CN108239648B

CN108239648B - Method for efficiently expressing rhizomucor miehei lipase

Info

Publication number: CN108239648B
Application number: CN201611217262.9A
Authority: CN
Inventors: 宣姚吉; 徐正军; 牛其文
Original assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Current assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Priority date: 2016-12-26
Filing date: 2016-12-26
Publication date: 2023-04-07
Anticipated expiration: 2036-12-26
Also published as: CN108239648A

Abstract

The invention relates to a method for efficiently expressing rhizomucor miehei lipase. In particular, the present invention provides a polynucleotide sequence selected from the group consisting of: (1) 1, nucleotide sequence 229-1035 of SEQ ID NO; and (2) the complement of the sequence of (1). The present invention also provides a polynucleotide sequence selected from the group consisting of: (1) A polynucleotide sequence encoding a polypeptide sequence of the formula: A-L1-B-L2-RML; wherein A may or may not be present and when present is a Rhizomucor miehei lipase leader peptide; l1 is present in the presence of A and is the cleavage site for proteases kex2 and ste 13; b is rhizomucor miehei lipase leader peptide; l2 is the enzyme cutting site of protease kex2 and ste 13; RML represents the mature peptide sequence of Rhizomucor miehei lipase; and (2) the complement of the sequence of (1).

Description

Method for efficiently expressing rhizomucor miehei lipase

Technical Field

The invention relates to a method for efficiently expressing rhizomucor miehei lipase.

Background

Lipase EC 3..1.1.3, triacylglycerol acyl hydrolase, it catalyzes the natural substrate oil and fat to hydrolyze and produce fatty acid, glycerin and monoglyceride or diester, apply to industries such as oil and fat processing, food, medicine, daily use chemicals, etc. extensively, it is one of the important industrial enzyme preparations. The catalytic activity of lipase is only dependent on its protein structure, so that lipases from different sources have different catalytic properties and catalytic activities.

Currently, about 2% of microorganisms are known to produce lipase, and the microorganisms capable of producing lipase include at least 65 genera, among which 28 genera of bacteria, 4 genera of actinomycetes, 10 genera of yeast, and 23 genera of other fungi (Hasan, 2006). Among them, rhizomucor miehei lipase has been widely used in the enzymatic synthesis of structural lipids (Structured lipids) due to its strong sn-1,3 selectivity and high activity, such as the synthesis of SOS, OPO, DAG, processing fish oil to enrich polyunsaturated fatty acids such as DHA (Pedersen, 1995), and the preparation of chiral pharmaceutical intermediates and certain biomaterials (Fujikih, 2009), etc.

The natural Rhizomucor miehei fatty acid has the defects of low yield, unstable components, difficult extraction and the like, so that the natural Rhizomucor miehei fatty acid cannot be industrially produced. Therefore, the research on RML mainly focuses on the enzymatic properties, the application range and the high-efficiency expression in genetically engineered bacteria. Among reports on RML, novoxin (Novo) was studied most thoroughly. In 1977 (Moskowitz, 1977), researchers at Novo performed extensive studies on natural RML, and the results showed that: RML is a glycoprotein which can extensively hydrolyze animal fats and vegetable oils and is stable at pH4-9 at room temperature for a certain period of time, with pH 6.0 being the most stable. In 1987, huge-jensen of Novo reports that two enzymes RML-A and RML-B with consistent high immunogenicity exist in natural Rhizomucor miehei fermentation liquor, and the relationship and the primary structure of the two enzymes are clarified. In 1988 (Boel, 1988), the company Novo, boel and Huge-jensen, found a cDNA sequence encoding RML, and determined that the RML precursor protein was composed of RML, a precursor peptide chain of 70 amino acid residues and a signal peptide of 24 amino acid residues, and that the enzyme cleaved peptide bond between MET-SER of zymogen protein to give 269 amino acid residues of RML. In 1989, huge-jensen inserted the precursor protein gene of RML into a vector of Aspergillus oryzae, and expressed the gene using the promoter of alpha-amylase gene and the terminator of glucoamylase to obtain extracellular-secreted rRML. The rRML obtained by using the expression vector has 70% of the N-terminal amino acid sequence identical to that of the natural enzyme, and the other 30% of the recombinant enzyme has one less serine threonine residue than that of the natural enzyme. In addition, the isoelectric point of the recombinant enzyme is 4.3, the isoelectric point of the recombinant enzyme is consistent with that of RML, the sugar content of the recombinant enzyme is 1.2%, and the immunological properties of the recombinant enzyme are also highly similar to those of natural enzyme. At present, RML sold in the market of Novo company is also gene modified lipase expressed by using Aspergillus oryzae as a vector by using genetic engineering technology, and the trade name of liquid enzyme is

20000L, immobilized enzyme is sold under the trade name Lipozyme RM IM. Although the fungus Aspergillus oryzae is an excellent expression vector, it secretes itself various non-target proteins, such as: the content of non-target protein amylase in the Lipozyme RM enzyme solution of Novo is much higher than that of target protein RML lipase, and in addition,there are also many other heteroproteins such as: proteases, etc., which have resulted in their use being greatly limited. Currently, even though Novo repurifies RML liquid to obtain the trade name &>

388 RML, but is expensive, further limiting its use in industry.

Disclosure of Invention

The first aspect of the invention provides a coding sequence of Rhizomucor miehei lipase, which is selected from the following sequences:

(1) 1, nucleotide sequence 229-1035 of SEQ ID NO; and

(2) (1) the complement of said sequence.

