CN100336906C - Lipase gene sequence and its application in yeast - Google Patents

Abstract

The present invention relates to a lipase gene sequence expressed effectively in yeast and saccharomycete with the gene sequence, which is characterized in that according to a lipase amino acid sequence, the gene sequence of codon with high application frequency in yeast is designed. The 5' terminal of the gene sequence is connected to a gene fragment which is matched with object yeast, and the 3' terminal of the gene sequence is connected to a restriction endonuclease site. The saccharomycete of the present invention can effectively secrete lipase protein, can be widely used for the fields of petroleum industries, paper making industries, biological medicine making industries, chemical industries, etc. and has the effect on degreasing, perfuming, washing, etc.

Description

Lipase gene sequence and application thereof in yeast

The present invention relates to a gene sequence capable of being expressed in yeast (specially in Pichia pastoris), its carrier for cloning and yeast for expressing it, its screening method and gene engineering lipase obtained from yeast gene engineering bacteria.

Lipases are esterases which are capable of hydrolyzing water-insoluble esters, such as long-chain triglycerides (fats). It hydrolyzes fat specifically into fatty acid and glycerin on water-fat interface, and catalyzes reverse reaction in non-aqueous medium. The lipase hydrolysis occurs in the ester bond of the grease, and the catalytic reaction is as follows:

the reaction may also be carried out on monoglycerides until hydrolysis to glycerol. (Ghosh&Saxena, 79(Pt 2): 119-. The lipase can catalyze esterification reaction or change ester bond in a non-aqueous medium, and has high specificity and mutual selectivity in esterification reaction and transesterification reaction. Lipases are widely found in various organ tissues and microorganisms of animals and plants. Microbial lipases are of a wide variety, and their biochemical properties and substrate specificity are related to the producer. The structural function of lipase has been studied mainly on Candida rugosa (also known as Candida cylindracea). Candida rugosa lipase is an extracellular lipase which is secreted under the induction of fatty acids and consists of several isomerases with different catalytic activities. The coding sequences of lipase gene family constitute several enzyme proteins with high homology and different substrate acting region structures. The enzyme protein expressed by Lip1 gene is one of the main. The gene synthesized by point mutation is expressed in both saccharomyces cerevisiae and pichia pastoris, and the expression amount after 208-hour fermentation in pichia pastoris is 150U/mL. (Lotti&Monticelli, Chem Phys Lipids, 93 (1-2): 143-, Degreasing in the textile and gelatin industries, etc., have very wide applications. Currently, lipase preparations produced from rhizonases, candida cylindracea, aspergillus, trichome, chromobacterium mucilaginosum, and the like have been supplied to the market. Among them, lipase (CRL) derived from Candida rugosa is highly preferred in the market. (Lotti&Tramontano, Protein Eng, 7 (4): 531-

It is a first object of the present invention to provide a lipase gene sequence which can be efficiently expressed in yeast.

The second objective of the invention is to provide a vector for cloning the gene sequence.

The third object of the present invention is to provide a recombinant plasmid of the above vector capable of expressing lipase secretion outside yeast cells.

The fourth object of the present invention is to provide a yeast having the above gene sequence.

The fifth object of the present invention is to provide a method for screening the above genetically engineered yeast.

The first object of the present invention is achieved by: a lipase gene sequence capable of being efficiently expressed in yeast, characterized in that: a gene sequence having a codon which is frequently used in yeastis designed based on the amino acid sequence of lipase, a gene fragment which is compatible with the target yeast is ligated to the 5 'end of the gene sequence, and a restriction enzyme site is ligated to the 3' end. In order to design a DNA sequence that can be expressed in a yeast by transformation from the amino acid sequence of Lip1 (a kind of Candida rugosa. lipase) (see FIG. 1), it is necessary to design a DNA sequence corresponding to the amino acid sequence of lipase using codons that are frequently used in the target yeast. On the basis, a gene segment matched with the target yeast must be connected to the 5' end of the target yeast, such as: CTCGAGAAAAGAGAGGCTGAAGCT, respectively; in order to clone the sequences into the corresponding vectors, restriction enzyme sites must be ligated to their 3' ends in order to achieve efficient, even highly efficient, expression of the genes in yeast.

① A DNA sequence corresponding to the amino acid sequence of Lip1 is designed according to the codon preference in Pichia pastoris ② in order to realize the expression in Pichia pastoris, the invention can connect a gene fragment GAATTCTCGAGAAAAGAGAGGCTGAAGCT at the 5 'end, ③ connects a restriction enzyme site TCTAGA at the 3' end of the sequence, because the site is not in the gene and the mis-cutting can not be caused during cloning ④ the 144 th position and the 1461 th position of the sequence are designed as C to avoid BamHI site, and the invention can form the following specific gene sequence (Tsp) after the above treatment:

