Methanol-tolerant lipase

Abstract

The present application provides novel polypeptides with lipase activity, which comprise the amino acid sequences shown in SEQ ID NO: 4 or 6, or sequences obtained by substituting, deleting or adding at least one amino acid to the above sequences. The present application also provides polynucleotides encoding the polypeptides, expression vectors containing the above polynucleotides and host cells. In addition, the present application also relates to the uses of the polypeptides with lipase activity. The polypeptides disclosed herein can rapidly prepare fatty acid methyl esters at high temperatures and high methanol concentrations, and are thus used for the preparation of biodiesel.

Description

Field of the Invention

[0001] This application belongs to the field of enzyme engineering. Specifically, it relates to polypeptides with lipase activity, nucleic acids encoding the same, expression vectors and host cells containing the encoding nucleic acids. This application also relates to screening methods and uses of the above polypeptides. Background of the Invention

[0002] Lipase (Lipase EC 3.1.1.3), namely triacylglycerol acylhydrolase, catalyzes the hydrolysis of natural substrate oils and fats to produce fatty acids, glycerol and glycerol monoesters or diesters. It is widely used in industries such as oil processing, food, medicine, and daily chemicals, and is one of the important industrial enzyme preparations.

[0003] It can catalyze the hydrolysis of glycerol ester bonds on oils and fats, and can also catalyze reactions such as transesterification, esterification, alcoholysis, and acidolysis. Lipases from microbial sources are widely used in industrial production due to their diverse functions, easy cultivation, and rich yields. Among them, the research and development of thermostable lipases have important commercial value. Currently, it is known that about 2% of microorganisms produce lipases. Microorganisms capable of producing lipases include at least 65 genera, including 28 genera of bacteria, 4 genera of actinomycetes, 10 genera of yeasts, and 23 genera of other fungi. Thermomyces lanuginosus is a fungus with a wide distribution and a relatively high upper growth temperature. It can produce a thermostable lipase with important industrial value, Thermomyces lanuginosus lipase (TL). TL can be used for fatty acid methyl esterification reactions to produce biodiesel. Since methanol is one of the substrates in the reaction process, further improving the methanol tolerance of TL may further make it more suitable for the methyl esterification reaction, such as reducing the dosage. Summary of the Invention

[0004] In a first aspect, there is provided a polypeptide with lipase activity, which comprises a sequence selected from the following or consists of a sequence selected from the following:

[0005] (a) The amino acid sequence shown in SEQ ID NO: 4 or 6, and

[0006] (b) A sequence obtained by substituting, deleting or adding at least one amino acid to the sequence described in (a), wherein the polypeptide variant obtained from (b) still retains lipase activity.

[0007] In one embodiment, the polypeptide of the present application comprises the amino acid sequence shown in SEQ ID NO: 4 or 6. In a preferred embodiment, the above polypeptide consists of the amino acid sequence shown in any one of SEQ ID NO: 4 and 6.

[0008] In a second aspect, there is provided a polynucleotide encoding the polypeptide described in the first aspect, which comprises a sequence selected from the following or consists of a sequence selected from the following:

[0009] (a) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 or 6, or a sequence containing at least one amino acid substitution, deletion or addition in SEQ ID NO: 4 or 6; and

[0010] (b) a nucleotide sequence that hybridizes with the nucleotide sequence in (a) under stringent conditions.

[0011] In one embodiment, the polynucleotide of the present application comprises the nucleotide sequence shown in SEQ ID NO: 3 or 5. In a preferred embodiment, the above polynucleotide consists of the nucleotide sequence shown in SEQ ID NO: 3 or 5.

[0012] In a third aspect, there is provided an expression vector comprising at least one polynucleotide described in the second aspect.

[0013] In certain embodiments, the expression vector of the present application further comprises a regulatory sequence for regulating the expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence. In a specific embodiment, the expression vector is a plasmid vector.

[0014] In a fourth aspect, there is provided a host cell comprising the polynucleotide of the second aspect or the expression vector of the third aspect.

[0015] In some embodiments, the host cell is Escherichia coli or yeast such as Pichia pastoris.

[0016] In a fifth aspect, there is provided the use of the polypeptide described in the first aspect for carrying out a fatty acid methylation reaction or for preparing biodiesel.

[0017] In some embodiments, the fatty acid methylation reaction or the preparation of biodiesel is carried out in a methanol environment with a concentration not higher than 30%, preferably 15% - 25%.

[0018] In a sixth aspect, there is provided a method for preparing biodiesel, which comprises contacting the polypeptide described in the first aspect with an oil raw material and a lower alcohol.

[0019] In some embodiments, the lower alcohol is selected from methanol, ethanol, propanol, butanol and pentanol or any combination thereof.

[0020] In some embodiments, the polypeptide described in the first aspect is contacted with the oil raw material and the lower alcohol in a lower alcohol environment with a concentration not higher than 30%, preferably 15% - 25%. In some embodiments, the lower alcohol is methanol.

[0021] In a seventh aspect, a method for screening the polypeptide of the present application is provided, which includes:

[0022] 1) Mutating the nucleotide sequence shown in SEQ ID NO: 1 to obtain a mutant sequence,

[0023] 2) Cloning the mutant sequence obtained in step 1) onto an expression vector, and then transforming or transducing it into a suitable host cell,

[0024] 3) Culturing the host cell in step 2), recovering the recombinant expression vector, and then transforming it into a yeast strain for culturing, and

[0025] 4) Screening for mutant polypeptides with higher methanol tolerance than the polypeptide shown in SEQ ID NO: 2.

[0026] In addition, the present application also provides the use of the nucleic acid sequence shown in SEQ ID NO: 1 for screening the polypeptides disclosed herein.