In a second aspect, the present invention provides a polynucleotide sequence selected from the group consisting of:

(1) A polynucleotide sequence encoding a polypeptide sequence represented by formula I:

A-L1-B-L2-RML (formula I)

In the formula (I), the compound is shown in the specification,

a may be present or absent and, when present, is a Rhizomucor miehei lipase leader peptide;

l1, present in the presence of A, is the cleavage site for proteases kex2 and ste 13;

b is rhizomucor miehei lipase leader peptide;

l2 is the enzyme cutting site of protease kex2 and ste 13; and

RML represents the mature peptide sequence of Rhizomucor miehei lipase; and

(2) (1) the complement of said sequence.

In one or more embodiments, a and L1 are present in the polypeptide sequence of formula I.

In one or more embodiments, the Rhizomucor miehei lipase leader peptide coding sequence is set forth in positions 1-210 of SEQ ID NO 1.

In one or more embodiments, the Rhizomucor miehei lipase mature peptide coding sequence is as shown in SEQ ID NO. 1 at positions 229-1035.

In one or more embodiments, the nucleotide sequences of L1 and L2 are each independently as set forth in SEQ ID NO. 1, positions 211-228, or SEQ ID NO. 3, positions 211-234.

In one or more embodiments, the Rhizomucor miehei lipase leader peptide is as shown in SEQ ID NO 2 positions 1-70.

In one or more embodiments, the Rhizomucor miehei lipase mature peptide is as shown in SEQ ID NO 2 positions 77-345.

In one or more embodiments, the amino acid sequences of L1 and L2 are each independently as set forth in positions 71-76 of SEQ ID NO. 2, or as set forth in positions 71-78 of SEQ ID NO. 4.

In one or more embodiments, the polypeptide sequence of formula I is as set forth in SEQ ID NO 2 or 4.

In one or more embodiments, the polynucleotide sequence is selected from the group consisting of:

(i) 1 or 3; and

(ii) (ii) (i) the complement of the polynucleotide sequence.

In a third aspect, the invention provides a nucleic acid construct comprising a polynucleotide sequence according to the invention.

In one or more embodiments, the nucleic acid construct comprises a polynucleotide sequence according to the first aspect of the invention.

In one or more embodiments, the nucleic acid construct comprises a polynucleotide sequence according to the second aspect of the invention.

In one or more embodiments, the nucleic acid construct is a cloning vector or an expression vector.

In one or more embodiments, the expression vector is a vector suitable for expression in pichia pastoris, including but not limited to pPIC, pPICZ, pAO, pGAP, and pGAPZ.

In one or more embodiments, the expression vector has the pAO815 plasmid as a backbone.

In a fourth aspect, the invention provides a genetically engineered host cell comprising a polynucleotide sequence or nucleic acid construct according to the invention.

In one or more embodiments, the cell is a yeast.

In one or more embodiments, the yeast is pichia pastoris.

In a fifth aspect, the present invention provides a method for preparing Rhizomucor miehei lipase, which comprises the steps of constructing an expression vector containing the polynucleotide sequence of the present invention, transferring the expression vector into a host cell, and culturing the host cell to express the lipase.

The fifth aspect of the invention provides a method for constructing an expression vector capable of improving the expression level of rhizomucor miehei lipase, which comprises the step of inserting a gene sequence of enzyme cutting sites of protease kex2 and ste13 between a coding sequence of rhizomucor miehei lipase leader peptide and a coding sequence of mature peptide into the expression vector.

In one or more embodiments, the inserted gene sequence further comprises a copy of the Rhizomucor miehei lipase leader peptide coding sequence and cleavage sites for proteases kex2 and ste13 in front of the Rhizomucor miehei lipase leader peptide coding sequence.

In one or more embodiments, the gene sequence has been optimized according to the codon preference of pichia pastoris.

In one or more embodiments, the expression vector is suitable for expression in yeast.

In one or more embodiments, the expression vector is suitable for expression in pichia pastoris.

In one or more embodiments, the nucleotide sequence of the cleavage site of the proteases kex2 and ste13 is as shown in SEQ ID NO. 1 from position 211 to 228 or as shown in SEQ ID NO. 3 from position 211 to 234.

In one or more embodiments, the amino acid sequence of the cleavage sites of the proteases kex2 and ste13 is as shown in SEQ ID NO. 2 at positions 71 to 76 or SEQ ID NO. 4 at positions 71 to 78.

In one or more embodiments, the gene sequence encodes an amino acid sequence as set forth in SEQ ID NO 2 or 4.

In one or more embodiments, the gene sequence is selected from the group consisting of:

(i) 1 or 3; and

(ii) (ii) (i) the complement of the polynucleotide sequence.

In a sixth aspect, the present invention provides a method for producing a Rhizomucor miehei lipase, said method comprising the step of fermenting a host cell of the invention.

Drawings

FIG. 1: pro/RML and 2 pro/RML.

FIG. 2: SDS-PAGE of pro/RML-1 and 2pro/RML-1, lane 2pro/RML-1; lane 2 is pro/RML-2.

Detailed Description

The Rhizomucor miehei gene with leader peptide is optimized according to the preference of pichia pastoris codon, enzyme cutting sites of protease kex2 and ste13 are added between the leader peptide and mature peptide, the gene is cloned into a pichia pastoris expression vector such as pAO815 to obtain a corresponding expression vector, the pichia pastoris strain is transformed, the obtained positive clone is subjected to shake flask fermentation, and the enzyme activity is measured to be 311U/ml. The invention also connects the leader peptide of the 2 copies of Rhizomucor miehei lipase gene with the mature peptide, and increases the enzyme cutting sites of protease kex2 and ste13 between the two leader peptides and between the second leader peptide and the mature peptide, clones the gene into a pichia expression vector such as pAOm-PLC to obtain a corresponding expression vector, converts the pichia strain, and carries out shake flask fermentation on the obtained positive clone to obtain the enzyme activity of 464U/ml, and the output of RML is improved by 50 percent compared with the expression mode of single leader peptide.