CTC GAG AAA AGA GAG GCT GAA GCTGCT CCA ACT GCT ACT TTG GCT AAC

GGT GAT ACT ATT ACT GGT TTG AAC GCT ATT ATT AAC GAA GCT TTT TTG GGT

ATT CCA TTT GCT GAA CCA CCA GTT GGT AAC TTG AGA TTT AAG GAC CCA GTT

CCA TAC TCT GGT TCT TTG GAT GGT CAA AAG TTT ACT TCT TAC GGT CCA TCT

TGT ATG CAA CAA AACCCA GAA GGT ACT TAC GAA GAA AAC TTG CCA AAG GCT

GCT TTG GAT TTG GTT ATG CAA TCT AAG GTT TTT GAA GCT GTT TCT CCA TCT

TCT GAA GAT TGT TTG ACT ATT AAC GTT GTT AGA CCA CCA GGT ACT AAG GCT

GGT GCT AAC TTG CCA GTT ATG TTG TGG ATT TTT GGT GGT GGT TTT GAA GTT

GGT GGT ACT TCT ACT TTT CCA CCA GCT CAA ATG ATT ACT AAG TCT ATT GCT

ATG GGT AAG CCA ATT ATT CAT GTT TCT GTT AAC TAC AGA GTT TCT TCT TGG

GGT TTT TTG GCT GGT GAT GAA ATT AAG GCT GAA GGT TCT GCT AAC GCT GGT

TTG AAG GAT CAA AGA TTG GGT ATG CAA TGG GTT GCT GAT AAC ATT GCT GCT

TTT GGT GGT GAT CCA ACT AAG GTT ACT ATT TTT GGT GAA TCT GCT GGT TCT

ATG TCT GTT ATG TGT CAT ATT TTG TGG AAC GAT GGT GAT AAC ACT TAC AAG

GGT AAG CCA TTG TTT AGA GCT GGT ATT ATG CAA TCT GGT GCT ATG GTT CCA

TCT GAT GCT GTT GAT GGT ATT TAC GGT AAC GAA ATT TTT GAT TTG TTG GCT TCT

AAC GCT GGT TGT GGT TCT GCT TCT GAT AAG TTG GCT TGT TTG AGA GGT GTT

TCT TCT GAT ACT TTG GAA GAT GCT ACT AAC AAC ACT CCA GGT TTT TTG GCT

TAC TCT TCT TTG AGA TTG TCT TAC TTG CCA AGA CCA GAT GGT GTT AAC ATT

ACT GAT GAT ATG TAC GCT TTG GTT AGA GAA GGT AAG TAC GCT AAC ATT CCA

GTT ATT ATT GGT GAT CAA AAC GAT GAA GGT ACT TTT TTT GGT ACT TCT TCT

TTG AAC GTT ACT ACT GAT GCT CAA GCT AGA GAA TAC TTT AAG CAA TCT TTT

GTT CAT GCT TCT GAT GCT GAA ATT GAT ACT TTG ATG ACT GCT TAC CCA GGT

GAT ATT ACT CAA GGT TCT CCA TTT GAT ACT GGT ATT TTG AAC GCT TTG ACT

CCA CAA TTT AAG AGA ATT TCT GCT GTT TTG GGT GAT TTG GGT TTT ACT TTG

GCT AGA AGA TAC TTT TTG AAC CAT TAC ACT GGT GGT ACT AAG TAC TCT TTT

TTG TCT AAG CAA TTG TCT GGT TTG CCA GTT TTG GGT ACT TTT CAT TCT AAC

GAT ATT GTT TTT CAA GAT TAC TTG TTG GGT TCT GGT TCT TTG ATT TAC AAC AAC

GCT TTT ATT GCT TTT GCT ACT GAT TTG GAC CCA AAC ACT GCT GGT TTG TTG

GTT AAG TGG CCA GAA TAC ACT TCT TCT TCT CAA TCT GGT AAC AAC TTG ATG

ATG ATT AAC GCT TTG GGT TTG TAC ACT GGT AAG GAT AAC TTT AGA ACT GCT

GGT TAC GAT GCT TTG TTT TCT AAC CCA CCA TCT TTT TTT GTT TAATCTAGA

the designed lipase gene sequence can be obtained by a PCR synthesis method (see figure 2): firstly, 27 primers are designed according to lipase genes, wherein CAG93F 1-F14 are forward primers, and CAG93R 1-R13 are reverse primers (see figure 3). After 27 primers are synthesized, different primers are finally synthesized into a full-length lipase gene sequence through DNA polymerase reaction (chain extension reaction and PCR reaction), enzyme digestion, ligation reaction and the like (see fig. 2-8).

The synthesis of primers, the synthesis of full-length lipase gene by PCR method, etc. in the present invention can be carried out by a special biotechnology company or institution.

The second object of the present invention is achieved in that the above lipase gene may constitute a recombinant plasmid in which a functional sequence of lipase gene is ligated into a yeast expression vector by restriction enzyme sites at 5 ' and 3 ' ends, and the synthesized lipase gene may be ligated into the vector by restriction enzyme sites at 5 ' and 3 ' ends thereof, and a pichia pastoris expression vector (pGAPZ α a or pz α a,Invitrogen) having a glyceraldehyde-3-phosphate dehydrogenase (GAP) or Alcohol Oxidase (AOX) gene promoter is selected for efficient expression of lipase gene in yeast, and the lipase synthesis gene (tsapp) is cloned into a vector pgz α a (see fig. 9) using an XhoI restriction enzyme site at 5 ' end and a picai restriction enzyme site at 3 ' end, and the pichia pastoris signal vector (pg563278) is cloned into a vector, and expressed in pGAPZ 35865A vector if a correct expression vector of pGAPZ peptide is formed by adding a part of a signal peptide coding sequence of α, the sequence is fused with a part of picai restriction enzyme site at 3 ' end, and pGAPZ promoter, pGAPZ expression vector is optimized for pGAPZ 35 α, pGAPZ expression of pGAPZ coding sequence, pg563226C, pg563514C is expressed as a gene, and pg3656357, pg567 is expressed in a vector.