[0027] The polypeptide of the present application has good methanol stability. In particular, the polypeptide of the present application has good methanol stability under high temperature (such as 50 °C) conditions. Among them, under the conditions of 50 °C and 30% methanol concentration, the methanol stability of G2-4D is 1.56 times higher than that of the wild type, and the methanol stability of mutant A12 is 1.64 times higher than that of the wild type. Brief Description of the Drawings

[0028] Figure 1 Shows the stability of TL, G2-4D and A12 under 50 °C and 30% methanol conditions.

[0029] Figure 2 Shows the stability of TL, G2-4D and A12 at 50 °C.

[0030] Figure 3 Shows the method for detecting lipase activity. Detailed Description of the Embodiments

[0031] The polypeptide of the present application

[0032] The present application provides a polypeptide with lipase activity, which comprises a sequence selected from the following or consists of a sequence selected from the following:

[0033] (a) The amino acid sequence shown in SEQ ID NO: 4 or 6, and

[0034] (b) A sequence obtained by substituting, deleting or adding at least one amino acid to the sequence described in (a), wherein the polypeptide variant obtained from (b) still retains lipase activity.

[0035] In certain embodiments, the number of the above-mentioned amino acid substitutions, deletions or additions is 1-30, preferably 1-20, more preferably 1-10, wherein the obtained polypeptide variant substantially retains the lipase activity of the unchanged protein.

[0036] In certain embodiments, the above polypeptide variant differs from the amino acid sequence shown in SEQ ID NO: 4 or 6 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions, deletions and / or additions. In a preferred embodiment, the above polypeptide variant differs from the amino acid sequence shown in SEQ ID NO: 4 or 6 by about 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.

[0037] In some embodiments, the above polypeptide consists of the amino acid sequence shown in SEQ ID NO: 4 or 6.

[0038] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more conservative amino acid substitutions occur in the amino acid sequence of the polypeptide. The polypeptide after the conservative substitution substantially retains the lipase activity of the original polypeptide. Those skilled in the art are familiar with the specific conservative substitutions that can be implemented.

[0039] In other embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be deleted from the C-terminus or N-terminus of the polypeptide, and still have lipase activity.

[0040] In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added to the C-terminus or N-terminus region of the polypeptide, and the obtained polypeptide variant still has lipase catalytic activity.

[0041] In addition, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added or deleted from a region other than the C-terminus or N-terminus of the polypeptide, as long as the modified polypeptide substantially retains the lipase activity of the original polypeptide.

[0042] As used herein, the term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids used in protein biosynthesis, as well as other amino acids such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, etc., which are known to those skilled in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications can include, for example, substituting chemical groups and moieties on the amino acid, or derivatization of the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties, such as the charge and charge-space characteristics of an amino acid. For example, an organic structure mimicking arginine (Arg or R) has a positively charged moiety located in a similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures to maintain the optimal spatial and charge interactions of an amino acid or amino acid functional group. Those skilled in the art can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0043] In some embodiments, variants of the amino acid sequence set forth in SEQ ID NO:4 have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO:4. In a preferred embodiment, the polypeptide variant has 99% or more homology to the sequence set forth in SEQ ID NO:1.

[0044] In some embodiments, variants of the amino acid sequence set forth in SEQ ID NO:6 have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO:6. In a preferred embodiment, the polypeptide variant has 99% or more homology to the sequence set forth in SEQ ID NO:2.

[0045] As used herein, "homology" is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percentage of homology. Methods and computer programs for alignment are well known in the art.

[0046] The "polypeptides" and "proteins" of the present application are used interchangeably herein and refer to polymers of amino acid residues, their variants, and synthetic and naturally occurring analogs. Thus, these terms apply to naturally occurring amino acid polymers and their naturally occurring chemical derivatives, as well as amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids (such as chemical analogs of the corresponding naturally occurring amino acids). Such derivatives include, for example, post-translational modifications and degradation products, including phosphorylated, glycosylated, oxidized, isomerized, and deaminated variants of polypeptide fragments shown in SEQ ID NO: 4 or 6.

[0047] Certain amino acid substitutions known as "conservative amino acid substitutions" can occur frequently in proteins without altering the conformation or function of the protein, which is an established principle in protein chemistry.

[0048] Conservative amino acid substitutions in the present application include, but are not limited to, substituting any one of these aliphatic amino acids with any other of glycine (G), alanine (A), isoleucine (I), valine (V), and leucine (L); substituting serine (S) for threonine (T) and vice versa; substituting aspartic acid (D) for glutamic acid (E) and vice versa; substituting glutamine (Q) for asparagine (N) and vice versa; substituting lysine (K) for arginine (R) and vice versa; substituting any one of these aromatic amino acids with any other of phenylalanine (F), tyrosine (Y), and tryptophan (W); and substituting methionine (M) for cysteine (C) and vice versa. Other substitutions may also be considered conservative, depending on the particular amino acid environment and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can often be interchanged, as can alanine (A) and valine (V). The relatively hydrophobic methionine (M) can often be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchanged at positions where the important feature of the amino acid residue is its charge and the different pKs of these two amino acid residues are not significant. In certain environments, still other changes can be considered "conservative" (see, for example, BIOCHEMISTRY at pp. 13-15, 2nd ed. Lubert Stryer ed. (Stanford University); Henikoff et al., Proc. Nat’l Acad. Sci. USA (1992) 89: 10915-10919; Lei et al., J. Biol. Chem. (1995) 270(20): 11882-11886).