Therefore, the invention provides a method for efficiently expressing rhizomucor miehei lipase and pichia pastoris for efficiently expressing rhizomucor miehei lipase.

In the invention, the Rhizomucor miehei lipase can be various lipases from Rhizomucor miehei known in the field, including various mutants of wild type Rhizomucor miehei lipase, as long as the lipase activity of the mutant has industrial application value. Generally, the Rhizomucor miehei lipase of the present invention refers to a mature peptide. In certain embodiments, the methods of the invention are used to express Mucor miehei lipase represented by the amino acid sequence at positions 77-345 of SEQ ID NO 2.

According to the invention, the enzyme cutting sites of protease kex2 and ste13 are inserted between the coding sequences of the rhizomucor miehei lipase leader peptide and the mature peptide coded by the vector for expressing the rhizomucor miehei lipase, so that the high expression of the rhizomucor miehei lipase in host cells through the expression vector can be realized. Several (e.g., 1 to 3) kex2 protease cleavage sites and several (e.g., 1 to 3) stel3 protease cleavage sites may be inserted. In certain embodiments, the amino acid sequences of the cleavage sites of proteases kex2 and ste13 can be as shown in SEQ ID NO 2 at positions 71 to 76. In certain embodiments, 1 kex2 and 2 stel3 cleavage sites can be inserted. In these embodiments, the amino acid sequence of an exemplary cleavage site is shown in SEQ ID NO 4, positions 71-78.

Various aspects of the invention will be described in detail below.

Polynucleotide sequences

The polynucleotide sequences of the invention are selected from:

A-L1-B-L2-RML (formula I)

In the formula (I), the compound is shown in the specification,

l1 is present in the presence of A and is the cleavage site for proteases kex2 and ste 13;

b is rhizomucor miehei lipase leader peptide;

l2 is the enzyme cutting site of protease kex2 and ste 13; and

RML represents the mature peptide sequence of Rhizomucor miehei lipase; and

(2) (1) the complement of said sequence.

In the present invention, the Rhizomucor miehei lipase leader peptide may be various leader peptides of the lipase obtained from Rhizomucor miehei. In certain embodiments, the leader peptide has the sequence shown in SEQ ID NO 2 positions 1-70.

In the present invention, the cleavage sites of the proteases kex2 and ste13 generally refer to the amino acid sequences shown in SEQ ID NO. 2 at positions 71 to 76. In certain embodiments, 1 kex2 and 2 stel3 cleavage sites can be inserted. In these embodiments, the amino acid sequence of an exemplary cleavage site is shown in SEQ ID NO 4 at positions 71-78.

Thus, L1 and L2 of the present invention may each independently be an amino acid sequence as shown in positions 71 to 76 of SEQ ID NO. 2 or an amino acid sequence as shown in positions 71 to 78 of SEQ ID NO. 4.

In certain embodiments of the invention, a and L1 are present in the polypeptide sequence of formula I encoded by the nucleotide sequence.

As a specific example, the polypeptide sequence of formula I can be shown as SEQ ID NO 2 or 4. As a specific example of the polynucleotide sequence, the present invention provides the polynucleotide sequence shown in SEQ ID NO. 1 or 3, also including the complementary sequences of SEQ ID NO. 1 and 3.

The present application also encompasses degenerate variants of the nucleotide sequence encoding a polypeptide of formula I of the present invention. As used herein, "degenerate variants" refers in the present invention to nucleotide sequences that encode identical amino acid sequences, but differ in nucleotide sequence.

In a preferred embodiment, the polynucleotide sequence of the invention, in particular the coding sequence for the mature peptide of Rhizomucor miehei lipase, has been optimized according to the preferences of the Pichia pastoris codons. For example, in certain embodiments, the coding sequence for the mature peptide is as set forth in nucleotides 77-345 of SEQ ID NO. 1.

Thus, in certain embodiments, the present invention also provides a coding sequence for a mature peptide of rhizomucor miehei lipase selected from the group consisting of:

(1) 1, nucleotide sequence 229-1035 of SEQ ID NO; and

(2) (1) the complement of said sequence.

The polynucleotide sequences of the present invention can be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Nucleic acid constructs

The invention also relates to nucleic acid constructs comprising one or more control sequences operably linked to a polynucleotide sequence of the invention.

As used herein, "operably linked" or the like refers to an arrangement of elements wherein the components are arranged in a configuration so as to perform their intended function. Thus, a given promoter operably linked to a coding sequence is capable of effecting expression of that coding sequence in the presence of the appropriate transcription factors and the like. The promoter need not be contiguous with the coding sequence, so long as it functions to direct expression of the sequence. Thus, for example, sequences which are not involved in translation but are transcribed may be present between the promoter sequence and the coding sequence, as may the transcribable intron; and the promoter sequence may still be considered "operably linked" to the coding sequence.

Polynucleotides encoding polypeptides of the invention can be manipulated in a variety of ways to ensure expression of the polypeptide. Manipulation of the polynucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate promoter sequence, a nucleotide sequence recognized by a host cell for expression of a polynucleotide encoding a polypeptide of formula I of the present invention. The promoter sequence comprises transcriptional control sequences which are linked to the expression of the polypeptide. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

In a yeast host, useful promoters may be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), saccharomyces cerevisiae galactokinase (GAL 1), saccharomyces cerevisiae alcohol dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, saccharomyces cerevisiae triose phosphate isomerase, saccharomyces cerevisiae 3-phosphoglycerate kinase, pichia pastoris alcohol oxidase. Other useful promoters for yeast host cells are described by Romanos et al, 1992, yeast 8.

