WO2020135763A9 - 用于表达外源基因的毕赤酵母突变株 - Google Patents

用于表达外源基因的毕赤酵母突变株 Download PDF

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WO2020135763A9
WO2020135763A9 PCT/CN2019/129359 CN2019129359W WO2020135763A9 WO 2020135763 A9 WO2020135763 A9 WO 2020135763A9 CN 2019129359 W CN2019129359 W CN 2019129359W WO 2020135763 A9 WO2020135763 A9 WO 2020135763A9
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pichia pastoris
gene
amino acid
pichia
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PCT/CN2019/129359
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French (fr)
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WO2020135763A1 (zh
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吴伟
戴小军
曹海生
周美凤
牛其文
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丰益(上海)生物技术研发中心有限公司
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Priority to JP2021538140A priority Critical patent/JP7430189B2/ja
Priority to US17/419,122 priority patent/US20220162543A1/en
Priority to EP19903304.4A priority patent/EP3910062A4/en
Publication of WO2020135763A1 publication Critical patent/WO2020135763A1/zh
Publication of WO2020135763A9 publication Critical patent/WO2020135763A9/zh

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Definitions

  • the present invention relates to a Pichia mutant strain for expressing foreign genes.
  • the Pichia pastoris expression system is a new type of foreign protein expression system developed in the early 1980s. Because it not only has the advantages of easy operation, easy culture, fast growth, high expression, and low cost of the prokaryotic expression system, it also has the characteristics of post-translational modification of foreign proteins that the prokaryotic expression system does not have, such as glycosylation. , Protein phosphorylation, etc. At the same time, it also avoids the defects of Saccharomyces cerevisiae (Saccharomyces cerevisiae) secretion efficiency is poor, the expression strain is not stable enough, and the expression plasmid is easy to lose. Therefore, the expression system has quickly become one of the best and most widely used foreign gene expression systems.
  • RCT's basic strain GS115 is derived from chemical mutagenesis of the original strain NRRL-Y 11430 (ATCC 76273), making it a histidine auxotrophic host (His-), which is convenient for clone screening.
  • His- histidine auxotrophic host
  • the alcohol oxidase gene AOX1 of GS115 is complete, and it has been able to use methanol to express most foreign proteins, but its background AOX1 will still be highly expressed, so the yield of some genes is affected.
  • One of the subsequent Pichia-derived strains is to knock out the AOX1 gene on the basis of GS115 and replace it with the Saccharomyces cerevisiae ARG4 gene to obtain KM71 (his4 arg4 aox1 ⁇ ::ARG4), whose AOX2 gene remains intact, so the methanol utilization rate is very low , It grows very slowly under the cultivation of methanol as the only carbon source.
  • the AOX2 gene was further knocked out, and the host MC100-3 (his4 arg4 aox1 ⁇ ::SARG4aox2 ⁇ ::Phis4) that could not use methanol was obtained.
  • Another direction modified on the basis of GS115 is to inactivate the host protease.
  • Pichia pastoris vacuolar protease B (Proteinase B, prb1) to obtain SMD1165 (his4 prb1).
  • knock out vacuolar aspartic protease (PEP4) to get SMD1168 (his4 pep4), which is used to activate other vacuolar proteases, including carboxypeptidase Y and protease B.
  • vacuolar protease B (Proteinase B, prb1) was further knocked out to obtain SMD1163 (his4 pep4 prb1). That is, PEP4 protease is more critical and is used for the activation of some proteases. If necessary, the vacuolar protease prb1 and carboxypeptidase can be further knocked out.
  • the present invention transforms Pichia pastoris CICC32806 to obtain Pichia pastoris which can be used to efficiently express various proteins, especially phospholipase and lipase.
  • the first aspect of the present invention is to provide a Pichia strain. Compared with Pichia strain GS115 or CICC32806, it contains one or more of the following 6 mutations: BQ9382_C1-2260, 308-310 EKK deletion, hypothesis Protein; BQ9382_C1-3800, E129K, 60S ribosomal subunit assembly/export protein LOC1; BQ9382_C1-5700, I312M, NADH dehydrogenase outside the mitochondria, Class II NAD(P)H: Quinone oxidoreductase; BQ9382_C2-3950, Q145X , An essential protein, with a binding partner Psr1p, used to fully activate the general stress response; BQ9382_C3-2220, E188K, hypothetical protein; and BQ9382_C3-4370, W196X, orotidine 5'-phosphate decarboxylase.
  • the strain includes 6 mutations as described above.
  • the Pichia pastoris strain is a Pichia pastoris strain with a deposit number of CGMCC No. 16670, and a Pichia pastoris strain with a deposit number of CGMCC No. 16669, and the deposit number is CGMCC No. 19221 Pichia pastoris (Pichia pastoris) strain.
  • the Pichia yeast strain provided by the present invention is a histidine and uracil double-deficient strain.
  • the present invention provides a Pichia pastoris strain with a deposit number of CGMCC No. 16670.
  • the present invention also provides a genetically engineered Pichia pastoris strain.
  • the Pichia pastoris strain is a genetically engineered Pichia pastoris strain with a deposit number of CGMCC No. 16670, and (a) is a histidine-deficient Type strain; and/or (b) contains a plasmid expressing a growth promoting factor, and/or a coding sequence of a growth promoting factor integrated in the genome, and/or expressing a growth promoting factor.
  • the genetically engineered Pichia strain is a Pichia pastoris strain with a deposit number of CGMCC No. 16669.
  • the Pichia strain described in any of the foregoing embodiments can be used as a basic strain to be genetically engineered to express foreign genes of interest. Therefore, the present invention also provides a genetically engineered Pichia pastoris strain, which is the Pichia pastoris strain described in any one of the foregoing embodiments that has been genetically engineered to contain a foreign gene or a vector containing the foreign gene , Including the CGMCC No. 16670 Pichia pastoris strain of CGMCC No.
  • the 16670 that has been genetically engineered to contain a foreign gene or a vector containing the foreign gene, the histidine and uracil double-deficient strains, The histidine single-deficient strain and/or the plasmid containing the growth promoting factor, the Pichia strains genetically engineered with the coding sequence of the growth promoting factor integrated in the genome and/or the genetically engineered expression of the growth promoting factor, and the deposit number It is the Pichia pastoris strain of CGMCC No. 16669. It should be understood that the foreign gene described here does not include the gene encoding the growth promoting factor, and can be genetically engineered to further contain any other foreign genes of interest on the basis of the expression gene containing the growth promoting factor. Source gene.