[0049] In certain embodiments, the polypeptides of the present application, such as the polypeptides shown in SEQ ID NO: 4 or 6 or variants thereof, are fused with heterologous polypeptides. In some embodiments, the fusion protein substantially retains the lipase activity of the polypeptide shown in SEQ ID NO: 4 or 6. In certain embodiments, the heterologous polypeptide is linked to the N-terminus of the polypeptide shown in SEQ ID NO: 4 or 6. In certain embodiments, the heterologous polypeptide is linked to the C-terminus of the polypeptide shown in SEQ ID NO: 4 or 6. In these embodiments, the heterologous polypeptide can be selected from purification tags (such as but not limited to: GST, MBP), epitope tags (such as but not limited to: Myc, FLAG), targeting sequences, signal peptides, etc. In a specific embodiment, the fusion protein comprises the polypeptide shown in SEQ ID NO: 4 or 6 and a tag, which binds to the C-terminus or N-terminus of the polypeptide shown in SEQ ID NO: 4 or 6, usually a peptide tag.

[0050] The polypeptides of the present application still have high enzyme activity under conditions of high methanol content, which is beneficial to the fatty acid methylation reaction. The "high methanol content" described herein is a methanol content of more than 14%, preferably not higher than 30% concentration, and more preferably 15%-25%. In some embodiments, the methanol content can be 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% and any value between the above two values.

[0051] Polynucleotide

[0052] The present application provides polynucleotides encoding the polypeptides disclosed herein, which comprise a sequence selected from or consist of a sequence selected from the following:

[0053] (a) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 or 6, or a sequence containing at least one amino acid substitution, deletion or addition in SEQ ID NO: 4 or 6; and

[0054] (b) A nucleotide sequence that hybridizes with the nucleotide sequence in (a) under stringent conditions.

[0055] In certain specific embodiments, the polynucleotides of the present application encode the polypeptides shown in SEQ ID NO: 4 or 6 and their functionally equivalent variants. In one embodiment, the polynucleotides of the present application have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the polynucleotides encoding the polypeptides shown in SEQ ID NO: 4 or 6 and their functionally equivalent variants.

[0056] In certain embodiments, the polynucleotides of the present application comprise nucleotide sequences having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the nucleotide sequences shown in SEQ ID NO: 3 or 5. In some embodiments, the polynucleotides of the present application comprise the nucleotide sequences shown in SEQ ID NO: 3 or 5.

[0057] In some embodiments, the polynucleotides of the present application consist of nucleotide sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the nucleotide sequences shown in SEQ ID NO: 3 or 5. In preferred embodiments, the polynucleotides of the present application consist of the nucleotide sequences shown in SEQ ID NO: 3 or 5. In more preferred embodiments, the polynucleotide consists of the nucleotide sequence shown in SEQ ID NO: 5.

[0058] In specific embodiments, the polynucleotide shown in SEQ ID NO: 3 encodes the amino acid sequence shown in SEQ ID NO: 4; the polynucleotide shown in SEQ ID NO: 5 encodes the amino acid sequence shown in SEQ ID NO: 6.

[0059] As used herein, the term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, cDNA or DNA, including DNA in single-stranded and double-stranded forms. The term generally refers to polynucleotide forms of nucleotides at least 10 bases in length, wherein the nucleotides are ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide.

[0060] In certain embodiments, the polynucleotides of the present application comprise nucleotide sequences that hybridize under stringent conditions to nucleotide sequences encoding the polypeptides shown in SEQ ID NO: 4 or 6 and their functionally equivalent variants, or consist of nucleotide sequences that specifically hybridize to nucleotide sequences encoding the polypeptides shown in SEQ ID NO: 4 or 6 and their functionally equivalent variants and encode polypeptides that are functionally equivalent to the polypeptides shown in SEQ ID NO: 4 or 6.

[0061] Those skilled in the art can routinely select the stringent conditions for DNA hybridization. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization usually depends on the ability of denatured DNA to reanneal when the complementary strands are in an environment below their melting temperature. The higher the degree of homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Higher relative temperatures tend to make the reaction conditions more stringent, while at lower temperatures, the stringency is lower. For a detailed description of the stringent conditions for hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0062] In certain embodiments, the stringent conditions employed for DNA hybridization include: 1) low ionic strength and high temperature during washing, such as 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 °C; 2) denaturing agents such as formamide during hybridization, such as 50% (v / v) formamide plus 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone in 50 mM sodium phosphate buffer, pH 6.5, and 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or (3) overnight hybridization at 42 °C in a hybridization solution containing 50% formamide, 5×SSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 mg / mL), 0.1% SDS, and 10% dextran sulfate, followed by washing in 0.2×SSC (sodium chloride / sodium citrate) at 42 °C for 10 minutes and then high-stringency washing in 0.1×SSC containing EDTA at 55 °C. Moderate stringency conditions can be determined as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989. Moderate stringency conditions include using washing solutions and hybridization conditions (such as temperature, ionic strength, and SDS percentage) with a stringency lower than those described above. For example, moderate stringency conditions include hybridization at 42 °C with at least about 16% v / v to at least about 30% v / v formamide and at least about 0.5 M to at least about 0.9 M salt, and washing at 55 °C with at least about 0.1 M to at least about 0.2 M salt. Moderate stringency conditions can also include hybridization at 65 °C with 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS, and washing at 60 - 65 °C with (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. A person skilled in the art will adjust the temperature, ionic strength, etc. according to factors such as the probe length. The stringency during nucleic acid hybridization depends on the length and degree of complementarity of the nucleic acid molecules, as well as other variables well known in the art. The greater the similarity or homology between two nucleotide sequences, the greater the Tm of the nucleic acid hybrid containing these sequences. The relative stability of nucleic acid hybridization (corresponding to a higher Tm) decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. Preferably, the minimum length of the hybridizable nucleic acid is at least about 12 nucleotides, preferably at least about 16 nucleotides, more preferably at least about 24 nucleotides, and most preferably at least about 36 nucleotides.

[0063] In some embodiments, the inventors of the present application are based on the lipase (TL) gene derived from Thermomyces lanuginosus, and according to the codon preference of Pichia pastoris, the sequence encoding the mature protein is codon-optimized, and the synthesized gene is expressed using Escherichia coli or Pichia pastoris as the expression host.

