the TAKA promoter. The plasmid pKB2 was transformed mto E coli DH5 cells E coli transformants containing the pKB2 plasmid were isolated and plasmid DNA was prepared for transformation and expression in Aspergillus.
Protoplasts were prepared from Aspergillus oryzae strain BANe3 in which the amdS gene of the host strain was deleted. Protoplast preparation and transformation were done as previously described (Chπstensen et ai, supra). Aspergillus oryzae transformants expressing acetamidase were selected based on their ability to utilize acetamide as the sole nitrogen source. A total of 42 transformants were generated and spore purified twice on selective plates The spore purified transformants were used for further analysis
The transformants were screened for lipase expression by cultivation in shake flasks (25 ml medium in 125 ml flasks) using a medium containing 50 g of maltose, 2.0 g of MgS04-7H20, 10 g of KH,P04, 2 0 g of K,S04, 2.0 g of citric acid, 10 g of yeast extract, 0 5 ml of trace metals solution, and 2 0 g of urea per liter The trace metals solution was comprised of 14.3 g of ZnS04-7H20, 2.5 g of CuS04-5H20, 0.5 g of NιCl,-6H20), 13.8 g of FeS04-7H20), 8.5 g of MnSO„-H20, and 3.0 g of citric acid per liter The pH of the medium was adjusted to 6.5 before sterilization by autoclaving. Flasks were inoculated with freshly harvested spores and incubated in an incubator at 34 °C and 200 φm Cultures were assayed for lipase activity daily after 48 hours of cultivation Since nothing was known about the suitable substrate for this lipase, enzyme activity was assayed by three methods: I) lipase plate assay using olive oil as the substrate, n) coloπmetπcally using p-mtrophenylbutyrate as the substrate and in) titration using tπbutyrm (tnglyceπde of butyric acid) A lipase plate assay was performed using a plate medium that contained the following. 0.1 M
Tns pH 9.0, 0 1 M CaCl2, 1% Tπton X-100, 0.5% olive oil, and 2.0% agarose. The medium was autoclaved and poured into 150 mm plates using 50-60 ml per plate After solidification of the agarose, 15 wells per plate were made and 25 ml of the culture broth was added to each well Culture broth from untransformed Aspergillus oryzae strain BANe3 was used as a control. The plates were incubated overnight at 37°C. The presence of lipase activity in the transformants was identified as clear zones around the well. Control wells loaded with culture broth from the untransformed strain did not show such cleaπng indicating the presence of lipase activity only in the transformants
Lipase activity was also measured colonmetπcally using p-nitrophenylbutyrate as a substrate. p-Nitrophenylbutyrate was prepared by adding 10 ml of this compound to 1.0 ml dimethylsulfoxide (DMSO) and 4.0 ml of 0.1 M Tns pH 9.0 buffer One hundred microliters of suitably diluted culture broth were added to each well The reaction was started by adding 100 ml of the p-nitrophenylbutyrate substrate and the absorbance was measured at 405 nm for 3-5 min. The enzyme activity was calculated from a curve made with a known amount of Humicola lanuginosa lipase as the standard. The untransformed strain produced little or no activity while different transformants produced lipase after 48 hours of cultivation.
Lipase activity was further determined by titration based on the hydrolysis of tπbutyπn catalyzed by the lipase. The liberation of butync acid was followed by alkaline titration in a pH-stat. The assay was performed on culture broths from selected transformants as well as from the untransformed control strain. The results showed that the untransformed control strain produced no lipase activity, while the transformants produced detectable lipase activity.
Example 8: Expression of the Absidia sporophora-variabihs Lipase Gene in Aspergillus oryzae.