  • the foreign gene is integrated into the genome of the strain.
  • the exogenous gene is a protein coding sequence used in industry, feed, or food.
  • the exogenous gene is the coding sequence of an enzyme.
  • the enzyme is selected from at least one of the following enzymes: lipase, protease, cellulase, amylase, phytic acid Enzymes, esterases, pectinases, galactosidases and phospholipases.
  • the present invention also provides a culture containing the Pichia pastoris strain according to any embodiment of the present invention and an optional culture medium.
  • the medium is a seed medium or a fermentation medium.
  • the medium is YPD medium or BMMY medium.
  • the present invention also provides an enzyme product containing the fermentation broth of Pichia pastoris or the lysate of the cells obtained by fermentation, the foreign gene of which is the coding sequence of the enzyme according to any one of the embodiments of the present invention, or The concentrate of the fermentation broth or lysate.
  • the present invention also provides the application of the enzyme product of the present invention in transesterification, wherein the enzyme product contains the fermentation broth of Pichia pastoris whose exogenous gene is the coding sequence of lipase according to any one of the embodiments of the present invention Or a lysate of cells obtained by fermentation, or a concentrate of the fermentation broth or lysate.
  • the present invention also provides the application of the enzyme product of the present invention in oil degumming, wherein the enzyme product contains the coding sequence of phospholipase (especially phospholipase C) according to any embodiment of the present invention.
  • the enzyme product contains the coding sequence of phospholipase (especially phospholipase C) according to any embodiment of the present invention.
  • the amino acid sequence of the lipase is an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 7 or 9, more preferably The amino acid sequence of the lipase is shown in SEQ ID NO: 7 or 9.
  • the amino acid sequence of the phospholipase is an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 2, more preferably, The amino acid sequence of the phospholipase is shown in SEQ ID NO: 2.
  • the present invention provides a method for preparing a Pichia yeast strain double deficient in histidine and uracil, the method comprising:
  • step (3) Perform mutagenesis on the mutant obtained in step (3), and screen to obtain that the foreign gene knocked in in step (3) does not have mutations, but the expression level of the foreign gene is relatively high or the activity of the expression product is relatively high Higher mutants;
  • the present invention also provides a method for preparing a Pichia yeast strain for expression of foreign genes, the method comprising:
  • step (3) Perform mutagenesis on the mutant obtained in step (3), and screen to obtain that the foreign gene knocked in in step (3) does not have mutations, but the expression level of the foreign gene is relatively high or the activity of the expression product is relatively high Higher mutants;
  • the present invention also provides the application of the Pichia pastoris strain described herein in constructing a strain expressing foreign genes.
  • the Pichia pastoris mutant strain of the present invention is an effective universal host for exogenous expression, and can efficiently express various proteins, especially phospholipase and lipase.
  • Figure 1 Comparison of PLC enzyme activity of m314-SPLC expressing PLC.
  • Figure 2 Comparative protein electrophoresis of m314-SPLC expressing PLC.
  • Figure 3 Comparison diagram of enzyme activity of m314H expressing RML.
  • Figure 4 Comparative protein electrophoresis of m314H expressing RML.
  • Figure 5 Comparison of enzyme activity of m315H expressing TL.
  • Figure 6 Comparative protein electrophoresis of m315H expressing TL.
  • Figure 7 Comparison of enzyme activity of m316H expressing TL.
  • Figure 8 Comparative protein electrophoresis of m316H expressing TL.
  • genetic engineering used in the present invention is also called gene splicing technology and DNA recombination technology. It is based on molecular genetics and modern methods of molecular biology and microbiology as a means to pre-design genes from different sources. The blueprint is to construct hybrid DNA molecules in vitro and then introduce them into living cells to change the original genetic characteristics of organisms, obtain new varieties, and produce new products.
  • mutation used in the present invention has a general meaning in the art, which refers to artificial measures to induce mutations in the genetic genes of strains, and then select new and excellent varieties from the mutated strains as required.
  • Mutagenic agents treat mutagenic materials to induce mutation, which is often referred to as chemical mutagenesis.
  • mutant used in the present invention refers to a Pichia strain derived from the yeast strain CICC32806 and containing modifications or changes at one or more positions, namely substitutions, insertions and/or deletions.
  • Pichia strain mutants can be obtained by various techniques well known in the art.
  • examples of techniques for altering the sequence of the yeast strain CICC32806 include, but are not limited to, site-directed mutagenesis, random mutagenesis, and genetic engineering.
  • homology and “identity” used in the present invention describe the degree of similarity between two or more amino acid sequences. Determine the percentage of "sequence identity" between two sequences in the following way: compare the two best aligned sequences on the comparison window, so that the part of the sequence in the comparison window can be compared with the reference sequence (which does not contain Additions or deletions) include additions or deletions (gaps) for optimal alignment of the two sequences. Calculate the percentage by determining the number of positions where the same amino acid residue appears in the two sequences to get the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100, Get the percentage of sequence identity. A sequence that is the same at each position compared to the reference sequence is considered to be the same as the reference sequence, and vice versa.
  • gene knockout used in the present invention refers to the use of a DNA fragment containing a certain known sequence to undergo homologous recombination with a gene with the same or similar sequence in the genome of the recipient cell, integrated into the genome of the recipient cell and expressed A technique for introducing foreign DNA. It changes the genetic genes of an organism with a known sequence but an unknown function, so that the function of a specific gene is lost, so that part of the function is blocked, and it can further affect the organism, and then infer the organism of the gene. Learn function.
  • Pichia pastoris is subjected to mutagenesis, and then auxotrophic mutants are screened out, thereby preparing a Pichia pastoris strain for expression of exogenous genes.
  • the present invention uses CICC32806 purchased from the China Industrial Microbial Culture Collection and Management Center (CICC) as the initial strain, and after UV mutagenesis, the uracil auxotrophic strain U7 is obtained by screening. Then through gene knockout, the HIS4 gene in U7 strain was inactivated, and histidine auxotrophic strain 7H3 was obtained. Then the PLC coding sequence was transferred into 7H3, and the recombinant Pichia pastoris 7H3-SPLC was obtained by screening.