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 linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, saccharomyces cerevisiae cytochrome C, saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase, pichia pastoris alcohol oxidase, and the like.

Carrier

The present invention also relates to vectors, including but not limited to expression vectors and cloning vectors, comprising a polynucleotide sequence of the present invention. For example, in certain embodiments, the nucleic acid constructs of the invention are expression vectors or cloning vectors.

In an expression vector, the various nucleic acids and control sequences may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites which allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, the nucleotide sequences of the present invention may be expressed by insertion of the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate expression vector. In making the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleotide sequence of interest. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

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 comprise any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids, or a transposon, which together contain the total DNA to be introduced into the genome of the host cell, may be used.

The vectors of the invention preferably comprise one or more selectable markers that allow for easy selection of transformed, transfected, transduced, or the like cells. Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophy to auxotrophs, and the like.

The vectors of the present invention preferably contain elements that permit integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome.

More than one copy of a polynucleotide of the invention may be inserted into a host cell to increase the yield of the gene product. An increase in the copy number of a polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including an amplifiable selectable marker gene with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene and, thus, additional copies of the polynucleotide can be screened for by culturing the cells in the presence of the appropriate selectable agent.

The expression vector of the present invention is more preferably selected from vectors that can be used for expression in Pichia pastoris. The vector of the present invention is preferably a series of vectors such as pPIC, pPICZ, pAO, pGAP or pGAPZ, which are used in commercially available Pichia pastoris.

Cloning vectors containing the polynucleotide sequences of the present invention are useful for replicating a sufficient number of plasmids of interest. Therefore, the cloning vector of the present invention has strong self-replicating elements such as replication initiation sites and the like. Typically, the cloning vectors of the present invention do not have expression elements.

Host cell

The invention also relates to recombinant host cells comprising a polynucleotide of the invention which are used for the recombinant production of the polypeptide. The vector comprising the polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as an extrachromosomal self-replicating vector as described earlier. The choice of host cell will depend to a large extent on the gene encoding the polypeptide and its source.

Preferably, the host cells suitable for use in the present invention are cells of the phylum Ascomycota, such as the genera Saccharomyces (Saccharomyces), pichia (Pichia), yarrowia (Yarrowia), candida (Candida), and Komagataella, among others.

In a most preferred aspect, the host cell is Pichia pastoris (Pichia pastoris), saccharomyces cerevisiae (Saccharomyces cerevisiae), yarrowia lipolytica (Yarrowia lipolytica), and the like. In a further most preferred aspect, the host cell is a Pichia pastoris (Pichia pastoris) cell.

Production method

After obtaining the coding sequence for the polypeptide, the polypeptide of the present invention can be produced by a method comprising: (a) Culturing a host cell comprising an expression vector that expresses a polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

In the production methods of the present invention, the cells can be cultured in a medium suitable for production of the polypeptide using methods known in the art. For example, a cell may be cultured by shake flask culture and 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. Cultivation takes place in a suitable medium comprising carbon and nitrogen sources and inorganic salts using methods known in the art. Suitable media are available from commercial suppliers or may be prepared according to the disclosed compositions. If the polypeptide is secreted into the culture medium, the polypeptide can be recovered directly from the culture medium. If the polypeptide is not secreted into the culture medium, it can be recovered from the cell lysate.

In certain embodiments, the host cell of the invention is a yeast, preferably pichia pastoris. Thus, production of the polypeptide can be carried out according to conventional yeast fermentation methods. For example, as a specific example of fermentation, the yeast cells of the invention may be obtained by activating them in liquid YPD, then inoculating them in BMGY, culturing overnight at 30 ℃ and 220rpm, transferring to BMMY medium with an initial OD600 of 6, an initial induction with 2% methanol, 1% after 24h and 32h, and 1% after 48h and 56h, respectively.

The polypeptides described herein can be recovered using methods known in the art. For example, the polypeptide can be recovered from the culture medium by conventional methods, including but not limited to centrifugation, filtration, ultrafiltration, extraction, chromatography, spray drying, freeze drying, evaporation, or precipitation, and the like.

The polypeptides of the invention can be purified by a variety of methods known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatofocusing, size exclusion), electrophoresis (e.g., isoelectric focusing), differential solubility (e.g., salting-out precipitation), SDS-PAGE, or extraction to obtain a substantially pure polypeptide.

The present invention will be illustrated below by way of specific examples. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (Cold Spring Harbor Laboratory Press, N.Y., 1989), or according to the manufacturer's recommendations. For the use and amounts of the reagents, the conventional use and amounts are used unless otherwise indicated.

Experimental Material

1. Laboratory strains and plasmids

The strain is as follows: pichia pastoris GS115 (Invitrogen, cat # C175-00), escherichia coli DH5a (TAKARA: catalog #. D9057A).

Plasmid: pAO815 plasmid (Invitrogen, cat # V180-20).

2. Culture media and solutions

Unless otherwise indicated, the chemical reagents used in this application are available from Biotechnology, inc., shanghai.

LB liquid medium: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH7.0.

LB solid medium: agar was added to LB liquid medium at a concentration of 1.5%.

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

YPD solid Medium: agar was added to LB liquid medium at a concentration of 2%.