[0064] The polynucleotides disclosed in the present application can be combined with other DNA sequences, such as promoters, polyadenylation signals, other restriction enzyme cleavage sites, multiple cloning sites, other coding segments, etc., such that their total lengths can vary significantly. Thus, it is contemplated that polynucleotide fragments of almost any length can be utilized; the total length is preferably limited by the convenience of preparation and use in the intended recombinant DNA protocol.

[0065] Any of a variety of well-known and available mature techniques in the art can be utilized to prepare, manipulate, and / or express polynucleotides and their fusions. For example, the polynucleotide sequences encoding the polypeptides or variants thereof of the present application can be used in recombinant DNA molecules to direct the expression of the polypeptides in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can also be used in the present application, and these sequences can be used to clone and express a given polypeptide. Those skilled in the art can also select a codon optimization strategy suitable for a particular host cell according to the characteristics of the host cell, and obtain other DNA sequences encoding substantially the same or functionally equivalent amino acid sequences.

[0066] In addition, those skilled in the art are aware that DNA sequences expressed in eukaryotes often also contain introns to maintain their stability, and the relevant intron sequences will be spliced by the eukaryotic editing system during the coding process to obtain the finally expressed sequence. Therefore, for DNA sequences having expression in eukaryotic systems, they should also include sequences containing introns, as long as the amino acid sequences finally encoded by the DNA sequences are substantially the same or functionally equivalent to the aforementioned amino acid sequences.

[0067] In addition, the polynucleotide sequences of the present application can be modified using methods well-known in the art, including but not limited to altering the cloning, processing, expression, and / or activity of the gene product.

[0068] In certain embodiments, the polynucleotides of the present application are produced by artificial synthesis, such as direct chemical synthesis or enzymatic synthesis. In alternative embodiments, the above polynucleotides are produced by recombinant techniques.

[0069] In a specific embodiment, the wild-type TL amino acid sequence used in this application is derived from NCBI (GenBank Accession: O59952). A DNA sequence SEQ ID NO:1 is designed according to the codon preference of Pichia pastoris, and this DNA sequence is cloned into the pAO815 plasmid to obtain the pAO-TL expression vector.

[0070] In certain embodiments, the sequence of the obtained polynucleotide can be determined by conventional methods, preferably such as the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74:5463-5467). Such polynucleotide sequencing can also be completed using commercially available sequencing kits.

[0071] Expression vector

[0072] This application provides an expression vector comprising the polynucleotide of this application.

[0073] The "expression vector" described in this application is a recombinant or synthetically produced nucleic acid construct that has a series of specific nucleic acid elements that permit the transcription of a particular nucleic acid in a host cell. The expression vectors used in this application can be plasmid vectors such as pPIC9K, pAO815, pUC57, pET-24a(+), pIRES2-EGFP, pcDNA3.1, pCI-neo, pDC516, pVAC, pcDNA4.0, pGEM-T, pDC315, or viral vectors such as adenovirus, adeno-associated virus, retrovirus, semliki forest virus (sFv) vectors, or other vectors well known in the art.

[0074] In certain embodiments, the polynucleotide sequence encoding the polypeptide shown in SEQ ID NO:4 or 6 and its variants is cloned into a vector to construct a recombinant vector containing the polynucleotide described in this application.

[0075] In a preferred embodiment, the expression vector used for cloning the polynucleotide is a plasmid vector. In a more preferred embodiment, the plasmid vector is pPIC9K or pAO815.

[0076] In a specific embodiment, the above expression vector further comprises a regulatory sequence for regulating the expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence.

[0077] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence required to effect the expression of an encoding sequence to which it is ligated. The nature of such regulatory sequences varies with the host organism. In prokaryotes, such regulatory sequences generally include a promoter, a ribosome binding site, and a terminator; in eukaryotes, such regulatory sequences generally include a promoter, a terminator, and in some cases, an enhancer. Thus, the term "regulatory sequence" includes all sequences that are minimally required for the expression of a gene of interest to be present, and may also include other sequences that are beneficial for the expression of the gene of interest, such as a leader sequence.

[0078] As used herein, the term "operably linked" refers to the situation where the sequences involved are in a relationship that allows them to function in the desired manner. Thus, for example, a regulatory sequence "operably linked" to an encoding sequence enables the expression of the encoding sequence under conditions compatible with the regulatory sequence.

[0079] In certain embodiments, expression vectors containing a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 4 or 6 and its variants and appropriate transcriptional / translational regulatory elements are constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The nucleotide sequence is operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of such promoters may include, but are not limited to: the lac or trp promoter of Escherichia coli; the PL promoter of phage λ; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses, and some other known promoters that can control the expression of genes in prokaryotic cells, eukaryotic cells, or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcriptional terminator, etc. Inserting an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs in length, and act on the promoter to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication origin, the polyomavirus enhancer on the late side of the replication origin, and the adenovirus enhancer, etc.

[0080] In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli, etc.

[0081] Host cell

[0082] The present application provides host cells comprising the polynucleotides or expression vectors disclosed herein.

[0083] In certain embodiments, a polynucleotide encoding the polypeptide shown in SEQ ID NO: 4 or 6 and its variants, or an expression vector containing the polynucleotide, is transformed or transfected into a host cell to obtain a genetically engineered host cell containing the polynucleotide or expression vector.

[0084] The host cells used herein can be any host cell well-known to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells, etc. Exemplary bacterial cells include any species in the genera Escherichia, Bacillus, Streptomyces, Salmonella, Pseudomonas, and Staphylococcus, including, for example, Escherichia coli, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, Pseudomonas fluorescens. Exemplary fungal cells include any species in the genus Aspergillus. Exemplary yeast cells include any species in the genera Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, including Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe. Exemplary insect cells include any species in Spodoptera litura or Drosophila, including Drosophila S2 and Spodoptera litura Sf9. Exemplary animal cells include CHO, COS, or melanoma, or any mouse or human cell line. Selecting a suitable host is within the ability of those skilled in the art.