  • CICC32806 purchased from the China Industrial Microbial Culture Collection and Management Center (CICC) as the initial strain, and after UV mutagenesis, the uracil auxotrophic strain U7 is obtained by screening. Then through gene knockout, the HIS4 gene in U7 strain was inactivated, and histidine
  • the present invention further implements mutagenesis on 7H3-SPLC, and obtains mutant m314-SPLC by screening. By knocking out the PLC gene and HIS4 gene in m314-SPLC, the m314H strain was obtained.
  • a histidine knockout vector can be constructed and transferred to the strain m314H of the present invention, in a medium containing YNB and histidine, a medium containing YNB and uracil, and a medium containing YNB, histidine and uracil
  • the strain was screened in the medium containing YNB, histidine and uracil, and the strain was selected as the double auxotrophic strain of histidine and uracil, which was named as strain m314HU.
  • the present invention also overexpressed three growth promoting genes in the m314HU strain and knocked in the URA3 gene to construct the strain m315H.
  • the present invention overexpresses the vacuolar protease A gene in the m314HU strain and knocks in the URA3 gene to construct the strain m316H.
  • BQ9382_C1-2260, 308EKKdel, hypothetical protein is the number given by GENEBANK, where C1: the first chromosome, 2260 is the gene number, and EKK at positions 308-310 is missing.
  • BQ9382_C1-3800 E129K, 60S ribosomal subunit assembly/export protein LOC1.
  • BQ9382_C1-3800 is the number given by GENEBANK, where C1: the first chromosome, 3800 is the gene number, and the E mutation at position 129 is K.
  • BQ9382_C1-5700 I312M, NADH dehydrogenase outside the mitochondria, class II NAD(P)H: quinone oxidoreductase.
  • BQ9382_C1-5700 is the number given by GENEBANK, where C1: the first chromosome, 5700 is the gene number, and the I mutation at position 312 is M.
  • BQ9382_C2-3950 is the number given by GENEBANK, where C2: the second chromosome, 3950 is the gene number, and Q at position 145 becomes the stop codon.
  • BQ9382_C3-2220 E188K, hypothetical protein.
  • BQ9382_C3-2220 is the number given by GENEBANK, where C3: the third chromosome, and 2220 is the gene number.
  • BQ9382_C3-4370 is the number given by GENEBANK, where C3: the third chromosome, 4370 is the gene number, and the 196th W mutation is a stop codon.
  • a person of ordinary skill in the art can use strategies including but not limited to homologous recombination, gene knockout and filling, zinc finger nuclease, TALE nuclease, Crisp/Cas9, etc., to combine one or the other of the above six mutations disclosed in the present invention. Multiple, introduced into the Pichia strain of interest.
  • the mutagenesis can be physical mutagenesis or chemical mutagenesis.
  • Physical mutagenesis includes ultraviolet mutagenesis, such as placing the Pichia strain under ultraviolet light for a period of time, for example, 60 to 120 seconds.
  • Chemical mutagenesis involves contacting the Pichia pastoris strain with a chemical mutagen such as nitrosoguanidine for a period of time, for example, 15 to 60 minutes.
  • a culture medium containing uracil and 5-fluoroorotic acid (5-FOA) such as a medium containing YNB (non-amino yeast nitrogen source)
  • 5-FOA 5-fluoroorotic acid
  • YNB non-amino yeast nitrogen source
  • the mutagenized strain can be cultured on a medium containing YNB, glycerol, agarose, uracil and 5-fluoroorotic acid, and cultured at 25-33°C in the dark for 3-8 days , And then pick out the single colony grown in this medium, and transfer it to a medium containing YNB and uracil (such as YNB, glycerol, agarose and uracil). The picking can only be on this medium The grown strains can obtain uracil-deficient strains.
  • the concentration of YNB can be 10-20g/L
  • the content of glycerol can be 0.5-2%
  • the content of agarose can be 1-3%
  • the concentration of uracil can be 30-100 ⁇ g/mL; when it is contained, 5 -FOA concentration can be 0.5-1.2mg/mL.
  • the present invention uses Pichia pastoris with the deposit number CICC32806 as the initial strain for mutagenesis. Therefore, in these embodiments, the uracil-deficient strain is a strain obtained by Pichia pastoris with deposit number CICC32806 after mutagenesis and screening for uracil auxotrophy.
  • the obtained uracil-deficient strain can be knocked out its histidine dehydrogenase gene (HIS4) by gene knockout, and screened in the medium containing histidine, and the strain can be selected in the medium containing histidine
  • HIS4 histidine dehydrogenase gene
  • the uracil-deficient strains transferred into the histidine knockout vector are cultured in MDS screening plates containing histidine (such as 10-50 ⁇ g/mL), and the obtained single colonies are respectively inoculated into YNB-containing In the medium and the medium containing YNB and histidine, the strains that can grow in the histidine-containing medium but cannot grow in the histidine-free medium can be found by comparison, which can be screened Obtain histidine-deficient strains.
  • histidine such as 10-50 ⁇ g/mL
  • the strains obtained after mutagenesis and histidine auxotrophic screening are transformed into an expression vector expressing PLC, and the strains obtained by screening on phospholipid plates are cultured with larger hydrolysis circles and transfected
  • the imported nucleic acid sequence (such as AOX promoter, signal peptide, PLC gene, transcription terminator, etc.) used to express PLC is not mutated in the mutant 7H3-SPLC.
  • the expression vector of PLC is transferred, for example, the HIS4 gene is also transferred at the same time, and the strain thus constructed is not a histidine auxotrophic strain.
  • the mutant can be subjected to further physical mutagenesis, such as ultraviolet irradiation, to screen out that the phospholipid plate culture has a larger hydrolysis circle and is transferred into the nucleic acid sequence for expressing PLC (such as the AOX promoter , Signal peptide, PLC gene, transcription terminator, etc.) without mutation m314-SPLC.
  • PLC such as the AOX promoter , Signal peptide, PLC gene, transcription terminator, etc.