The coding region of the A. sporophora-variabihs lipase was amplified using the original genomic clone as the template for Pwo polymerase (Boehringer-Mannheim Biochemicals, Indainapohs, IN) in a PCR reaction which contained the following: 61 ml sterile water, 10 ml of diluted template DNA (ca. 5 ng/ml), 1 ml pnmer 1 (ca. 30 pmol. dATGATGCATTCTCATTTTGTAGTCTTATTG, SEQ ID NO:19), 1 ml primer 2 (ca. 25 pmol dGCTTAATTAATTATAAACAGAGACCAGTGTTCATGTCAAG, SEQ ID NO:20), 16 ml dATP, dCTP, dGTP, and dTTP mix (200 mmol final concentration), 10 ml of Pwo buffer (10X solution; Boehr ger-Mannheim Biochemicals), and 1 ml Pwo polymerase (5 units). Amplification conditions were as follows: First cycle at 95 °C for 5 minutes, 45 °C for 2 minutes, and 67 °C for 5 minutes; cycles 2 through 30 at 95°C for 2 minutes, 45°C for 1 minute, and 67°C for 2 minutes; and a soak cycle at 4°C. The amplified lipase gene segment was digested with Pad and isolated by preparative agarose gel electrophoresis, excised and purified using Prep-a-Gene reagents (BioRad Laboratories, Hercules, CA). The purified fragment was ligated with pBANe6 which had been cleaved with Swal and Pad to generate the lipase expression vector pRaMB19 (Figure 11). pRaMB19 was subsequently used to transform an alkaline protease-deficient Aspergillus oryzae host JaL142 using standard methods (Chπstensen et al. 1988. Bio/Technology 1419-1422) Transformant colonies were purified twice through condiospores and tested for lipase expression by streaking on tributyπn agar plates containing 130 g of maltodextrin, 3 g of MgS04-7H20, 5 g of KH,P04, 4 g of citric acid, 6 g of K2S04, 0.5 ml of trace metals (described in Example 7), 5 g of yeast extract, 166 ml of 1 M urea, 35.3 ml of 1 M NaN03, 25 g of Noble agar, and 10 g of triburyrm pH 4.5 per liter. After a 48 hour incubation at 30 °C, 80 of 84 transformants showed distinct cleaπng zones on the tributynn agar plates indicating production of extracellular lipase activity. Ten of these transformants were further tested in shake flask cultures of MY50 medium containing 50 g of maltodextrin, 2 g of MgS04-7H20, 10 g of KH2P04J 2 g of citric acid, 2 g of K2S04, 0.5 ml of trace metals solution, 10 g of yeast extract, 2 g of urea pH 6.0 per liter. After incubating the shake flask cultures at 37 °C for 48 hours, culture filtrates from each were assayed for extracellular lipase activity using »-nιtrophenyl butyrate as the substrate as described in Example 7. Cultures of untransformed control cells produced no detectable lipase activity, whereas the pRaMB19 transformants produced detectable lipase activity.
Deposit of Biological Materials
The following biological materials have been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, 1815 University Street, Peoria, Illinois, 61604 on January 18, 1996, and given the following accession numbers.
Deposit Accession Number
E. coh DH 1 OB (pZL-NL 1 ) - Absidia blakesleeana NRRL B -21520
E. coli DH 1 OB (pZL-NL61 ) - Absidia corymbifera NRRL B-21521 E. coli DH10B (pZL-NL95) - Absidia griseola-iguchn NRRL B-21522
E. coli DH 1 OB (pZL-NL124) - Absidia sporophora-variablihs NRRL B-21523

Claims

What is claimed is
1 . An isolated nucleic acid sequence encoding a polypeptide having lipase activity selected from the group consisting of: (a) a nucleic acid sequence which encodes a polypeptide endogenous to an Absidia strain with an ammo acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO 8, or SEQ ID NO: 10,
(b) a nucleic acid sequence endogenous to an Absidia strain which is capable of hybridizing under medium stringency conditions with (i) the nucleic acid sequence set forth in SEQ ID NO. l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or (n) any of their complementary strands;
(c) a nucleic acid sequence which is capable of hybridizing under medium stringency conditions with (I) the nucleic acid sequence set forth in SEQ ID NO: l , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or (n) any of their complementary strands, (d) a nucleic acid sequence encodmg a polypeptide having lipase activity with an amino acid sequence which has at least 65% identity with the amino acid sequence set forth in SEQ ID NO:2, SEQ ID N0 4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10;
(e) an allelic form of (a), (b), (c), or (d); and
(f) a fragment of (a), (b), (c), or (d)
2 . The nucleic acid sequence of claim 1 , wherein the nucleic acid sequence is capable of hybridizing under medium stringency conditions to (I) the nucleic acid sequence set forth in SEQ ID NO 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, or (n) any of their complementary strands, or a fragment thereof
3 . The nucleic acid sequence of claim 2, wherein the nucleic acid sequence is capable of hybridizing under medium stringency conditions to (I) the nucleic acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, or (n) any of their complementary strands
4 . The nucleic acid sequence of claim 3, wherein the nucleic acid sequence is capable of hybridizing under medium stringency conditions to the nucleic acid sequence set forth in SEQ ID NO 1 or its complementary strand 5 The nucleic acid sequence of claim 3, wherein the nucleic acid sequence is capable of hybridizing under medium stringency conditions to the nucleic acid sequence set forth m SEQ ID NO 3 or its complementary strand