In order to clone the above lipase gene into a vector to form a recombinant plasmid, the vector of the present invention must have two sites of XhoI and XbaI, and the specific vector may be pBluescript II SK (+), pGAPZ α A or pGAPZ α B or pGAPZ α C, pPICZ α A or pPICZ α B or pPICZ α C. pBluescript II SK (+).

The third object of the present invention is achieved by: the gene sequence is cloned into a vector, and the cloned recombinant plasmid is transformed into Pichia pastoris (Pichia pastoris). The preferred Pichia pastoris (P. pastoris) expression system of the present invention has many advantages over the host strain mold used in Van Gorcom et al (US Pat: 5436156, 1995; US Pat: 5863533, 1999). Firstly, the growth and reproduction period of the mould is more than that of pichia pastoris growth root: secondly, the vegetative growth of moulds is achieved by the elongation growth of the tips of the mycelium, which growth characteristics are particularly suitable for solid cultures and not for liquid fermentations as are common in modern fermentation processes, which are generally less efficient, longer and more costly, thus increasing the cost of obtaining large quantities of fermented product considerably compared to liquid fermentations. In contrast, pichia pastoris increases trophozoite by fission, a reproductive characteristic well suited for liquid fermentation; third, the mold trophosome needs to provide enough, relatively complex carbon-nitrogen organic nutrients during growth and development, which also increases the fermentation cost, and the pichia pastoris trophosome mainly utilizes cheap simple nutrients such as methanol, glucose, ammonia water and the like. Obtaining the same amount of nutrients pichia pastoris is much cheaper than the nutrients consumed by moulds. High cell density, low cost fermentation methods for yeast have been established (Siegel R.S., Biotechnol.Bioeng, 34: 403-; fourth, the peculiar mold flavor of the mold can reduce the palatability, and the sauce flavor generated by fermentation of the pichia pastoris has good food calling effect; fifth, pichia pastoris contains a large variety of organic compounds that promote growth, such as oligosaccharides, nucleotides, various amino acids, short peptides, etc., which are superior or absent from molds; sixth, in eukaryotic expression systems, yeasts, including Pichia pastoris, are certainly the most extensively studied, and are easier, more convenient and more efficient than molds in performing molecular biology manipulations. Yeast has been used as a good eukaryotic expression system to successfully and efficiently express a plurality of exogenous gene products with biological activity. Seventh, Pichia pastoris has very good safety, has been used as single cell protein to be widely used, yeast culture medium does not contain toxic substances and pyrogen, so recombinant yeast expressed lipase can be directly used in the form of yeast culture without separation and purification, can reduce the production cost of lipase; eighth, the expressed lipase is secreted into the culture medium under the guidance of the signal peptide, which directly exposes the lipase without breaking yeast cells, and provides the possibility of directly utilizing the yeast culture containing the lipase in industrial production, and lays the foundation for the industrial large-scale and low-cost fermentation production of the lipase by utilizing the recombinant yeast.

The recombinant expression plasmids pGAPZ α A, pGAPZ α B, pGAPZ α C and pPICZ α A, pPICZ α B, pPICZ α C containing the lipase synthetic gene adopted by the invention can transform yeast, and are used for realizing the integration of lipase gene fragments on yeast chromosomes to achieve the purpose of effectively or efficiently secreting and expressing exogenous genes (see figure 11).

The reason for this is that the recombinant expression plasmid pGAPZ α A containing synthetic gene has partial sequence of Pichia pastoris GAP gene, and after the recombinant expression plasmid enters yeast cell, through in vivo homologous recombination, pGAPZ α A can be directionally integrated onto Pichia pastoris genomic DNA, and the promoter can start the expression of lipase synthetic gene without the existence of exogenous inducer methanol.

Before the recombinant expression plasmid pGAPZ α A containing the lipase synthetic gene is transformed into Pichia pastoris, the circular recombinant expression plasmid is cut by endonuclease to be linearized, different expression plasmids and different endonuclease sites can be selected, the recombinant expression plasmid pGAPZ α A is digested by endonuclease BspHI, the electrophoresis detection shows that the enzyme cutting is negative, the completely linearized recombinant expression plasmid pGAPZ α A is purified and recovered, and the plasmid is preserved at-20 ℃ for later use (see figure 9).

Pichia pastoris is transformed by an electric or chemical transformation method, a recipient strain (P. Pastoris protease A deficient strain AMD1168H) is prepared, 50-80 μ l of the recipient strain and 5-90 μ g of linearized recombinant plasmid pGAPZ α A DNA are mixed uniformly, electric excitation (voltage: 1500V; capacitance; 25 μ F; resistance; 200 Ω) is carried out, and recombinants are screened (see FIG. 10).

In the above-mentioned invention, the tool enzyme, vector, expression system and the like required for the recombinant plasmid pBluescript II SK (+) or pGAPZ α A containing a lipase synthetic gene are provided by a specific biotechnology company (e.g., Promega, Japan TaKaRa, Invitrogen).

The expression vector pGAPZ α A originally has Zeocin resistance gene and GAP promoter sequence, and after the recombinant expression plasmid containing lipase gene is transformed into Pichia pastoris and recombined onto chromosome, the transgenic engineering bacterium obtains resistance to Zeocin⁺) In addition, the enzyme activity of the resistant yeast recombinant is measured by a method such as pH measurement, temperature measurement, and heat resistance measurement. The strain which is currently preserved in China center for type culture Collection of Wuhan university in Wuhan City with the preservation number of No.M201002 and the preservation date of 2000 years and months is an excellent strain screened by the method.