  • the strain m314H of the present invention has been deposited at the China General Microbial Culture Collection and Management Center (CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101) on October 31, 2018, and is classified as Pasteur Pichi Yeast (Pichia pastoris), the deposit number is CGMCC No. 16670.
  • CGMCC China General Microbial Culture Collection and Management Center
  • the present invention also includes histidine and uracil double auxotrophic strains.
  • a histidine knockout vector can be constructed and transferred to the strain m314H of the present invention, in a medium containing YNB and histidine, a medium containing YNB and uracil, and a medium containing YNB, histidine and urine.
  • the strain was screened in the pyrimidine medium, and the strain that could only grow in the medium containing YNB, histidine and uracil was selected, namely the histidine and uracil double auxotrophic strain, named strain m314HU.
  • the growth-promoting gene can be transferred into the strain m314HU and the URA3 gene can be knocked in to construct a strain expressing the growth-promoting gene.
  • the growth promotion gene is preferably a growth promotion gene derived from yeast itself, including but not limited to Protein required general stress response (Genbank no: XM_002491428.1), Mitochondrial external NADH dehydrogenase (Genbank no: XM_002490375.1), Vacuolar protease A (Genbank no: XM_002493288.1).
  • One or more of the growth promoting genes can be transferred.
  • the present invention also includes single-histidine-deficient strains that have successfully transferred the growth promoting gene.
  • the single-histidine-deficient strain obtained in the present invention is named strain m315H herein.
  • the strain m315H of the present invention has been deposited in the China General Microbial Culture Collection Management Center (CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101) on October 31, 2018, and is classified and named as Pasteur Pichi Yeast (Pichia pastoris), the deposit number is CGMCC No. 16669.
  • CGMCC China General Microbial Culture Collection Management Center
  • the vacuolar protease A (Genbank no: XM_002493288.1) was transformed into the strain m314HU and the URA3 gene was knocked in to construct a single histidine-deficient strain overexpressing the vacuolar protease A gene.
  • the single-histidine-deficient strain obtained in the present invention is named strain m316H herein.
  • the strain m316H of the present invention has been deposited at the China General Microbial Culture Collection Management Center (CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101), and is classified as Pichia pastoris ( Pichia pastoris), the deposit number is CGMCC No.19221.
  • CGMCC China General Microbial Culture Collection Management Center
  • Pichia pastoris Pichia pastoris
  • the auxotrophic Pichia pastoris of the present invention can be used as basic strains to construct host strains for expressing foreign genes.
  • a foreign gene refers to a gene with a function of interest that is transferred into a host strain from the outside, regardless of whether the gene is from another species or exists in the host genome.
  • the foreign gene can be a gene encoding any protein of interest.
  • the protein of interest includes, but is not limited to, various proteins used in the fields of industry, feed or food, including but not limited to various lipases, proteases, cellulases, amylases, phytases, esterases, fruit Pectinases, galactosidases and phospholipases.
  • the histidine auxotrophic Pichia pastoris described herein can be used to construct a strain expressing exogenous phospholipase C.
  • the amino acid sequence of the phospholipase is an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 2; or is an amino acid sequence that is identical to SEQ ID NO: under high stringency conditions. :1.
  • the amino acid sequence of the phospholipase C is shown in SEQ ID NO: 2, and preferably, the coding sequence thereof is shown in SEQ ID NO: 1.
  • the histidine auxotrophic Pichia pastoris described herein can be used to construct a strain expressing exogenous lipase.
  • the amino acid sequence of the lipase is an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 7 or 9; or is an amino acid sequence that is identical to SEQ ID NO: 7 or 9 under high stringency conditions.
  • ID NO: 6 or 8 the amino acid sequence encoded by the polynucleotide hybridized with its cDNA sequence or its full-length complement.
  • high stringency conditions means that so-called specific hybrids are formed under these conditions, and non-specific hybrids are not formed.
  • high stringency conditions include typical washing conditions for Southern hybridization, that is, corresponding to 1x SSC, 0.1% SDS at 60°C, preferably 0.1x SSC, 0.1% SDS at 60°C, more preferably 0.1x SSC, 0.1% SDS The washing is performed once, preferably twice or three times, at a salt concentration and temperature of 68°C.
  • the amino acid sequence of the lipase is as shown in SEQ ID NO: 7 or 9, and its coding sequence is preferably as shown in SEQ ID NO: 6 or 8.
  • the strain of the present invention can be used to express phospholipase C or phospholipase C whose amino acid sequence has one or more (for example, within 10) amino acid residue mutations compared with SEQ ID NO: 2, 7 or 9
  • Lipase includes substitution, insertion or deletion of one or more amino acid residues.
  • the above-mentioned one or more amino acid disabled substitutions, insertions or deletions are conservative mutations that maintain the normal function of the protein (that is, the activity of the mutant phospholipase C or lipase does not substantially change).
  • a typical example of a conservative mutation is a conservative substitution, where if the substitution site is an aromatic amino acid, the substitution occurs between Phe, Trp, and Tyr; if it is a hydrophobic amino acid, the substitution occurs between Leu, Ile, and Val ; If it is a polar amino acid, the substitution takes place between Gln and Asn; if it is a basic amino acid, it takes place between Lys, Arg, and His; if it is an acidic amino acid, it takes place between Asp and Glu; and If it is an amino acid with a hydroxyl group, the substitution occurs between Ser and Thr.
  • substitutions considered to be conservative substitutions particularly include substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn, substitution of Asn, Glu or Gln for Asp, and Ser Or Ala for Cys, Asn, Glu, Lys, His, Asp or Arg for Gln, Gly, Asn, Gln, Lys or Asp for Glu, Pro for Gly, Asn, Lys, Gln, Arg or Tyr for His, Replace Ile with Leu, Met, Val or Phe, replace Leu with Ile, Met, Val or Phe, replace Lys with Asn, Glu, Gln, His or Arg, replace Met with Ile, Leu, Val or Phe, replace Met with Trp, Tyr , Met, Ile or Leu for Phe, Thr or Ala for Ser, Ser or Ala for Thr, Phe or Tyr for Trp, His, Phe or Trp for
  • the conventional backbone vector for expressing foreign genes in Pichia pastoris can be used to construct an expression vector suitable for the present invention for transforming the histidine auxotrophic Pichia pastoris described herein.