6 . The nucleic acid sequence of claim 3, wherein the nucleic acid sequence is capable of hybridizing under medium stringency conditions to the nucleic acid sequence set forth in SEQ ID NO 5 or its complementary strand
7 . The nucleic acid sequence of claim 3, wherein the nucleic acid sequence is capable of hybridizing under medium stringency conditions to the nucleic acid sequence set forth in SEQ ID
NO 7 or its complementary strand
8 . The nucleic acid sequence of claim 3, wherein the nucleic acid sequence is capable of hybridizing under medium stringency conditions to the nucleic acid sequence set forth SEQ ID NO 9 or its complementary strand
9 . The nucleic acid sequence of claim 2, wherein the nucleic acid sequence encodes a polypeptide having lipase activity obtained from Absidia
10 The nucleic acid sequence of claim 9, wherein the nucleic acid sequence encodes a polypeptide having lipase activity obtained from Absidia blakesleeala
11 . The nucleic acid sequence of claim 9, wherein the nucleic acid sequence encodes a polypeptide having lipase activity obtained from Absidia corymbifera
12 . The nucleic acid sequence of claim 9, wherein the nucleic acid sequence encodes a polypeptide having lipase activity obtained from Absidia griseola
13 . The nucleic acid sequence of claim 9, wherein the nucleic acid sequence encodes a polypeptide having lipase activity obtained from Absidia griseola var iguchii
14 . The nucleic acid sequence of claim 9, wherein the nucleic acid sequence encodes a polypeptide having lipase activity obtained from Absidia reflexa 15 . The nucleic acid sequence of claim 9, wherein the nucleic acid sequence encodes a polypeptide having lipase activity obtained from Absidia sporophora-variabihs
16 . The nucleic acid sequence of claim 1 , wherein the nucleic acid sequence encodes an amino acid sequence with at least 65% identity with any of the amino acid sequences set forth in SEQ ID
NO.2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO: 10, or a fragment thereof.
17 . The nucleic acid sequence of claim 16, wherein the nucleic acid sequence encodes an ammo acid sequence with at least 70% identity with any of the ammo acid sequences set forth in SEQ ID NO-2, SEQ ID N0 4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO: 10.
18 . The nucleic acid sequence of claim 17, wherein the nucleic acid sequence encodes an ammo acid sequence with at least 75% identity with any of the amino acid sequences set forth in SEQ ID NO.2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO: 10.
19 . The nucleic acid sequence of claim 18, wherein the nucleic acid sequence encodes an amino acid sequence with at least 80% identity with any of the amino acid sequences set forth in SEQ ID NO.2, SEQ ID NO 4, SEQ ID NO:6, SEQ ID NO.8, and SEQ ID NO- 10.
20 . The nucleic acid sequence of claim 19, wherein the nucleic acid sequence encodes an amino acid sequence with at least 85% identity with any of the ammo acid sequences set forth in SEQ ID NO 2, SEQ ID NO .4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO: 10.
21 . The nucleic acid sequence of claim 20, wherein the nucleic acid sequence encodes an ammo acid sequence with at least 90% identity with any of the ammo acid sequences set forth m SEQ ID NO-2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID N0 8, and SEQ ID NO: 10.
22 . The nucleic acid sequence of claim 21 , wherem the nucleic acid sequence encodes an ammo acid sequence with at least 95% identity with any of the ammo acid sequences set forth in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO:6, SEQ ID NO.8, and SEQ ID NO.10.
23 . The nucleic acid sequence of claim 22, wherein the nucleic acid sequence encodes an ammo acid sequence with at least 95% identity with the ammo acid sequence set forth in SEQ ID NO.2. 24 . The nucleic acid sequence of claim 23, wherein the nucleic acid sequence encodes an ammo acid sequence with the ammo acid sequence set forth in SEQ ID NO.2.
25 . The nucleic acid sequence of claim 24, wherein the nucleic acid sequence is set forth m SEQ ID NO:l
26 . The nucleic acid sequence of claim 22, wherem the nucleic acid sequence encodes an ammo acid sequence with at least 95% identity with the amino acid sequence set forth in SEQ ID NO:4
27 . The nucleic acid sequence of claim 26, wherein the nucleic acid sequence encodes an amino acid sequence with the ammo acid sequence set forth in SEQ ID NO:4.
28 . The nucleic acid sequence of claim 27, wherein the nucleic acid sequence is set forth in SEQ ID NO:3
29 . The nucleic acid sequence of claim 22, wherein the nucleic acid sequence encodes an amino acid sequence with at least 95% identity with the ammo acid sequence set forth in SEQ ID NO:6.
30 . The nucleic acid sequence of claim 29, wherem the nucleic acid sequence encodes the amino acid sequence set forth m SEQ ID NO:6.
31 . The nucleic acid sequence of claim 30, wherein the nucleic acid is set forth in SEQ ID NO:5
32 . The nucleic acid sequence of claim 22, wherein the nucleic acid sequence encodes an amino acid sequence with at least 95% identity with the am o acid sequence set forth m SEQ ID NO:8.