The fifth object of the present invention is achieved by: yeast cells YPD were cultured in a liquid for 92 hours, and centrifuged to remove the cells, thereby obtaining a supernatant. Through enzyme activity determination, the enzyme activity of the supernatant is 120-160U/mL at the pH of 7.2 and the temperature of 30-40 ℃.

Through determination, the lipase expression quantity of the Pichia pastoris engineering bacteria reaches 120-160U/mL when the fermentation culture is carried out for 92 hours, the PH is 7.2, and the temperature is between30 and 40 ℃. According to foreign reports, the enzyme activity of the fermentation liquor obtained after the high-density fermentation for 280 hours is 150U/mL. The modification of lipase gene was proved to be successful.

The recombinant lipase gene engineering strain provided by the invention is fermented and cultured, and the coded lipase gene is expressed and secreted into fermentation liquor. Can produce lipase through fermentation and induction with simple culture medium on a shaking table. The specific method comprises the following steps:

(1) seed activation: inoculating the recombinant lipase gene engineering strain preserved on the inclined plane to a YPD plate, and performing static culture at 30 ℃ for 48 hours.

(2) First-stage seed: the activated strain was inoculated in 5ml YPD liquid medium and cultured with shaking in water bath at 30 ℃ and 230rpm for 18 hours.

(3) Secondary seeds: 0.1ml of the bacterial suspension was inoculated into 50ml LYPD liquid medium and cultured in water bath at 30 ℃ and 260rpm for 72 hours with shaking.

The lipase generated by the expression of the lipase gene sequence is modified by various chemical modification groups, such as glycosylation, phosphorylation, N-terminal amination, protein internal cutting and the like; the basic function of the protein gene sequence may be unchanged by adding a signal peptide and a fusion peptide before or after the signal peptide to improve the transport after the expression. These are generally referred to as functional organisms of the gene sequences of the invention.

In summary, the following steps: according to the protein amino acid structure of Lip1 in Candida rugosa, the full-length gene sequence of lipase is designed in vitro so that the lipase is suitable for being expressed in Pichia pastoris; through gene synthesis, recombinant expression vector construction, transformation and expression screening, the Pichia pastoris genetically engineered bacteria for efficiently expressing lipase can be obtained, and a foundation is provided for industrial large-scale fermentation production of lipase.

(1) The invention selects the codon of the lipase gene according to the preference degree of the selected expression host Pichia pastoris on genetic codes, designs a DNA sequence by using codons with higher use frequency in the Pichia pastoris, and ensures that the obtained gene sequence can be correctly and efficiently expressed in the Pichia pastoris.

(2) On the basis, the artificial modification of the gene of the lipase gene is carried out, and a partial signal peptide sequence and a restriction endonuclease site for yeast secretory expression are increased; the modified lipase gene sequence can be correctly transcribed, translated and expressed in yeast, and the lipase is secreted into fermentation liquor. Meanwhile, according to the characteristics of an expression host, namely Pichia pastoris, a partial signal peptide sequence which can enable lipase genes to be secreted to the outside is designed, and a correct reading frame can be formed with an expression vector when a recombinant expression plasmid is constructed, so that the lipase genes can be correctly secreted to the outside; through the modification, a lipase gene sequence which can be correctly and efficiently expressed in Pichia pastoris and secreted to the outside of cells is generated.

(3) The lipase gene sequence is synthesized by PCR, and a recombinant expression plasmid which contains the synthetic gene and can be integrated and expressed in Pichia pastoris is further constructed by adopting an expression vector pGAPZ α A.

(4) And transforming the recombinant expression plasmid into pichia pastoris toobtain a series of pichia pastoris gene engineering bacteria for expressing lipase.

(5) Screening the engineering strains to obtain the engineering strain for efficiently expressing the lipase synthetic gene.

Because the invention selects the lipase with good activity and a proper yeast system, especially Pichia pastoris, as the basis of DNA design, the invention realizes the effective expression of the active lipase and obtains a simplified, effective, industrialized and large-scale production method of the genetic engineering lipase. The invention realizes the yeast in vitro expression and in vitro secretion of the lipase by adopting the method of designing the 5' end sequence of the gene sequence as the corresponding segment on the carrier.

FIG. 1: candida rugosa lipase Lip1 mature protein amino acid sequence:

FIG. 2: schematic diagram of synthesis of Tsp gene sequence ("←" forward primer; "→" reverse and primer)

FIG. 3: base sequence of primer (F represents forward primer, R represents reverse primer)

FIG. 4: synthetic CAG93-44F, CAG93(Fhc01), CAG93-44 CAG93F1, CAG93-44 CAG93F2 and CAG93-44R fragment base sequences

FIG. 5: full-automatic sequencing map of synthesized lipase gene (CAG93-44F fragment)

FIG. 6: full-automatic sequencing map of synthesized lipase gene (CAG93-44 CAG93F1 fragment)

FIG. 7: full-automatic sequencing map of synthesized lipase gene (CAG93-44 CAG93F2 fragment)

FIG. 8: full-automatic sequencing map of synthesized lipase gene (CAG93-44R fragment)