  • Such backbone vectors include but are not limited to pPIC3, pPIC9, pPIC9k, pHIL-D1, pAO804, pAO815, and pPSC3K.
  • a typical Pichia pastoris expression vector contains the alcohol oxidase-1 (AOX1) gene promoter and transcription terminator (5'AOX1 and AOXTT), which are separated by a multiple cloning site (MCS), where foreign genes can be inserted .
  • AOX1 alcohol oxidase-1
  • MCS multiple cloning site
  • Such vectors can also contain histidine alcohol dehydrogenase gene (HIS4) selection marker and 3'AOX1 region.
  • HIS4 histidine alcohol dehydrogenase gene
  • the 5'AOX1, AOXTT, 3'AOX1 and HIS4 of the vector can recombine with the homologous gene on the chromosome individually or together, so that the entire vector and the foreign gene to be expressed can be inserted into On the chromosome of the recipient bacteria, the foreign gene is expressed under the control of the 5'AOX1 promoter.
  • the AOX1 promoter can be substituted.
  • Suitable promoters include, but are not limited to, inducible and constitutive promoters.
  • the construction method of the vector is well known in the art. For example, after PCR amplification to obtain the target gene, the PCR product and the backbone vector are subjected to corresponding restriction endonuclease digestion, and the digested fragment of the PCR product is connected with the digested fragment of the vector by DNA ligase, and the linker is transferred Into Escherichia coli, after culturing in a suitable medium, a commercially available plasmid extraction kit is used to extract a plasmid for transforming the histidine auxotrophic Pichia pastoris.
  • the transformation method of Pichia pastoris is also well known in the art.
  • the constructed expression vector is digested with restriction enzymes to obtain a linearized vector.
  • the competent cells of Pichia pastoris can be transformed by electroporation, and then spread on a suitable plate (such as MDS screening plate) and cultivated for several days. Afterwards, the transformants are picked on a suitable plate, and the required recombinant strains are selected according to the biological activity of the foreign protein expressed.
  • the foreign protein is a phospholipase (such as phospholipase C), and the transformant will be cultured on a phospholipid plate.
  • phospholipid plates contain 1 to 3% YNB, 1 to 3% phospholipids, and 1 to 3% agar. Since phospholipase can hydrolyze phospholipids, the activity of phospholipase expressed by transformants can be determined according to the size of the hydrolysis circle. Picking the transformant with relatively large hydrolysis circle can obtain the excellent histidine auxotrophic Pichia pastoris.
  • the present invention also includes histidine auxotrophic Pichia pastoris containing the foreign gene to be expressed.
  • the foreign gene is usually integrated into the genome of the Pichia pastoris.
  • the histidine auxotrophic Pichia yeast containing the foreign gene can stably express the foreign gene.
  • the exogenous gene is a coding sequence for phospholipase or lipase.
  • the exogenous gene is a coding sequence for phospholipase C, RML lipase, or TL lipase.
  • the histidine auxotrophic Pichia pastoris is transformed into an expression vector containing foreign genes constructed using pPIC9.
  • the expression vector containing the exogenous gene constructed using pPIC9 contains the nucleotide sequence shown in SEQ ID NO: 1, 6 or 8.
  • the histidine auxotrophic Pichia pastoris herein can be cultivated using conventional culture media and methods in the art.
  • the medium may be a conventional BMGY medium. It can be cultured at 28 ⁇ 32°C and 180 ⁇ 300rpm.
  • a certain amount of methanol can be added to the culture medium to induce expression.
  • the fermentation broth is centrifuged and the supernatant is filtered to obtain the fermentation broth containing the target protein expressed by the foreign gene.
  • the fermentation broth can be further concentrated by conventional methods.
  • the upper tank initial medium may be a basic fermentation medium, which contains calcium sulfate, potassium dihydrogen phosphate, anhydrous magnesium sulfate, ammonium sulfate, emulsified silicone oil defoamer and glycerin, and is added There are PTMs, namely copper sulfate pentahydrate, sodium iodide, manganese sulfate monohydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, zinc chloride pentahydrate, ferrous sulfate heptahydrate, boric acid, concentrated sulfuric acid and biotin.
  • the concentration or content of the components in the basic fermentation medium is well known in the art.
  • the concentration of calcium sulfate can be 0.5 ⁇ 1.5 g/L
  • the concentration of potassium dihydrogen phosphate can be 30 ⁇ 40 g/L
  • the concentration of anhydrous magnesium sulfate can be 10 ⁇ 13 g/L
  • the concentration of ammonium sulfate can be 6 ⁇ 12g/L
  • the concentration of emulsified silicone oil defoamer can be 0.1 ⁇ 0.5ml/L
  • the concentration of glycerin can be 30 ⁇ 70g/L.
  • the concentration of copper sulfate pentahydrate can be 5 ⁇ 6.5g/L
  • the concentration of sodium iodide can be 60 ⁇ 100mg/L
  • the concentration of manganese sulfate monohydrate can be 2.0 ⁇ 4.0g/L
  • molybdic acid dihydrate The concentration of sodium can be 0.2 ⁇ 0.4g/L
  • the concentration of cobalt chloride hexahydrate can be 0.4 ⁇ 0.6g/L
  • the concentration of zinc chloride pentahydrate can be 18 ⁇ 22g/L
  • the concentration of ferrous sulfate heptahydrate It can be 60-70 g/L
  • the concentration of boric acid can be 0.01-0.03 g/L
  • the concentration of concentrated sulfuric acid can be 19.0-19.5 ml/L
  • the concentration of biotin can be 0.3-0.5 g/L.
  • the fermentation broth can be subjected to centrifugal treatment or plate and frame filtration treatment to remove the bacteria, and the supernatant is subjected to microfiltration and ultrafiltration treatment, and the buffer is replaced and concentrated. Further, an appropriate protective agent can be added and stored at 4°C as an enzyme preparation after it is completely dissolved.
  • the present application also provides an enzyme product containing the fermentation broth of the Pichia strain containing the exogenous gene encoding the enzyme described in any of the embodiments herein or the lysate of the cells obtained by fermentation, or the Concentrate of fermentation broth or lysate.
  • the Pichia yeast strain contains an expression vector containing the coding sequence of the enzyme constructed using pPIC9.