MGYS solid culture medium: 1.34% Yeast Nitrogen source base (YNB) ammonium sulfate-free, 1% Glycerol, 1M sorbitol, 4X 10 ^-5 % D-biotin, 2% agar.

BMMY-olive oil screening culture medium: the component A comprises: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) ammonium sulfate free of amino acids, 4X 10 ^-5 % D-biotin, 0.5% methanol (added after sterilization), 0.1M citric acid-sodium citrate buffer pH6.6,2% agar. And B component: component B olive oil substrate solution:the amount of the PVA solution was measured to 4% by volume, 50ml of olive oil was added, emulsification was carried out for 3min at 8000rpm of a high-speed homogenizer, and after suspension for 1min, further emulsification was carried out for 3min to prepare a substrate solution. 100ml of the sterilized A component was mixed with 12ml of the B component, and 1ml of 0.1% rhodamine B was added.

BMGY liquid Medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) ammonium sulfate free of amino acids, 1% glycerol, 4X 10 ^-5 % D-biotin, 0.1M citric acid-sodium citrate buffer pH6.6.

BMMY liquid medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) ammonium sulfate free of amino acids, 0.5% methanol (added after sterilization), 4X 10 ^-5 % D-biotin (added after sterilization), 0.1M citric acid-sodium citrate buffer pH6.6.

Improved Bradford method protein concentration determination kit (purchased from Shanghai biological engineering Co., ltd.)

Restriction enzymes HindIII, ecoRI, avrII (from Nippon Biotechnology (Beijing) Ltd.)

PCR enzyme: taKaRa Taq, prime

HS DNA Polymerase (available from Bao bioengineering (Dalian) Co., ltd.)

T4DNA ligase (from Fuzyme Tess Co., ltd.)

Example 1: construction of Rhizomucor miehei lipase gene Pichia pastoris expression strain with leader peptide and protease cutting point

According to the amino acid sequence (GenBank: A02536.1) of the Rhizomucor miehei lipase gene and the codon preference of pichia pastoris, a DNA sequence of pro/RML is designed, and enzyme cutting sites of protease kex2 and ste13 (the coding sequence of which is shown as the 211 th to 228 th bases of SEQ ID NO: 1) are added between a leader peptide (the coding sequence of which is shown as the 1 st to 210 th bases of SEQ ID NO: 1) and a mature peptide (the coding sequence of which is shown as the 229 th to 1035th bases of SEQ ID NO: 1). The DNA sequence is shown as SQE ID NO. 1, and the coded amino acid sequence is shown as SEQ ID NO. 2.

The sequence SQE ID NO:1 is provided for Shanghai Biometrics limited company for whole gene synthesis and is directly cloned into a pAO815 expression vector to obtain a Pichia pastoris expression vector pAO-pro/RML containing the sequence SQE ID NO: 1.

Linearizing pAO-pro/RML by SalI, preparing competent cells of Pichia pastoris GS115 strain by using a LiAC method, transforming the linearized pAO-pro/RML fragment into the competent cells of GS115 by electrotransformation, coating a transformant on a MGYS (MGYS) plate, culturing for 3 days at 30 ℃, selecting a large number of monoclones on the plate on a BMMY-olive oil screening plate, and selecting positive clones with best activity performance from the BMMY-pro/RML plate, wherein the positive clones are named as pro/RML-1.

Meanwhile, the original gene sequence of the Rhizomucor miehei lipase gene shown in SQE ID NO:5 is provided to Shanghai Bionical Limited company for complete gene synthesis and direct cloning into pAO815 expression vector. The Pichia expression vector pAO-pro/NOP-RML containing the sequence SQE ID NO. 5 was obtained (the amino acid sequence of the encoded pro/NOP-RML is shown in SEQ ID NO. 6). The pAO-pro/NOP-RML is linearized by SalI, competent cells of Pichia pastoris GS115 strain are prepared by a LiAC method, the linearized pAO-pro/NOP-RML fragment is transformed into GS115 competent cells by electrotransformation, the transformed cells are coated on MGYS plates, the MGYS plates are cultured for 3 days at the temperature of 30 ℃, a large number of monoclones on the plates are selected on BMMY-olive oil screening plates, and positive clones with best activity performance are selected from the BMMY-pro/NOP-RML fragments and named as pro/NOP-RML-1.

Example 2: construction of Rhizomucor miehei lipase gene Pichia pastoris expression strain with 2-copy leader peptide and protease cleavage point

Taking a leader peptide sequence of an amino acid sequence (GenBank: A02536.1) of the Rhizomucor miehei lipase gene, designing and obtaining two copies of DNA sequences 2rmlPRO of the tandem Rhizomucor miehei lipase gene leader peptide sequence according to the preference of pichia pastoris codons, adding protease kex2 and 2 ste13 enzyme cutting sites at the tail ends of the two leader peptides, as shown in SQE ID NO:3, and coding the amino acid sequence as shown in SEQ ID NO: 4.

The SQE ID NO 3 sequence was subjected to whole gene synthesis by Shanghai Biometrics Ltd to obtain pUC57-2PRO vector.

The mature peptide sequence of RML was cut out from pAO-proRML using HindIII and EcoRI, and ligated to the HindIII-EcoRI-digested pmAO-PLC vector (the construction method is described in example 1 of CN 201510946696.1) to obtain pAOmu-RML vector. The two copies of leader peptide 2PRO sequence on pUC57-2PRO vector were excised with AvrII and HindIII and ligated to the pmAO-RML vector digested with AvrII and HindIII to obtain pmAO-2PRO/RML vector. Linearizing pAOmu-2pro/RML by SalI, preparing competent cells of Pichia pastoris GS115 strain by using a LiAC method, transforming the linearized pmAO-2pro/RML fragment into GS115 competent cells by electrotransformation, coating the transformed product on an MGYS plate, culturing for 3 days at 30 ℃, selecting a large number of monoclones on the plate on a BMMY-olive oil screening plate, and selecting positive clones with best activity performance from the BMMY-2 pro/RML fragment, wherein the positive clones are named as 2pro/RML-1.