[0085] In a specific embodiment, the host cell used in the present application is Escherichia coli. In a preferred embodiment, the expression vector carrying the polynucleotide sequence of the present application is transformed into Escherichia coli DH5α strain for induced expression. In another specific embodiment, the expression vector carrying the polynucleotide sequence of the present application is transformed into Pichia pastoris cells for expression. Pichia pastoris that can be used in the present application includes GS115, M314, etc., which are suitable for general yeast transformation methods.

[0086] Any technique known in the art can be used to introduce the expression vector into the host cell, including transformation, transduction, transfection, viral infection, gene gun or Ti-mediated gene transfer. Specific methods include calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofection or electroporation, etc. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). As an example, when the host is a prokaryote such as Escherichia coli, competent cells can be harvested after the exponential growth phase and transformed by electroporation well-known in the art.

[0087] Screening and preparation methods of the polypeptides of the present application

[0088] The polypeptides of the present application can be screened and prepared by any suitable method known to those skilled in the art.

[0089] In some embodiments, the polypeptides disclosed herein are obtained by site-directed mutagenesis of TL, and the results can be verified by methods such as sequencing, and can also be used to guide the directed modification of TL.

[0090] In some embodiments, the polypeptides of the present application can also be produced by recombinant techniques or chemically synthesized. Methods for producing recombinant peptides are known in the art. Chemical synthesis methods of peptides are also well-known to those skilled in the art. For example, the polypeptides and their variants of the present application can be produced by directed peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963)). Protein synthesis can be carried out manually or by automation. For example, automated synthesis can be achieved using a 431A peptide synthesizer from Applied Biosystems (Perkin Elmer). Optionally, different fragments can be synthesized chemically separately and combined using chemical methods to prepare the desired molecule. This method is so common in the art that sequence synthesis can even be entrusted to professional companies.

[0091] In some embodiments, the screening methods of the polypeptides disclosed herein include:

[0092] 1) Mutate the nucleotide sequence shown in SEQ ID NO: 1 to obtain a mutant sequence,

[0093] 2) Clone the mutant sequence obtained in step 1) onto an expression vector, and then transform or transduce it into a suitable host cell,

[0094] 3) Culture the host cell in step 2), recover the recombinant expression vector, and then transform the product into a yeast strain for culture, and

[0095] 4) Screen for mutant polypeptides with higher methanol tolerance than the polypeptide shown in SEQ ID NO:2.

[0096] In some embodiments, sequence the verified mutants to determine the mutated sequences.

[0097] A suitable host cell refers to a host cell suitable for the expression of a vector or a polynucleotide of interest. A suitable culture medium refers to a culture medium suitable for the growth of the host cell or for inducing its expression.

[0098] In certain embodiments, various conventional culture media can be selected according to the host cell used. Culture under conditions suitable for the growth of the host cell. Preferably, the engineered host cell can be cultured in a conventional nutrient medium modified to activate the promoter to screen for transformants or amplify the polynucleotides of the present application. Transform the appropriate host cell and, when the host cell has grown to an appropriate cell density, induce the selected promoter by a suitable method (such as temperature shift or chemical induction), and culture the cells for a further period of time to allow it to produce the polypeptide of interest or a fragment thereof.

[0099] In certain embodiments, the polypeptide produced by the host cell can be coated intracellularly, or expressed on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. For example, the expressed polypeptide or a fragment thereof can be recovered and purified from the recombinant cell culture by the following methods well known in the art: conventional refolding treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods. As an illustrative example, affinity chromatography purification of proteins containing peptide tags (such as His-tag, etc.) at the C-terminus or N-terminus is a conventional method for obtaining highly pure polypeptide preparations.

[0100] Application of the polypeptide with lipase activity

[0101] The polypeptide of the present application is a polypeptide with lipase activity and can be used to produce biodiesel by the fatty acid methylation reaction. In some embodiments, the polypeptide of the present application is contacted with an oil raw material and a lower alcohol to prepare biodiesel.

[0102] In some embodiments, the reaction system is an environment with a high methanol content. For example, the methanol content is above 14%, preferably not higher than 30% in concentration, more preferably 15 - 25%, specifically 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% and any value between the above two values.

[0103] In some embodiments, the above-mentioned lower alcohols are selected from methanol, ethanol, propanol, butanol, and pentanol or any combination thereof. In a preferred embodiment, the lower alcohol is methanol.

[0104] The polypeptides disclosed herein have high methanol tolerance activity. For example, they can have high methanol tolerance activity at high temperatures (such as 50°C) and high methanol concentrations (such as 30% methanol concentration). Thus, the polypeptides disclosed herein can rapidly prepare fatty acid methyl esters at high temperatures and high methanol concentrations. Furthermore, the polypeptides disclosed herein can be used for the preparation of biodiesel at high temperatures and high methanol concentrations.

[0105] The polypeptides disclosed herein can maintain their activity for a long time at 50°C and 30% methanol concentration. Therefore, those skilled in the art can very easily foresee that at methanol concentrations lower than 30%, for example, at 15% - 25% methanol concentration, the polypeptides disclosed herein can maintain the activity of the enzyme for a longer time. Thus, the polypeptides disclosed herein can also be at methanol concentrations lower than 30%.

[0106] For example, at 15% - 25% methanol concentration, they can rapidly prepare fatty acid methyl esters. Furthermore, the polypeptides disclosed herein can be used for the preparation of biodiesel under the above conditions.

[0107] Examples

[0108] Experimental materials

[0109] 1) Experimental strains and plasmids

[0110] Strains: Pichia pastoris GS115 (Invitrogen), Escherichia coli DH5α (TAKARA).

[0111] Plasmid: pAO815, purchased from Invitrogen, catalog number V18020.