  • the enzyme preparation may also contain glycerin and a preservative.
  • the preservative may be a conventional preservative such as potassium sorbate.
  • the amounts of glycerin and preservatives can be conventional amounts in the art. For example, 40-70% glycerol and 0.1-0.8% potassium sorbate can be added to account for the weight of the enzyme product.
  • the enzyme preparation contains phospholipase C or lipase.
  • the enzyme preparation containing phospholipase C described herein can be used for oil degumming.
  • Degumming can be implemented by a conventional method, including the step of contacting the fat to be degummed with the phospholipase C-containing enzyme preparation described herein. For example, take crude oil and heat to a certain temperature (such as 50 ⁇ 5°C), add a certain amount of pure water and enzyme solution, high-speed shear (such as 10000r/min), and then stir at a certain temperature (such as 750r/min) , React for 1 to 5 hours. Finally, the reaction mixture can be heated to 80-90°C and maintained for a period of time, the enzymes can be inactivated, and the degummed oil can be obtained by centrifugation.
  • a certain temperature such as 50 ⁇ 5°C
  • high-speed shear such as 10000r/min
  • React such as 750r/min
  • the present application also provides a degumming method, which includes the step of contacting the fat to be degummed with the phospholipase C-containing enzyme preparation described herein; and the phospholipase C-containing enzyme preparation described herein is used in oil degumming Applications.
  • the application also relates to the application of the lipase-containing enzyme preparation described herein in transesterification.
  • a transesterification method which includes the step of contacting a reaction substrate with the lipase-containing enzyme preparation of the present invention.
  • This application also provides selected from the sequence shown in Genbank accession number XM_002491428.1, the sequence shown in Genbank accession number XM_002490375.1, the sequence shown in Genbank accession number XM_002493288.1 and XM_002491428.1, XM_002490375.
  • 1 or XM_002493288.1 has at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% of any one or more of the sequence or its expression vector in improving the presence of foreign genes in the host Application in expression in cells, or application in promoting the growth of host cells containing exogenous genes, or application in preparing host cells with increased expression or growth ability of exogenous genes.
  • the sequence with certain sequence identity is also from yeast, more preferably from Pichia pastoris.
  • the host cell is yeast, more preferably Pichia pastoris, and more preferably the m314H strain described herein or its histidine and uracil double-deficient mutant or histidine single-deficient mutant.
  • Tools well known in the art can be used to calculate the sequence identity between two or more sequences, and these tools can come from various online tools provided by NCBI.
  • Example 1 Obtainment of uracil auxotrophic Pichia pastoris CICC32806-U7 strain
  • YNB-Uracil-FOA (13.4g/L YNB, 1% glycerol, 2% agarose, 50 ⁇ g/mL uracil (Uracil), 0.75mg/mL 5-fluoroorotic acid (5-FOA)
  • YNB-Uracil-FOA 1% glycerol, 2% agarose, 50 ⁇ g/mL uracil (Uracil), 0.75mg/mL 5-fluoroorotic acid (5-FOA)
  • HIS-AF/R amplified HIS-A fragment HIS-BF/R amplified HIS-B fragment, URA3-1F/2R amplified URA3 fragment, and then ligated into pSP72 plasmid in turn to construct a good
  • the histidine knockout vector pHISA-URA3-HISB, the primer sequence is as follows:
  • HIS-A-F 5'CCGCTCGAGTCACCTCAGCCAGATCAAAGT 3'(SEQ ID NO: 10);
  • HIS-A-R 5'ACATGCATGCCTTTGGACAACTCTTTCTGCC 3'(SEQ ID NO: 11);
  • HIS-B-F 5'CGGGGTACCCCTGGTTGATAAAGTTGCAT 3'(SEQ ID NO: 12);
  • HIS-B-R 5'GGCGAGCTCAGGTGTCTTCAAAGCGACTC 3'(SEQ ID NO: 13);
  • URA3-1F ACATGCATGCCTGCAGAAATGGGGAGATAACCACC (SEQ ID NO: 14);
  • URA3-2R CGGGGTACCACTAGTGGTTTTCTGGGGGTATTTGCTG (SEQ ID NO: 15).
  • the knockout vector was linearized with XhoI and SacI, transformed into CICC32806-U7 by electroporation, and spread on the MDS screening plate containing histidine (20 ⁇ g/mL), and the single colonies that grew were transferred to YNB solids.
  • Medium YNB-HIS solid medium.
  • the histidine auxotrophic mutant cannot grow on YNB solid medium, but can grow on YNB-HIS solid medium.
  • Strains with the correct phenotype were again subjected to single colony marking on YNB solid medium and YNB-HIS solid medium, and single colony strains that could only grow on YNB-HIS solid medium were picked.
  • the histidine-deficient Pichia pastoris CICC32806-7H3 strain was obtained.
  • amino acid sequence of this PLC is:
  • PLC_F TACGTATGGTCAGCTGAGGACAAGC
  • PLC_R CCTAGGTTACCTGTCACCGTAAGTGTCGAAC
  • PLC_R CCTAGGTTACCTGTCACCGTAAGTGTCGAAC
  • the PCR product was purified by Axygen PCR product purification kit (AP-PCR-50), digested with NEB's SnaBI and AvrII restriction enzymes, and then purified by Axygen PCR product purification kit again.
  • the pPIC9K plasmid was digested with the same restriction endonuclease, and the digested product was also purified.
  • Fermentas T4 DNA ligase according to the product instructions, the PCR product digested fragments and pPIC9k vector digested fragments were ligated, the ligation was transferred into E. coli DH5 ⁇ by heat shock method, and cultured overnight on an LB plate containing ampicillin. The next day, a single clone was picked and cultured in LB liquid medium.
  • the plasmid was extracted using Axygen plasmid extraction kit and sent to Shanghai Shenggong for sequencing.
  • the correctly sequenced recombinant expression vector was digested and linearized with Bgl II restriction endonuclease, and then transformed into Pichia pastoris CICC32806-7H3, SMD1168 competence by electroporation according to the standard transformation method of Pichia pastoris (Shixuan Wu&Geoffrey J Letchworth, 2004)
  • the cells were spread on the selection medium MDS selection plate and cultured at 28°C for three days.