FIG. 9: schematic diagram of recombinant vector construction

FIG. 10: recombinant plasmid pBluescript II SK (+) electrophoretogram containing synthetic lipase gene Tsp

Description of the drawings: the first lane is: marker (from Dalibao bioengineering company DL2000, DL15000)

The second lane is: XbaI + XhoI double cleavage of Pbluescript II SK (+) (results of the cleavage with XbaI + XhoI)

The second lane is: XbaI + XhoI double cleavage of Pbluescript II SK (+) Tsp

FIG. 11 shows the electrophoretogram of recombinant plasmid pGAZ α A containing synthetic lipase gene Tsp

The first lane shows the XbaI + XhoI double digestion results of pGAZ α A + Tsp

The second lane shows the XbaI + XhoI double digestion of pGAZ α A

The third lane is: marker (from DL2000, DL15000)

Example 1: gene sequence design and artificial synthesis of encoding lipase

The invention designs a gene sequence with codons with higher use frequency in yeast by adopting codons with high preference on the basis of an amino acid sequence (shown in figure 1) of Candida rugosa lipase Lip1, and connects a gene fragment GAATTCTCGAGAAAAGAGAGGCTGAAGCT to a 5' end; connecting a restriction enzyme site TCTAGA at the 3' end of the sequence; the following specific gene sequence Tsp can be formed by replacing codons that are used less frequently in the target yeast with codons that are used more frequently in the lipase gene sequence:

CTC GAG AAA AGA GAG GCT GAA GCTGCT CCA ACT GCT ACT TTG GCT AAC

GGT GAT ACT ATT ACT GGT TTG AAC GCT ATT ATT AAC GAA GCT TTT TTG GGT

ATT CCA TTT GCT GAA CCA CCA GTT GGT AAC TTG AGA TTT AAG GAC CCA GTT

CCA TAC TCT GGT TCT TTG GAT GGT CAA AAG TTT ACT TCT TAC GGT CCA TCT

TGT ATG CAA CAA AAC CCA GAA GGT ACT TAC GAA GAA AAC TTG CCA AAG GCT

GCT TTG GAT TTG GTT ATG CAA TCT AAG GTT TTT GAA GCT GTT TCT CCA TCT

TCT GAA GAT TGT TTG ACT ATT AAC GTT GTT AGA CCA CCA GGT ACT AAG GCT

GGT GCT AAC TTG CCA GTT ATG TTG TGG ATT TTT GGT GGT GGT TTT GAA GTT

GGT GGT ACT TCT ACT TTT CCA CCA GCT CAA ATG ATT ACT AAG TCT ATT GCT

ATG GGT AAG CCA ATT ATT CAT GTT TCT GTT AAC TAC AGA GTT TCT TCT TGG

GGT TTT TTG GCT GGT GAT GAA ATT AAG GCT GAA GGT TCT GCT AAC GCT GGT

TTG AAG GAT CAA AGA TTG GGT ATG CAA TGG GTT GCT GAT AAC ATT GCT GCT

TTT GGT GGT GAT CCA ACT AAG GTT ACT ATT TTT GGT GAA TCT GCT GGT TCT

ATG TCT GTT ATG TGT CAT ATT TTG TGG AAC GAT GGT GAT AAC ACT TAC AAG

GGT AAG CCA TTG TTT AGA GCT GGT ATT ATG CAA TCT GGT GCT ATG GTT CCA

TCT GAT GCT GTT GAT GGT ATT TAC GGT AAC GAA ATT TTT GAT TTG TTG GCT TCT

AAC GCT GGT TGT GGT TCT GCT TCT GAT AAG TTG GCT TGT TTG AGA GGT GTT

TCT TCT GAT ACT TTG GAA GAT GCT ACT AAC AAC ACT CCA GGT TTT TTG GCT

TAC TCT TCT TTG AGA TTG TCT TAC TTG CCA AGA CCA GAT GGT GTT AAC ATT

ACT GAT GAT ATG TAC GCT TTG GTT AGA GAA GGT AAG TAC GCT AAC ATT CCA

GTT ATT ATT GGT GAT CAA AAC GAT GAA GGT ACT TTT TTT GGT ACT TCT TCT

TTG AAC GTT ACT ACT GAT GCT CAA GCT AGA GAA TAC TTT AAG CAA TCT TTT

GTT CAT GCT TCT GAT GCT GAA ATT GATACT TTG ATG ACT GCT TAC CCA GGT

GAT ATT ACT CAA GGT TCT CCA TTT GAT ACT GGT ATT TTG AAC GCT TTG ACT

CCA CAA TTT AAG AGA ATT TCT GCT GTT TTG GGT GAT TTG GGT TTT ACT TTG

GCT AGA AGA TAC TTT TTG AAC CAT TAC ACT GGT GGT ACT AAG TAC TCT TTT

TTG TCT AAG CAA TTG TCT GGT TTG CCA GTT TTG GGT ACT TTT CAT TCT AAC

GAT ATT GTT TTT CAA GAT TAC TTG TTG GGT TCT GGT TCT TTG ATT TAC AAC AAC

GCT TTT ATT GCT TTT GCT ACT GAT TTG GAC CCA AAC ACT GCT GGT TTG TTG

GTT AAG TGG CCA GAA TAC ACT TCT TCT TCT CAA TCT GGT AAC AAC TTG ATG

ATG ATT AAC GCT TTG GGT TTG TAC ACT GGT AAG GAT AAC TTT AGA ACT GCT

GGT TAC GAT GCT TTG TTT TCT AAC CCA CCA TCT TTT TTT GTT TAATCTAGA。

the newly designed gene of the present invention can be artificially synthesized by PCR (see FIG. 2) by the following steps:

27 primers were designed and synthesized based on the newly designed gene sequence (see FIG. 3), including 14 forward primers: CAG93F 1-F14, and 13 reverse primers CAG93R 1-R13. Each primer has an average length of 80nt, wherein each pair of primers has an average 20nt of repetitive sequence between F/F, F/R, R/F, F/R, R/F, F/R, R/F, F/R, R/F, F/R and R/R: the 5 'end of CAG93F1 is added with Xho I site, and the 5' end of CAG93R1 primer is added with Xba I site.