Example 3: shaking flask fermentation of rhizomucor miehei lipase gene pichia pastoris expression strain

Taking pro/NOP-RML-1, pro/RML-1 and 2pro/RML-1 strains, firstly activating in liquid YPD, inoculating in BMGY, culturing overnight at 30 ℃ and 220rpm, then transferring into BMMY culture medium, initially inducing with 2% methanol, supplementing 1% after 24h and 32h, supplementing 1% after 48h and 56h, sampling for 72h, taking olive oil as a substrate, and carrying out lipase activity detection.

The fermentation enzyme activity is shown in figure 1. In FIG. 1, the fermentation enzyme activity of pro/NOP-RML-1 is 229 + -11U/ml, the fermentation enzyme activity of pro/RML-1 is 310 + -35U/ml, and the fermentation enzyme activity of 2pro/RML-1 is 464 + -4U/ml.

protein electrophoresis of pro/RML-1 and 2pro/RML-1 broths is shown in FIG. 2, with the arrow indicating the RML target protein. FIG. 2 shows that the amount of the protein of interest 2pro/RML-1 is significantly higher than pro/RML-1, by about 50%.

Sequence listing

<110> Fengyi (Shanghai) Biotechnology research and development center, inc

<120> method for efficiently expressing Rhizomucor miehei lipase

<130> 165681

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 1038

<212> DNA

<213> Artificial sequence

<220>

<223> pro/RML nucleotide sequence

<400> 1

gttccaatca agagacaatc taattccact gtcgattctt tgcctccatt gattccttct 60

agaactagtg caccttcatc ctctccatct acaactgacc ctgaggctcc agctatgtca 120

agaaatggtc cacttccttc tgatgttgag accaagtacg gaatggccct gaatgctact 180

tcttatccag attctgtcgt tcaagctatg aaaagagagg ctgaagcttc catcgacgga 240

ggtattagag ccgctacttc tcaggaaatc aacgaactta cttactatac aactttgtca 300

gctaattctt actgtagaac tgttattcct ggtgctactt gggattgcat acattgtgac 360

gccactgaag atttaaagat aattaaaacc tggtctactt tgatttacga cactaacgct 420

atggttgcta gaggagattc cgagaagact atttatatcg tgtttagagg ttcttcatct 480

attcgtaatt ggatcgctga tttgacattc gttccagtct cttaccctcc agtttctggt 540

actaaggttc acaaaggatt tcttgattct tatggtgaag ttcaaaacga gttggttgct 600

actgtcttgg atcagtttaa acaataccca tcttataagg ttgctgtcac tggtcactct 660

ttgggaggtg ctactgcctt gctgtgtgct ttaggtttat accagagaga ggaaggattg 720

tcttcaagta acctattctt gtacactcaa ggtcagccta gagttggaga tccagcattt 780

gctaattatg tggtttctac tggtattcca tatagacgta ctgttaacga aagagacata 840

gtaccacact tgcctccagc tgccttcgga tttctgcatg ccggtgaaga gtactggatc 900

acagataatt ctcctgaaac cgttcaagtg tgtacatctg atttagagac ttccgactgc 960

tctaacagta ttgttccatt tacttcagtt cttgatcatt tgtcttattt tggaattaac 1020

accggtttgt gtacttaa 1038

<210> 2

<211> 345

<212> PRT

<213> Artificial sequence

<220>

<223> pro/RML amino acid sequence

<400> 2

Val Pro Ile Lys Arg Gln Ser Asn Ser Thr Val Asp Ser Leu Pro Pro

1 5 10 15

Leu Ile Pro Ser Arg Thr Ser Ala Pro Ser Ser Ser Pro Ser Thr Thr

20 25 30

Asp Pro Glu Ala Pro Ala Met Ser Arg Asn Gly Pro Leu Pro Ser Asp

35 40 45

Val Glu Thr Lys Tyr Gly Met Ala Leu Asn Ala Thr Ser Tyr Pro Asp

50 55 60

Ser Val Val Gln Ala Met Lys Arg Glu Ala Glu Ala Ser Ile Asp Gly

65 70 75 80

Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu Leu Thr Tyr Tyr

85 90 95

Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val Ile Pro Gly Ala

100 105 110

Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp Leu Lys Ile Ile

115 120 125

Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala Met Val Ala Arg

130 135 140

Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg Gly Ser Ser Ser

145 150 155 160

Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro Val Ser Tyr Pro

165 170 175

Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu Asp Ser Tyr Gly

180 185 190

Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp Gln Phe Lys Gln

195 200 205

Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser Leu Gly Gly Ala

210 215 220

Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg Glu Glu Gly Leu

225 230 235 240

Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln Pro Arg Val Gly

245 250 255

Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly Ile Pro Tyr Arg

260 265 270

Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu Pro Pro Ala Ala

275 280 285

Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile Thr Asp Asn Ser

290 295 300

Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu Thr Ser Asp Cys

305 310 315 320

Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp His Leu Ser Tyr

325 330 335

Phe Gly Ile Asn Thr Gly Leu Cys Thr

340 345

<210> 3

<211> 1278

<212> DNA

<213> Artificial sequence

<220>

<223> 2pro/RML nucleotide sequence

<400> 3

gtgcccataa agagacaatc caactccaca gtcgattccc ttccaccatt aattccttcc 60

aggacatcag caccttcttc ttctccttct accaccgacc ctgaagcacc tgctatgtca 120