[0112] 2) Media and solutions

[0113] LB liquid medium: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0.

[0114] LB solid medium: Add agar at a concentration of 1.5% to the LB liquid medium.

[0115] YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose.

[0116] YPD solid medium: Add 2% agar to LB liquid medium.

[0117] BMMY - olive oil screening medium: Component A: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base (YNB) with ammonium sulfate and no amino acids, 1% methanol (added after sterilization), 4×10 - 5% D - biotin (added after sterilization), 0.1M citric acid - sodium citrate buffer pH 6.6, 2% agar. Component B: Component B olive oil substrate solution: Measure 150 ml of 4% PVA solution, add 50 ml of olive oil, emulsify with a high - speed homogenizer at 8000 rpm for 3 min, pause for 1 min and then emulsify for another 3 min to prepare the substrate solution. Mix 100 ml of sterilized Component A with 12 ml of Component B, and add 1 ml of 0.1% rhodamine B.

[0118] BMGY liquid medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base (YNB) with ammonium sulfate and no amino acids, 1% glycerol, 4×10 - 5% D - biotin, 0.1M citric acid - sodium citrate buffer pH 6.6.

[0119] Improved Bradford method protein concentration assay kit (purchased from Shanghai Sangon Biological Engineering Co., Ltd.);

[0120] Restriction endonucleases SacII and EcoRI (purchased from New England Biolabs (Beijing) Co., Ltd.);

[0121] PCR enzyme: TaKaRa Taq, HSDNA Polymerase (purchased from Takara Bio Inc. (Dalian));

[0122] T4 DNA ligase (purchased from Fulenzyme Co., Ltd.).

[0123] 3) Sequences and primers

[0124] SEQ ID: No.1 is the coding sequence of TL;

[0125] SEQ ID: No.2 is the amino acid sequence of TL;

[0126] SEQ ID: No.3 is the coding sequence of G2 - 4D;

[0127] SEQ ID: No.4 is the amino acid sequence of G2 - 4D;

[0128] SEQ ID: No.5 is the coding sequence of A12

[0129] SEQ ID: No. 6 is the amino acid sequence of A12;

[0130] SEQ ID: No. 7 is the α - mating factor leader peptide sequence;

[0131] SEQ ID: No. 8 is the TL - 1 sequence:

[0132] 5’ - GCGCCTAGGCCGCGGCGAAACGATGAGATTTCCTTCAATTTTTAC - 3’;

[0133] SEQ ID: No. 9 is the TL - 2 sequence:

[0134] 5’ - CCGGAATTCTTACAAGCAAGTACCAATCAG - 3’;

[0135] SEQ ID: No. 10 is the AOX1 - 5 sequence:

[0136] 5’ - GACTGGTTCCAATTGACAACG - 3’;

[0137] SEQ ID: No. 11 is the AOX1 - 3 sequence:

[0138] 5’ - GGCAAATGGCATTCTGACATCCTC - 3’.

[0139] Example 1: Construction of the TL mutant library and screening of mutants

[0140] The amino acid sequence of TL is derived from the wild - type Thermomyces lanuginosus lipase amino acid sequence, and the wild - type amino acid sequence is from NCBI (GenBank Accession: O59952), and its coding sequence is as shown in SEQ ID NO: 2. According to the codon preference of Pichia pastoris, the DNA sequence SEQ ID NO: 1 was designed, and the α - mating factor leader peptide sequence was added. This sequence is from the DNA sequence of the commercial vector pPIC9K, and its sequence is as shown in SEQ ID NO: 7. It was sent to Shanghai Sangon Biological Engineering Co., Ltd. for whole - gene synthesis, and this DNA sequence was cloned into the pAO815 plasmid to obtain the pAO - TL expression vector.

[0141] Using the pAO-TL vector as a template, error-prone PCR was performed with TaKaRa Taq enzyme and primer pair TL-1 (SEQ ID NO:8) / TL-2 (SEQ ID NO:9) (adding 0.3 mM of MnCl2 additionally during PCR), and a set of mutant amplicon fragments with a size of approximately 755 bp was obtained. The obtained fragments were cloned into pAO-TL through SacII and EcoRI restriction sites, and the resulting vector was transformed into Escherichia coli DH5α strain. A total of 1×10 6 TL mutants were obtained.

[0142] The plasmid library was transformed into Escherichia coli DH5α strain. All Escherichia coli clones were washed into LB liquid medium (containing 100 μg / ml ampicillin) and cultured at 37 °C for 4 h. The plasmid was extracted and linearized with SalI, and a fragment of approximately 8.5 kb was recovered. Taking 500 ng of the vector, the vector was transformed into competent cells of Pichia pastoris GS115 strain by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 °C for 3 days to obtain a Pichia pastoris mutant library of TL. Single colonies on the plates were picked and transferred to BMMY-olive oil screening plates. Two clones with large color-changing circles were selected and numbered G2-4D and A12 respectively.

[0143] The pAO-TL plasmid was linearized with SalI, and a fragment of approximately 8.5 kb was recovered. Taking 500 ng of the vector, the vector was transformed into competent cells of Pichia pastoris GS115 strain by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 °C for 3 days. Single colonies on the plates were picked and transferred to BMMY-olive oil screening plates. The clone with a large color-changing circle was selected as the wild-type TL expression strain.

[0144] Example 2: Performance detection of G2-4D and A12 mutants

[0145] Taking G2-4D and A12 mutant strains and the wild-type TL expression strain, first activate them in liquid YPD, and then inoculate them into BMGY medium and culture them overnight at 30 °C with shaking at 220 rpm. Transfer the culture to BMMY medium with an initial OD600 of 6 and culture it at 30 °C with shaking at 220 rpm for 72 h. The specific process is as follows:

[0146] First, induce with 2% methanol, add 1% methanol at 24 h and 32 h respectively, add 1% methanol at 48 h and 56 h respectively, and sample at 72 h. The obtained samples were ultrafiltered, desalted, and concentrated 40 times with an ultrafiltration tube with a molecular weight cut-off of 10 kDa. The treated samples were added to the buffer (20 mM citric acid-sodium citrate buffer (pH 6.6)).