  • Example 4 Obtaining the m314-SPLC strain by UV mutagenesis
  • Example 5 7H3-SPLC, m314-SPLC and SMD1168-SPLC shake flask fermentation
  • Example 2 and Example 3 it can be known that the amino acid sequence of the PLC gene contained in the three strains 7H3-SPLC, m314-SPLC and SMD1168-SPLC are exactly the same and the copy number is single copy. Inoculate these three bacteria into 50 mL of BMGY medium, cultivate overnight at 30°C and 240 rpm, and collect 200 OD bacterial cells by centrifugation. Resuspend and wash the bacteria in sterile water twice, and then resuspend the bacteria in BMMY medium. 2% methanol was added to the BMMY medium, and the expression was induced at 30°C and 240 rpm. Add 0.5mL methanol to 50mL medium every 12h. After 3 days of induction, the fermentation broth was centrifuged at 8000 rpm and 4°C, and the supernatant of the fermentation broth was taken for enzyme activity determination and protein electrophoresis detection.
  • pNPPC method phospholipase enzyme activity unit Under the conditions of temperature of 37°C and pH value of 7.6, the amount of enzyme that catalyzes the release of 1 ⁇ mol phosphocholine from the substrate in 1 minute is 1 phospholipase activity unit (U).
  • reaction buffer 0.1M boric acid-sodium borate buffer (pH 7.6), 20mM pNPPC, 1% Triton-X-100, 1mM CaCl 2 .
  • the specific steps of the determination Take two clean centrifuge tubes, one of which is used as a sample tube and the other is used as a blank control tube. Add 600 ⁇ L of reaction buffer to each centrifuge tube, add 25 ⁇ L of the enzyme to be tested to the sample tube, leave the blank tube alone, put the two tubes in a constant temperature water bath at 37°C for 15min at the same time, immediately add 500 ⁇ L 0.5M sodium hydroxide solution Stop the reaction, add 25 ⁇ L of the enzyme solution to be tested to the blank tube, measure the absorbance at 405nm, and use the blank tube to correct the zero point.
  • Polyacrylamide gel electrophoresis analysis use a 0.22 ⁇ m filter membrane to filter the supernatant. After concentrating the same amount of supernatant to the same volume using a Milipore 10KDa ultrafiltration concentration tank, take the same volume of concentrated enzyme solution for polyacrylamide coagulation. Gel electrophoresis analysis.
  • the protein expression ability of the m314-SPLC strain obtained by UV mutagenesis was increased by 127% compared with that before the mutagenesis.
  • the nucleotide sequence (AOX-His) used to knock out the PLC gene is as follows:
  • the AOX-His gene fragment was amplified by the PCR method, and the m314-SPLC strain was transformed by electroporation and spread on the phospholipase screening plate containing histidine. Select the transformants without hydrolysis circles and streak them on the plates without histidine and those with histidine, and select the correct phenotype (grow on the plates with histidine, and on the plates without histidine).
  • the transformant that did not grow on the surface) was named m314H.
  • the strain m314H has been deposited at the China General Microbial Culture Collection and Management Center (CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101) on October 31, 2018, and is classified as Pichia pastoris (Pichia pastoris), the deposit number is CGMCC No. 16670.
  • Example 7 Evaluation of m314H strain using RML gene as reporter gene
  • the RML lipase gene is used to evaluate the enhancement effect of the m314H strain on the expression level of other proteins.
  • the RML gene used is codon-optimized and synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.
  • the nucleotide sequence is:
  • the amino acid sequence of RML is:
  • pNPP method lipase enzyme activity unit Under the conditions of temperature of 40°C and pH value of 8.0, the sample hydrolyzes the substrate p-nitrophenyl palmitate and releases 1 ⁇ mol of p-nitrophenol (pNP) per minute. The amount of enzyme is 1 unit of enzyme activity (U).
  • Substrate buffer Take 5.3mL 0.2mol/L NaH 2 PO 4 solution and 94.7mL 0.2mol/L Na 2 HPO 4 solution, mix and add about 280mL water, add 0.92g sodium deoxycholate, 0.44g gum arabic Stir to dissolve the powder, adjust the pH to 8.0 with H 3 PO 4 or NaOH, dilute to 400 mL, and store at 4°C.
  • Substrate pNPP solution (0.0795mol/L, 3mg/L): Weigh 0.03 g of p-nitrophenyl palmitate (pNPP), add 10 mL of isopropanol, stir to dissolve, and store at 4°C.
  • pNPP p-nitrophenyl palmitate
  • the specific steps of the determination take 1 mL of pNPP solution and 9 mL of substrate buffer solution and mix. Take two clean centrifuge tubes, one as a sample tube and one as a blank control tube. Add 600 ⁇ L of reaction buffer to each centrifuge tube, add 25 ⁇ L of the enzyme solution to be tested to the sample tube, do not add the blank tube, put the two tubes in a constant temperature water bath at 40°C for 15min at the same time, immediately add 500 ⁇ L of absolute ethanol to stop the reaction, blank Add 25 ⁇ L of the enzyme solution to be tested to the tube and centrifuge at 12000rpm for 2min. Measure the absorbance at 405nm, and use a blank tube to correct the zero point.
  • Polyacrylamide gel electrophoresis analysis use a 0.22 ⁇ m filter membrane to filter the supernatant. After concentrating the same amount of supernatant to the same volume using a Milipore 10KDa ultrafiltration concentration tank, take the same volume of concentrated enzyme solution for polyacrylamide coagulation. Gel electrophoresis analysis.
  • Example 5 Summarizing the results of Example 5 and Example 7, when the m314H strain obtained by UV mutagenesis was used to express PLC and RML, its protein expression ability was increased by 127% and 92% compared with the original strain 7H3.
  • Example 8 Overexpression of growth promoting genes in m314H strain
  • HIS-A-F/R amplified HIS-A fragment
  • HIS-B-F/R amplified HIS-B fragment
  • pSP72 plasmid ligated into pSP72 plasmid to construct histidine knockout vector pHISA-HISB.
  • the knockout vector was linearized with XhoI and SacI, transformed into m314H by electroporation, and spread on the MDS screening plate containing histidine, Uracil and 5-FOA, and the single colonies that grew were transferred to YNB-HIS. On solid medium, YNB-Uracil and YNB-Uracil-HIS solid medium.