agaaacggac ctttgccatc agatgttgaa acgaagtacg gtatggcttt aaacgctacc 180

tcttacccag acagtgtcgt tcaggctatg aaacgagagg ctgaggctga agctgttcca 240

atcaaacgtc aatctaattc tactgttgac tcactgccac ccctgattcc ctctcgtaca 300

agtgctccat ctagtagtcc ttctactact gatccagagg cccctgccat gtcaagaaat 360

gggccattgc caagtgatgt tgaaactaaa tatggcatgg ccttgaatgc cacttcatat 420

cccgattcag tagtacaggc catgaagagg gaggctgaag ccgaagcttc catcgacgga 480

ggtattagag ccgctacttc tcaggaaatc aacgaactta cttactatac aactttgtca 540

gctaattctt actgtagaac tgttattcct ggtgctactt gggattgcat acattgtgac 600

gccactgaag atttaaagat aattaaaacc tggtctactt tgatttacga cactaacgct 660

atggttgcta gaggagattc cgagaagact atttatatcg tgtttagagg ttcttcatct 720

attcgtaatt ggatcgctga tttgacattc gttccagtct cttaccctcc agtttctggt 780

actaaggttc acaaaggatt tcttgattct tatggtgaag ttcaaaacga gttggttgct 840

actgtcttgg atcagtttaa acaataccca tcttataagg ttgctgtcac tggtcactct 900

ttgggaggtg ctactgcctt gctgtgtgct ttaggtttat accagagaga ggaaggattg 960

tcttcaagta acctattctt gtacactcaa ggtcagccta gagttggaga tccagcattt 1020

gctaattatg tggtttctac tggtattcca tatagacgta ctgttaacga aagagacata 1080

gtaccacact tgcctccagc tgccttcgga tttctgcatg ccggtgaaga gtactggatc 1140

acagataatt ctcctgaaac cgttcaagtg tgtacatctg atttagagac ttccgactgc 1200

tctaacagta ttgttccatt tacttcagtt cttgatcatt tgtcttattt tggaattaac 1260

accggtttgt gtacttaa 1278

<210> 4

<211> 425

<212> PRT

<213> Artificial sequence

<220>

<223> 2pro/RML amino acid sequence

<400> 4

Val Pro Ile Lys Arg Gln Ser Asn Ser Thr Val Asp Ser Leu Pro Pro

1 5 10 15

Leu Ile Pro Ser Arg Thr Ser Ala Pro Ser Ser Ser Pro Ser Thr Thr

20 25 30

Asp Pro Glu Ala Pro Ala Met Ser Arg Asn Gly Pro Leu Pro Ser Asp

35 40 45

Val Glu Thr Lys Tyr Gly Met Ala Leu Asn Ala Thr Ser Tyr Pro Asp

50 55 60

Ser Val Val Gln Ala Met Lys Arg Glu Ala Glu Ala Glu Ala Val Pro

65 70 75 80

Ile Lys Arg Gln Ser Asn Ser Thr Val Asp Ser Leu Pro Pro Leu Ile

85 90 95

Pro Ser Arg Thr Ser Ala Pro Ser Ser Ser Pro Ser Thr Thr Asp Pro

100 105 110

Glu Ala Pro Ala Met Ser Arg Asn Gly Pro Leu Pro Ser Asp Val Glu

115 120 125

Thr Lys Tyr Gly Met Ala Leu Asn Ala Thr Ser Tyr Pro Asp Ser Val

130 135 140

Val Gln Ala Met Lys Arg Glu Ala Glu Ala Glu Ala Ser Ile Asp Gly

145 150 155 160

Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu Leu Thr Tyr Tyr

165 170 175

Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val Ile Pro Gly Ala

180 185 190

Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp Leu Lys Ile Ile

195 200 205

Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala Met Val Ala Arg

210 215 220

Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg Gly Ser Ser Ser

225 230 235 240

Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro Val Ser Tyr Pro

245 250 255

Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu Asp Ser Tyr Gly

260 265 270

Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp Gln Phe Lys Gln

275 280 285

Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser Leu Gly Gly Ala

290 295 300

Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg Glu Glu Gly Leu

305 310 315 320

Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln Pro Arg Val Gly

325 330 335

Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly Ile Pro Tyr Arg

340 345 350

Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu Pro Pro Ala Ala

355 360 365

Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile Thr Asp Asn Ser

370 375 380

Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu Thr Ser Asp Cys

385 390 395 400

Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp His Leu Ser Tyr

405 410 415

Phe Gly Ile Asn Thr Gly Leu Cys Thr

420 425

<210> 5

<211> 1038

<212> DNA

<213> Artificial sequence

<220>

<223> pro/NOP-RML nucleotide sequence

<400> 5

gtgccaatca agagacaatc aaacagcacg gtggatagtc tgccacccct catcccctct 60

cgaacctcgg caccttcatc atcaccaagc acaaccgacc ctgaagctcc agccatgagt 120

cgcaatggac cgctgccctc ggatgtagag actaaatatg gcatggcttt gaatgctact 180

tcctatccgg attctgtggt ccaagcaatg aaaagagagg ctgaagctag cattgatggt 240

ggtatccgcg ctgcgacctc gcaagaaatc aatgaattga cttattacac tacactatct 300

gccaactcgt actgccgcac tgtcattcct ggagctacct gggactgtat ccactgtgat 360

gcaacggagg atctcaagat tatcaagact tggagcacgc tcatctatga tacaaatgca 420

atggttgcac gtggtgacag cgaaaaaact atctatatcg ttttccgagg ttcgagctct 480

atccgcaact ggattgctga tctcaccttt gtgccagttt catatcctcc ggtcagtggt 540

acaaaagtac acaagggatt cctggacagt tacggggaag ttcaaaacga gcttgttgct 600

actgttcttg atcaattcaa gcaatatcca agctacaagg ttgctgttac aggtcactca 660

ctcggtggtg ctactgcgtt gctttgcgcc ctgggtctct atcaacgaga agaaggactc 720

tcatccagca acttgttcct ttacactcaa ggtcaaccac gggtaggcga ccctgccttt 780

gccaactacg ttgttagcac cggcattcct tacaggcgca cggtcaatga acgagatatc 840