[0147] Adjust the enzyme solutions of TL, G2-4D and A12 to 1 mg / ml. Take one portion and place it in a 50°C water bath, and add 30% methanol to another portion and place it in a 50°C water bath. Samples are taken at 4 h, 22 h, and 44 h to measure the enzyme activity. Using the enzyme activity of the untreated enzyme solution (the initial enzyme activities of TL, G2-4D and A12 are 8067 U / ml; 8489 U / ml; and 8111 U / ml respectively) as a control, the relative enzyme activity is recorded as 100%. The method for measuring enzyme activity is as Figure 3 shown, where the substrate solution is as follows:

[0148] Measure 150 ml of 4% PVA solution, add 50 ml of olive oil, emulsify with a high-speed homogenizer at 8000 rpm for 3 min, pause for 1 min and then emulsify for 3 min to prepare the substrate solution (this solution needs to be prepared and used immediately).

[0149] The results are as Figure 1 and 2 shown. After TL, G2-4D and A12 are placed at 50°C for 44 h, the enzyme activity remains very stable without obvious changes. However, after adding 30% methanol, the residual enzyme activity of wild-type TL is only 55% after being placed for 22 h, while the residual enzyme activities of G2-4D and A12 are 84% and 95% respectively. After being placed for 44 h, the residual enzyme activity of wild-type TL is only 25%, while the residual enzyme activities of G2-4D and A12 are 64% and 66% respectively. Compared with TL, the methanol tolerance of G2-4D and A12 is significantly improved, so that fatty acid methyl esters can be rapidly prepared at high temperature and high methanol concentration, and then used for the preparation of biodiesel.

[0150] Example 3: Sequence analysis of G2-4D and A12 mutants

[0151] Inoculate the G2-4D and A12 strains into 3 ml of YPD liquid medium and culture overnight at 30°C to extract genomic DNA. Using the genomic DNA of the G2-4D and A12 strains as templates, use HSDNA polymerase and primer pair AOX1-5 (SEQ ID NO:10) / AOX1-3 (SEQ ID NO:11) for PCR amplification to obtain the DNA sequence of TL in the G2-4D and A12 strains. Send the obtained sequence to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing.

[0152] The DNA sequencing results of TL of the G2-4D strain are as shown in SEQ ID NO:3, and its coding sequence is as shown in SEQ ID NO:4. Among them, asparagine at position 33 is mutated to lysine, glycine at position 38 is mutated to cysteine, aspartic acid at position 111 is mutated to alanine, serine at position 231 is mutated to glutamic acid, and asparagine at position 233 is mutated to arginine.

[0153] The DNA sequencing result of the TL of strain A12 is shown in SEQ ID NO:5, and its coding sequence is shown in SEQ ID NO:6. Among them, asparagine at position 33 is mutated to lysine, glycine at position 38 is mutated to threonine, aspartic acid at position 111 is mutated to leucine, asparagine at position 233 is mutated to serine, and leucine at position 259 is mutated to valine.