When synthesizing a newly designed lipase gene by PCR, firstly synthesizing 5 segments as a target, then adding a new primer, prolonging small segments by PCR, amplifying large segments by PCR of the small segments obtained by PCR reaction, and finally synthesizing 5 large segments into a long chain, wherein the steps are as follows:

firstly, F3/R13, F6/R10, P9/R7, F12/R4 and F14/R2 are used for carrying out extension reaction on five pairs of primers, wherein the reaction system comprises 5 mul of primers F3 or F6, F9, F12, F14(0.01 OD/mul), 5 mul of primers R13 or R10, R7, R4 and R2(0.01 OD/mul), 10 mul of 10xTaKaRa LA Buffer, 16 mul of dNTP, H₂O63 mu l; after the temperature is preserved for 5min at 94 ℃, the mixture is cooled to 55 ℃ within 10min, 1 mu l of TaKaRa LA Taq enzyme is added, the temperature is preserved for 5min, and the temperature is preserved for 5min at 72 ℃ again, thus obtaining DNA extension solution.

And in the second step, the DNA extension solution obtained in the first step is used as a template, F2/R12, F5/R9, F8/R6, F11/R3 and F13/R1 are used as primer pairs to carry out PCR reaction, and a longer fragment is obtained through amplification. The reaction system is primer F2 or F5, F8, F11, F13(0.01 OD/mul) 1 mul, primer R12 or R9, R6, R3, R1(0.01 OD/mul) 1 mul, DNA extension solution 1 mul, dNTP 10 mul, 10xTaKaRa LA Buffer10 mul, TaKaRa Ex Taq enzyme 1 mul, H₂O76. mu.l. At 94 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C30 cycles of PCR reaction are carried out in total under the condition of 30s of PCR reaction; the obtained PCR product was precipitated with ethanol and dissolved in 100. mu.l of TE to obtain DNA fragments A1, B1, C1, D1 and F1.

The third step is to take the DNA fragments A1, B1, C1, D1 and F1 obtained in the second step as templates, respectively take F1/R11, F4/R8, F7/R5, F10/R3 and F13/R1 as primers to amplify longer fragments, and the reaction system comprises 5 mul of primers F1 or F4, F7, F11 and F13(0.01 OD/mul), 5 mul of primers R11 or R8, R5, R3 and R1(0.01 OD/mul), 5 mul of 10xTaKaRa LA Buffer10 mul, dNTP 16 mul, H₂O63 mu l; after the temperature is preserved for 5min at 94 ℃, the mixture is cooled to 55 ℃ within 10min, TaKaRaLA Taq enzyme 1 μ l is added, the temperatureis preserved for 5min, and the temperature is preserved for 5min at 72 ℃ to obtain a new DNA extension solution, wherein the extension solution comprises DNA fragments CAG93-44F, CAG93(Fhc01), CAG93-44 CAG93F1, CAG93-44 CAG93F2 and CAG 93-44R. (see FIG. 4)

And fourthly, carrying out PCR reaction by taking the DNA extension solution obtained in the third step as a template and respectively taking F1/R1 as primers to amplify the full-length DNA chain. The reaction system comprises 1. mu.l of primer F1(0.01 OD/. mu.l), 1. mu.l of primer R1(0.01 OD/. mu.l), 1. mu.l of DNA extension solution, 10. mu.l of dNTP, 1. mu.l of 10xTaKaRa LA Buffer, 1. mu.l of TaKaRa Ex Taq enzyme, and 1. mu.l of H₂O76. mu.l. Performing 30 cycles under the PCR reaction conditions of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 30 s; the obtained PCR product was precipitated with ethanol and dissolved in 100. mu.l of TE.

The fragments CAG93-44F, CAG93-44 CAG93F1, CAG93-44 CAG93F2 and CAG93-44R are sequenced by a DNA full-automatic sequencer, and the result is the same as the designed sequence (see FIGS. 5-8).

The modification of the gene can be realized by using the known PCR technology, the site-directed mutagenesis technology, the chemical synthesis after gene design and the like. In addition to gene design and primer design, the invention can be completed by professional biotechnology companies (Dalianbao bioengineering company) for primer synthesis, PCR synthesis of newly designed genes, sequencing and the like.

Example 2: construction of recombinant plasmid pBluescript II SK (+) -Tsp containing synthetic lipase gene

The synthetic lipase gene obtained in example 1 must be cloned into an appropriate vector plasmid for storage and utilization. The invention selects an escherichia coli carrier plasmid pBluescript II SK (+), constructs a recombinant plasmidpBluescript II SK (+) -Tsp containing a synthetic lipase gene. The method comprises the following specific steps:

1. strain and carrier: escherichia coli strain JM109, plasmid pBluescript II DK (+) and the like were purchased from Promega corporation.