gttcctcatc ttccacctgc tgcttttggt tttctccacg ctggcgagga gtattggatt 900

actgacaata gcccagagac tgttcaggtc tgcacaagcg atctggaaac ctctgattgc 960

tctaacagca ttgttccctt cacaagtgtt cttgaccatc tctcgtactt tggtatcaac 1020

acaggcctct gtacttaa 1038

<210> 6

<211> 345

<212> PRT

<213> Artificial sequence

<220>

<223> pro/NOP-RML amino acid sequence

<400> 6

Val Pro Ile Lys Arg Gln Ser Asn Ser Thr Val Asp Ser Leu Pro Pro

1 5 10 15

Leu Ile Pro Ser Arg Thr Ser Ala Pro Ser Ser Ser Pro Ser Thr Thr

20 25 30

Asp Pro Glu Ala Pro Ala Met Ser Arg Asn Gly Pro Leu Pro Ser Asp

35 40 45

Val Glu Thr Lys Tyr Gly Met Ala Leu Asn Ala Thr Ser Tyr Pro Asp

50 55 60

Ser Val Val Gln Ala Met Lys Arg Glu Ala Glu Ala Ser Ile Asp Gly

65 70 75 80

Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu Leu Thr Tyr Tyr

85 90 95

Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val Ile Pro Gly Ala

100 105 110

Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp Leu Lys Ile Ile

115 120 125

Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala Met Val Ala Arg

130 135 140

Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg Gly Ser Ser Ser

145 150 155 160

Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro Val Ser Tyr Pro

165 170 175

Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu Asp Ser Tyr Gly

180 185 190

Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp Gln Phe Lys Gln

195 200 205

Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser Leu Gly Gly Ala

210 215 220

Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg Glu Glu Gly Leu

225 230 235 240

Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln Pro Arg Val Gly

245 250 255

Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly Ile Pro Tyr Arg

260 265 270

Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu Pro Pro Ala Ala

275 280 285

Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile Thr Asp Asn Ser

290 295 300

Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu Thr Ser Asp Cys

305 310 315 320

Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp His Leu Ser Tyr

325 330 335

Phe Gly Ile Asn Thr Gly Leu Cys Thr

340 345

Claims

1. A polynucleotide molecule, wherein the polynucleotide sequence of said polynucleotide molecule is selected from the group consisting of:

A-L1-B-L2-RML (formula I)

In the formula (I), the compound is shown in the specification,

a is rhizomucor miehei lipase leader peptide;

l1 is the enzyme cutting site of protease kex2 and ste 13;

b is rhizomucor miehei lipase leader peptide;

l2 is the enzyme cutting site of protease kex2 and ste 13; and

RML represents a mature peptide sequence of Rhizomucor miehei lipase, and the coding sequence of the RML is shown as the 229 th to 1035 th nucleotide sequence of SEQ ID NO. 1; and

(2) (1) the complement of said sequence.

2. The polynucleotide molecule of claim 1, wherein the amino acid sequences of L1 and L2 are each independently as set forth in SEQ ID No. 4 from position 71 to 78.

3. The polynucleotide molecule of claim 1, wherein the polypeptide of formula I has the sequence shown in SEQ ID No. 4.

4. The polynucleotide molecule of claim 1, wherein said polynucleotide sequence is selected from the group consisting of:

(i) 3, the polynucleotide sequence shown in SEQ ID NO; and

(ii) (ii) (i) the complement of the polynucleotide sequence.

5. A nucleic acid construct comprising the polynucleotide sequence of the polynucleotide molecule of any one of claims 1-4.

6. The nucleic acid construct of claim 5, wherein said nucleic acid construct is a cloning vector or an expression vector.

7. The nucleic acid construct of claim 6, wherein the expression vector is a vector suitable for expression in Pichia pastoris.

8. The nucleic acid construct of claim 7, wherein the vector is selected from the group consisting of pPIC, pPICZ, pAO, pGAP, and pGAPZ.

9. The nucleic acid construct of claim 7, wherein the expression vector has the plasmid pAO815 as a backbone.

10. A genetically engineered host cell comprising a polynucleotide sequence of a polynucleotide molecule of any one of claims 1 to 4 or a nucleic acid construct of any one of claims 5 to 9.

11. The host cell of claim 10, wherein the cell is a yeast cell.

12. The host cell of claim 11, wherein the yeast is a pichia pastoris cell.

13. A method for producing a Rhizomucor miehei lipase, said method comprising the steps of constructing an expression vector comprising a polynucleotide sequence of a polynucleotide molecule of any one of claims 1-4, transferring said expression vector into a host cell, and culturing said host cell to express said lipase.

14. A method of producing rhizomucor miehei lipase comprising the step of fermenting the host cell of any one of claims 10-12.