[0154] It can be understood that although this application is described in a certain form, this application is not limited to the content shown and described in this specification. It is obvious to those skilled in the art that various changes can be made without departing from the scope of this application. These changes are all within the scope claimed in this application. Sequence Listing <110> Wilmar (Shanghai) Biotechnology R & D Center Co., Ltd. <120> A Methanol-Tolerant Lipase Mutant <130> 123123 <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 810 <212> DNA <213> Thermomyces lanuginosus <400> 1 gaagtctctc aagacttgtt caaccagttc aacttgttcg ctcaatactc tgccgctgcc 60 tactgtggta agaacaatga tgctccagct ggtactaaca ttacctgtac tggtaacgct 120 tgtccagaag ttgagaaggc tgatgctacc ttcctgtact ccttcgaaga ctctggagtt 180 ggagatgtta ctggtttcct ggccttggat aacactaaca agttgatcgt tctgtccttc 240 agaggttcca gatccatcga gaactggatt ggtaacttga actttgactt gaaggagatc 300 aacgacatct gttctggatg tcgtggtcac gatggattta cctcctcttg gagatctgtt 360 gctgatacct tgagacagaa ggtcgaagat gctgtcagag aacatccaga ctatagagtt 420 gtcttcactg gtcactcctt gggaggtgcc ttggctactg ttgctggtgc tgacttgcgt 480 ggtaatggtt atgacattga tgtcttctcc tacggtgctc caagagttgg taatcgtgcc 540 ttcgctgagt ttctgaccgt ccaaactgga ggtactttgt acagaattac ccatactaac 600 gacattgttc caagattgcc accacgtgag ttcggatact ctcattcctc tccagagtac 660 tggatcaagt ctggaacctt ggttccagtc actcgtaacg acatcgtcaa gattgaaggt 720 attgatgcca ctggaggtaa caatcaacca aacattccag acattccagc tcacttgtgg 780 tactttggtc tgattggtac ttgcttgtaa 810 <210> 2 <211> 269 <212> PRT <213> Thermomyces lanuginosus <400> 2 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Asp 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro 245 250 255 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 3 <211> 810 <212> DNA <213> Thermomyces lanuginosus <400> 3 gaagtctctc aagacttgtt caaccagttc aacttgttcg ctcaatactc tgccgctgcc 60 tactgtggta agaacaatga tgctccagct ggtactaaga ttacctgtac ttctaacgct 120 tgtccagaag ttgagaaggc tgatgctacc ttcctgtact ccttcgaaga ctctggagtt 180 ggagatgtta ctggtttcct ggccttggat aacactaaca agttgatcgt tctgtccttc 240 agaggttcca gatccatcga gaactggatt ggtaacttga actttgactt gaaggagatc 300 aacgacatct gttctggatg tcgtggtcac gcgggattta cctcctcttg gagatctgtt 360 gctgatacct tgagacagaa ggtcgaagat gctgtcagag aacatccaga ctatagagtt 420 gtcttcactg gtcactcctt gggaggtgcc ttggctactg ttgctggtgc tgacttgcgt 480 ggtaatggtt atgacattga tgtcttctcc tacggtgctc caagagttgg taatcgtgcc 540 ttcgctgagt ttctgaccgt ccaaactgga ggtactttgt acagaattac ccatactaac 600 gacattgttc caagattgcc accacgtgag ttcggatact ctcattcctc tccagagtac 660 tggatcaagt ctggaacctt ggttccagtc gagcgtcgtg acatcgtcaa gattgaaggt 720 attgatgcca ctggaggtaa caatcaacca aacattccag acattccagc tcacttgtgg 780 tactttggtc tgattggtac ttgcttgtaa 810 <210> 4 <211> 269 <212> PRT <213> Thermomyces lanuginosus <400> 4 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 Lys Ile Thr Cys Thr Cys Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Asp 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Ala Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Glu Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro 245 250 255 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 5 <211> 810 <212> DNA <213> Thermomyces lanuginosus <400> 5 gaagtctctc aagacttgtt caaccagttc aacttgttcg ctcaatactc tgccgctgcc 60 tactgtggta agaacaatga tgctccagct ggtactcgta ttacctgtac tacgaacgct 120 tgtccagaag ttgagaaggc tgatgctacc ttcctgtact ccttcgaaga ctctggagtt 180 ggagatgtta ctggtttcct ggccttggat aacactaaca agttgatcgt tctgtccttc 240 agaggttcca gatccatcga gaactggatt ggtaacttga actttgactt gaaggagatc 300 aacgacatct gttctggatg tcgtggtcac cttggattta cctcctcttg gagatctgtt 360 gctgatacct tgagacagaa ggtcgaagat gctgtcagag aacatccaga ctatagagtt 420 gtcttcactg gtcactcctt gggaggtgcc ttggctactg ttgctggtgc tgacttgcgt 480 ggtaatggtt atgacattga tgtcttctcc tacggtgctc caagagttgg taatcgtgcc 540 ttcgctgagt ttctgaccgt ccaaactgga ggtactttgt acagaattac ccatactaac 600 gacattgttc caagattgcc accacgtgag ttcggatact ctcattcctc tccagagtac 660 tggatcaagt ctggaacctt ggttccagtc actcgttcgg acatcgtcaa gattgaaggt 720 attgatgcca ctggaggtaa caatcaacca aacattccag acattccagc tcacctttgg 780 tactttggtc tgattggtac ttgcttgtaa 810 <210> 6 <211> 269 <212> PRT <213> Thermomyces lanuginosus <400> 6 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 Lys Ile Thr Cys Thr Thr Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Asp 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Leu Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Thr Arg Ser Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro 245 250 255 Ala His Val Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 7 <211> 267 <212> DNA <213> Artificial Sequence () <400> 7 atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagctgct 60 ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120 tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180 aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240 tctcttgaga aaagagaggc tgaagct 267 <210> 8 <211> 45 <212> DNA <213> Artificial sequence () <400> 8 gcgcctaggc cgcggcgaaa cgatgagatt tccttcaatt tttac 45 <210> 9 <211> 30 <212> DNA <213> Artificial sequence () <400> 9 ccggaattct tacaagcaag taccaatcag 30 <210> 10 <211> 21 <212> DNA <213> Artificial sequence () <400> 10 gactggttcc aattgacaac g 21 <210> 11 <211> 24 <212> DNA <213> Artificial sequence () <400> 11 ggcaaatggc attctgacat cctc 24

Claims

1. A polypeptide having lipase activity, which is selected from the amino acid sequences shown in SEQ ID NO: 4 or 6.

2. A polynucleotide encoding the polypeptide according to claim 1.

3. The polynucleotide according to claim 2, which is selected from the nucleotide sequences shown in SEQ ID NO: 3 or 5.

4. An expression vector comprising at least one polynucleotide according to claim 2 or 3.

5. The expression vector according to claim 4, which further comprises a regulatory sequence for regulating the expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence.

6. The expression vector according to claim 5, wherein the expression vector is a plasmid.

7. A host cell comprising the polynucleotide according to claim 2 or 3, or the expression vector according to claim 5 or 6, and the host cell is not a plant cell.

8. The host cell according to claim 7, wherein the host cell is yeast or Escherichia coli.

9. Use of the polypeptide according to claim 1 for carrying out fatty acid methylation reaction or for preparing biodiesel.

10. The use according to claim 9, wherein the polypeptide is used for carrying out fatty acid methylation reaction or for preparing biodiesel in a methanol environment with a concentration not higher than 30%.

11. The use according to claim 9, wherein the polypeptide is used for carrying out fatty acid methylation reaction or for preparing biodiesel in a methanol environment with a concentration of 15% - 25%.

12. A method for preparing biodiesel, which comprises contacting the polypeptide according to claim 1 with an oil raw material and a lower alcohol, wherein the lower alcohol is selected from methanol, ethanol or any combination thereof.

13. The method according to claim 12, wherein the contacting is carried out by contacting the polypeptide according to claim 1 with an oil raw material and a lower alcohol in a lower alcohol environment with a concentration not higher than 30%.

14. The method according to claim 12, wherein the contacting is carried out by contacting the polypeptide according to claim 1 with an oil raw material and a lower alcohol in a lower alcohol environment with a concentration of 15% - 25%.

15. The method according to claim 13 or 14, wherein the lower alcohol is methanol.

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