2. Enzyme and kit: restriction enzymes and various modifying enzymes are products of TaKaRa company.

3. Biochemical reagents: IPTG and X-Gal are both products of Sigma company.

4. Culture medium: LB Medium

5. The synthesized Tsp gene sequence is subjected to double enzyme digestion by XhoI and XbaI, and is connected with a vector pBluescript II SK (+) subjected to double enzyme digestion by XhoI and XbaI at 16 ℃ overnight to transform escherichia coli DH5 α, positive recombinants are selected after plating, a recombinant plasmid pBluescript II SK (+) -Tsp is obtained, and the complete sequence of the modified lipase gene is confirmed by DNA sequencing analysis.

6. Preserving the positive recombinants in glycerol, amplifying the positive recombinants when needed, extracting recombinant plasmids, and digesting the recombinant plasmids by XhoI and Xba I to obtain a large amount of lipase genes (Tsp).

Example 3 construction of recombinant expression plasmid pGAPZ α A-Tsp containing synthetic Lipase Gene

The lipase gene Tsp synthesized in the embodiment 1 of the invention can be correctly expressed in a eukaryotic expression system, since pichia pastoris is a more successful expression system at present, therefore, a recombinant expression plasmid pGAPZ α A-Tsp containing the synthetic lipase gene needs to be constructed so as to express the lipase in the pichia pastoris, the specific steps are as follows:

1. strain and vector Yeast strain Pichia pastoris SMD1168H, plasmid pGAPZ α A was purchased from Invitrogen corporation.

2. Enzyme and kit: restriction enzymes and various modifying enzymes are products of TaKaRa company.

3. Biochemical reagents: IPTG, X-Gal, SDS, ammonium persulfate, acryloyl and methylenebisacrylamide (the reagents used in the series should be changed) are all products of Sigma, and the others are domestic analytically pure.

4. The recombinant plasmid pBluescript II SK (+) -Tsp is cut by XhoI and XbaI, DNA fragment of about 1.65kb is recovered by electrophoresis, the vector plasmid pGAPZ α A is cut by XhoI and XbaI at the same time, large fragment of 3.1kb is recovered by electrophoresis, then the small fragment of 1.65kb is connected with the large fragment of 3.1kb, an expression vector pGAPZ α A-Tsp for yeast transformation is obtained, a target gene with partial secretion signal sequence is inserted into the downstream of the signal peptide sequence of the expression vector, and forms a correct reading frame with the signal peptide, thus constructing the recombinant expression vector which can be stably integrated on the yeast chromosome through homologous recombination between the partial gene sequence of P.pastro on the vector and the chromosome genome.

Example 4: transformation of Pichia pastoris and screening of genetically engineered bacteria (China center for type culture Collection, Wuhan university, Wuhan City, accession No. M201002)

The recombinant expression vector pGAPZ α A-Tsp containing the lipase gene obtained in example 3 was further transformed into P.pastoris, and the genetically engineered bacterium highly expressing the lipase gene was selected by resistance selection and enzyme activity assay

1. The yeast strain Pichia pastoris SMD1168H, plasmid pGAPZ α A were purchased from Invitrogen corporation, and the recombinant expression vector pGAPZ α A-Tsp containing lipase synthetic gene was from example 3.

2. Enzyme and kit: restriction enzymes and various modifying enzymes are products of TaKaRa company.

3. Biochemical reagents: zeocin is a product of French company, and others are made in China.

4. Culture medium: the yeast complete medium was YPD (1% yeast extract, 2% peptone, 2% glucose); yeast transformation medium RDB (18.6% sorbitol, 2% glucose, 1.34% Yeast);

5. the recombinant yeast fermentation medium is YPD.

6. The recombinant yeast expression vector is first digested with endonuclease BspHI and purified through PEG process, and the linearized pGAPZ α A is extracted with phenol, ethanol precipitated, washed twice with 70% ethanol, freeze dried, dissolved in sterile water and stored at-20 deg.c for further use.

7. 5ml of SMD1168H bacterial suspension was inoculated into 50ml of YPD liquid medium and cultured overnight at 30 ℃. 0.1-0.5 ml of overnight-cultured bacterial liquid is taken to be inoculated into 500ml of fresh YPD liquid culture medium, and then the mixture is cultured overnight at 30 ℃ until OD600 is 1.3-1.5. The cells were centrifuged at 1500Xg for 5 minutes at +4 ℃ and resuspended in 500ml of sterile water at 0 ℃. Centrifuged as above and resuspended in 250ml of sterile water at 0 ℃. Centrifuged as above and resuspended in 20ml of 1M sorbitol solution at 0 ℃. Centrifugation was performed as above, and resuspended in 1ml of 0 ℃ 1M sorbitol solution and placed on ice (used on the day).

8. Injecting 80 mu l of the prepared cells and 5-90 mu g of linearized DNA into a 0 ℃ electric shock pool (electric shock or electric excitation), uniformly mixing, and placing on ice for 5 minutes. Cells were pulsed according to yeast pulse parameters. (voltage: 1500V; capacitance: 25. mu.F; resistance: 200. omega.). 1ml of 1M sorbitol solution at 0 ℃ is rapidly added into an electric shock cell and mixed evenly. Then the liquid in the pool is transposed to 1.5ml EP tube, and cultured for 1-2 hours at 30 ℃. 150 ul of YPDS solid medium containing Zeocin at 1000 ug/ml was applied and cultured at 30 ℃ for 2-3 days.