33 . The nucleic acid sequence of claim 32, wherein the nucleic acid sequence encodes the amino acid sequence set forth in SEQ ID NO:8.
34 . The nucleic acid sequence of claim 33. wherein the nucleic acid sequence is set forth in SEQ ID NO.7
35 . The nucleic acid sequence of claim 22, wherein the nucleic acid sequence encodes an amino acid sequence with at least 95% identity with the ammo acid sequence set forth m SEQ ID NO: 10. 36 . The nucleic acid sequence of claim 35, wherein the nucleic acid sequence encodes the amino acid sequence set forth in SEQ ID NO' 10
37 . The nucleic acid sequence of claim 36, wherein the nucleic acid sequence is set forth in SEQ ID NO 9
38 The nucleic acid sequence of claim 1 , wherein the nucleic acid sequence is endogenous to an Absidia strain and is capable of hybridizing under medium stringency conditions to the nucleic acid sequence set forth SEQ ID NO 1 or its complementary strand
39 The nucleic acid sequence of claim 1, which comprises the Absidia hpase-encoding nucleic acid sequence contained in the plasmid pZL-NLl contained in Escherichia coh NRRL B-21520
40 . The nucleic acid sequence of claim 1, which comprises the Absidia hpase-encoding nucleic acid sequence contained in the plasmid pZL-NL61 contained in Escherichia coh NRRL B-21521
41 The nucleic acid sequence of claim 1, which comprises the Absidia hpase-encodmg nucleic acid sequence contained in the plasmid pZL-NL95 contained in Escherichia coh NRRL B-21522
42 The nucleic acid sequence of claim 1, which comprises the Absidia hpase-encoding nucleic acid sequence contained in the plasmid pZL-NL124 contained in Escherichia coh NRRL B-21523
43 . A nucleic acid construct compπsing the nucleic acid sequence of claim 1 operably linked to one or more control sequences capable of directing the expression of the polypeptide in a suitable expression host.
44 . A recombinant expression vector comprising the nucleic acid construct of claim 43, a promoter, and transcriptional and translational stop signals
45 The vector according to claim 44, further comprising a selectable marker
46 . A recombinant host cell comprising the nucleic acid construct of claim 43
47 . The cell according to claim 46, wherein the nucleic acid construct is contained on a vector 48 . The cell according to claim 46, wherein the nucleic acid construct is integrated into the host cell genome.
49 . The cell according to claim 46, wherein the host cell is a bacterial cell.
50 . The cell according to claim 49, wherein the bacterial cell is a Bacillus, Streptomyces, or Pseudomonas cell.
51 . The cell according to claim 46, wherein the host cell is a fungal cell.
52 . The cell according to claim 51 , wherein the fungal cell is a filamentous fungal cell.
53 . The cell according to claim 52, wherein the filamentous fungal cell is a cell of a species of Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.
54 . The cell according to claim 46, wherein the fungal cell is a yeast cell.
55 . The cell according to claim 54, wherein the yeast cell is a cell of a species of Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia.
56 . A method for producing a polypeptide having lipase activity comprising (a) cultivating the host cell of claim 46 under conditions conducive to expression of the polypeptide; and (b) recovering the polypeptide.
PCT/US1997/000598 1996-01-24 1997-01-21 Nucleic acids encoding polypeptides having absidia lipase activity WO1997027276A2 (en)

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AU17477/97A AU1747797A (en) 1996-01-24 1997-01-21 Nucleic acids encoding polypeptides having absidia lipase activity
EP97904764A EP0876489A2 (en) 1996-01-24 1997-01-21 Nucleic acids encoding polypeptides having absidia lipase activity
JP52690697A JP3333521B2 (en) 1996-01-24 1997-01-21 Nucleic acid encoding a polypeptide having abscisdia lipase activity

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US60/013,616 1996-01-24

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EP1555322A1 (en) 2000-04-28 2005-07-20 Novozymes A/S Lipolytic enzyme variant
WO2005100582A2 (en) 2004-03-25 2005-10-27 Novozymes Inc. Methods for degrading or converting plant cell wall polysaccharides
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WO2015158237A1 (en) 2014-04-15 2015-10-22 Novozymes A/S Polypeptides with lipase activity and polynucleotides encoding same
WO2017005816A1 (en) * 2015-07-06 2017-01-12 Novozymes A/S Lipase variants and polynucleotides encoding same
WO2017093318A1 (en) 2015-12-01 2017-06-08 Novozymes A/S Methods for producing lipases
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EP3219806A1 (en) 2004-03-25 2017-09-20 Novozymes, Inc. Methods for degrading or converting plant cell wall polysaccharides
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AU1747797A (en) 1997-08-20
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JPH11510395A (en) 1999-09-14
JP3333521B2 (en) 2002-10-15

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