9. Pick up Zeo from conversion plate with sterile toothpick⁺Recombinants were streaked on YPD solid medium containing 1000. mu.g/ml Zeocin, and single clones were isolated.

10. Zeocin prepared from the above 9⁺Phenotypic recombinants approximately 26 strains were further subjected to lipase enzyme activity assay. And screening out a strain with the enzyme activity of 120-160U/mL at the pH of 7.2 and the temperature of 30-40 ℃.

Claims (4)

1. A lipase gene sequence, characterized in that the structure of said sequence is as follows:

CTC GAG AAA AGA GAG GCT GAA GCT GCT CCA ACT GCT ACT TTG GCT AACGGT GAT ACT ATT ACT GGT TTG AAC GCT ATT ATT AAC GAA GCT TTT TTG GGT ATTCCA TTT GCT GAA CCA CCA GTT GGT AAC TTG AGA TTT AAG GAC CCA GTT CCATAC TCT GGT TCT TTG GAT GGT CAA AAG TTT ACT TCT TAC GGT CCA TCT TGT ATGCAA CAA AAC CCA GAA GGT ACT TAC GAA GAA AAC TTG CCA AAG GCT GCT TTGGAT TTG GTT ATG CAA TCT AAG GTT TTT GAA GCT GTT TCT CCA TCT TCT GAA GATTGT TTG ACT ATT AAC GTT GTT AGA CCA CCA GGT ACT AAG GCT GGT GCT AACTTG CCA GTT ATG TTG TGG ATT TTT GGT GGT GGT TTT GAA GTT GGT GGT ACT TCTACT TTT CCA CCA GCT CAA ATG ATT ACT AAG TCT ATT GCT ATG GGT AAG CCA ATTATT CAT GTT TCT GTT AAC TAC AGA GTT TCT TCT TGG GGT TTT TTG GCT GGT GATGAA ATT AAG GCT GAA GGT TCT GCT AAC GCT GGT TTG AAG GAT CAA AGA TTGGGT ATG CAA TGG GTT GCT GAT AAC ATT GCT GCT TTT GGT GGT GAT CCA ACTAAG GTT ACT ATT TTT GGT GAA TCT GCT GGT TCT ATG TCT GTT ATG TGT CAT ATTTTG TGG AAC GAT GGT GAT AAC ACT TAC AAG GGT AAG CCA TTG TTT AGA GCTGGT ATT ATG CAA TCT GGT GCT ATG GTT CCA TCT GAT GCT GTT GAT GGT ATT TACGGT AAC GAA ATT TTT GAT TTG TTG GCT TCT AAC GCT GGT TGT GGT TCT GCT TCTGAT AAG TTG GCT TGT TTG AGA GGT GTT TCT TCT GAT ACT TTG GAA GAT GCT ACTAAC AAC ACT CCA GGT TTT TTG GCT TAC TCT TCT TTG AGA TTG TCT TAC TTG CCAAGA CCA GAT GGT GTT AAC ATT ACT GAT GAT ATG TAC GCT TTG GTT AGA GAAGGT AAG TAC GCT AAC ATT CCA GTT ATT ATT GGT GAT CAA AAC GAT GAA GGTACT TTT TTT GGT ACT TCT TCT TTG AAC GTT ACT ACT GAT GCT CAA GCT AGA GAATAC TTT AAG CAA TCT TTT GTT CAT GCT TCT GAT GCT GAA ATT GAT ACT TTG ATGACT GCT TAC CCA GGT GAT ATT ACT CAA GGT TCT CCA TTT GAT ACT GGT ATTTTG AAC GCT TTG ACT CCA CAA TTT AAG AGA ATT TCT GCT GTT TTG GGT GAT TTGGGT TTT ACT TTG GCT AGA AGA TAC TTT TTG AAC CAT TAC ACT GGT GGT ACT AAGTAC TCT TTT TTG TCT AAG CAA TTG TCT GGT TTG CCA GTT TTG GGT ACT TTT CATTCT AAC GAT ATT GTT TTT CAA GAT TAC TTG TTG GGT TCT GGT TCT TTG ATT TACAAC AAC GCT TTT ATT GCT TTT GCT ACT GAT TTG GAC CCA AAC ACT GCT GGT TTGTTG GTT AAG TGG CCA GAA TAC ACT TCT TCT TCT CAA TCT GGT AAC AAC TTGATG ATG ATT AAC GCT TTG GGT TTG TAC ACT GGT AAG GAT AAC TTT AGA ACT GCTGGT TAC GAT GCT TTG TTT TCT AAC CCA CCA TCT TTT TTT GTT TAATCTAGA

CTCGAGAAAAGAGAGGCTGAAGCT is the gene segment which is connected with the 5' end and matched with the target yeast; the sequence connecting the 3' end stop codon and the restriction endonuclease site is: TAATCTAGA are provided.

2. A recombinant vector pBluescript II SK (+) -Tsp comprising the gene sequence of claim 1, wherein Tsp is the lipase gene sequence of claim 1.

3. A recombinant vector pGAPZ α a-Tsp comprising the gene sequence of claim 1, wherein Tsp is the lipase gene sequence of claim 1.

4. A yeast transformed with the recombinant vector of claim 3, wherein the expression vector pGAPZ α A-Tsp is transformed into Pichia pastoris.

Images