  • the histidine and uracil double auxotrophic transformant can only grow on the YNB-Uracil-HIS solid medium as a single colony strain.
  • the histidine and uracil double auxotrophic Pichia m314HU strain was finally obtained.
  • the URA3 fragment was amplified with primers URA3-1F/2R.
  • the synthesis of Protein required general stress response (Genbank no: XM_002491428.1), Mitochondrial external NADH dehydrogenase (Genbank no: XM_002490375.1), Proteinase A(Genbank no: XM_002493288.1)
  • Three growth-promoting gene fragments, and then these three gene fragments and URA3 fragments were sequentially ligated into the pSP72 plasmid to construct the overexpression vectors pURA3-Stress, pURA3-NADH and pURA3-ProA.
  • the strain m315H has been deposited in the China General Microbial Culture Collection and Management Center (CGMCC, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101) on October 31, 2018, and is classified as Pichia pastoris (Pichia pastoris), the deposit number is CGMCC No. 16669.
  • CGMCC China General Microbial Culture Collection and Management Center
  • Example 9 Evaluation of m315H strain using TL gene as reporter gene
  • the TL lipase gene is used to evaluate the enhancement effect of the m315H strain on the expression level of other proteins.
  • the TL gene used is codon-optimized and synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.
  • the nucleotide sequence is as follows:
  • the TL amino acid sequence is:
  • the primers were designed according to the synthesized TL gene sequence to construct pPIC9K-TL expression vector. After linearization with restriction enzyme BglII, the 7H3, m314H and m315H strains were transformed by electroshock respectively. After screening by lipase plate, they were selected to have hydrolysis circle and the TL gene was Single-copy transformants, numbered 7H3-STL, m314-STL and m315-STL, were selected for shake flask fermentation. After the fermentation broth is concentrated by ultrafiltration, the same volume of concentrated enzyme solution is taken, and the lipase activity determination and polyacrylamide gel electrophoresis analysis are carried out by the pNPP method lipase determination method.
  • Polyacrylamide gel electrophoresis analysis use a 0.22 ⁇ m filter membrane to filter the supernatant. After concentrating the same amount of supernatant to the same volume using a Milipore 10KDa ultrafiltration concentration tank, take the same volume of concentrated enzyme solution for polyacrylamide coagulation. Gel electrophoresis analysis.
  • Example 7 and Example 9 when m314H obtained by UV mutagenesis of strain 7H3 was used to express PLC, RML and TL, its protein expression ability was increased by 127% compared with the original strain 7H3. , 92% and 86%, which proved that the ability of the strain m314H to express protein exogenously was significantly higher than that of the original strain 7H3, and it has excellent characteristics; 3 kinds of growth promoting genes were overexpressed in m314H, and the strain m315H was constructed, and it was found to express TL At this time, the protein expression level of m314H was increased by 39%, and the protein expression level of the strain was increased again.
  • the URA3 fragment was amplified with primers URA3-1F/2R; the vacuolar protease A (Genbank no: XM_002493288.1) gene fragment was synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd., and the primer proAF: CGAGTTTCTCCGTATCTAAT And proAR: TCCTCATCTATACCCCAGG amplifies the Proteinase A gene fragment.
  • the expanded URA3 fragment and the Proteinase A fragment were co-transformed into the m314HU obtained in Example 8 by electric shock.
  • the electrotransformation material was spread on the MDS screening plate containing histidine, and the successful transfer of all transformed genes was verified by PCR to obtain the m316H strain.
  • the strain m316H has been deposited at the China General Microbial Culture Collection and Management Center (CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101), and is classified as Pichia pastoris (Pichia pastoris) , The deposit number is CGMCC No.19221.
  • Example 11 Evaluation of m316H strain using TL gene as reporter gene
  • the pPIC9K-TL expression vector constructed in Example 9 was linearized by restriction enzyme BglII and then electroporated to transform m316H strain. After lipase plate screening, the transformant with hydrolysis circle and single copy of TL gene was selected, numbered m316 -STL, compare the selected transformants with the 7H3-STL, m314-STL and m315-STL obtained in Example 9 in shake flask fermentation. After the fermentation broth is concentrated by ultrafiltration, the same volume of concentrated enzyme solution is taken, and the lipase activity determination and polyacrylamide gel electrophoresis analysis are carried out by the pNPP method lipase determination method.
  • Polyacrylamide gel electrophoresis analysis use a 0.22 ⁇ m filter membrane to filter the supernatant. After concentrating the same amount of supernatant to the same volume using a Milipore 10KDa ultrafiltration concentration tank, take the same volume of concentrated enzyme solution for polyacrylamide coagulation. Gel electrophoresis analysis.
  • Example 12 Genome sequencing of the Pichia yeast strain of the present invention and comparison with the existing Pichia yeast genome
  • the 31806, m314, m315 and m316 strains mentioned in the present invention were submitted to Suzhou Jinweizhi Biotechnology Co., Ltd. for genome sequencing. After Jin Weizhi extracts the strain's genome, it is interrupted and filled in, and the 3'end is added with an A and then connected to the linker containing the Index sequence.
  • the sequencing library constructed according to this strategy is bridged to the sequencing chip and then subjected to Illumina HiSeq sequencing. After re-sequencing off-machine data is processed, the original data is obtained, filtered to remove joints, decontaminated, and then compared with the reference genome.
  • the repetitive sequences caused by PCR amplification in each library were removed, and then the sequencing depth and coverage relative to the reference genome, single nucleotide variation (SNV), and insertion/deletion (InDels )Wait.
  • SNV single nucleotide variation
  • InDels insertion/deletion
  • some standard biological information analysis such as mutation annotation and functional enrichment can also be performed.
  • the coverage rate is 99.99% and above, the average depth is 400 ⁇ and above, and the depth distribution is more than 200 ⁇ , accounting for more than 99.95%.
  • the starting strains CICC32806 and GS115 of the present invention After genome sequencing and comparison with the existing Pichia pastoris genome, the starting strains CICC32806 and GS115 of the present invention have 1125 SNV events and 830 INDEL event reports in the genome. Obviously, the starting strains of the present invention are significantly different from GS115.
  • the genomes of the m314H, m315H and m316H strains of the present invention and the GS115 or CICC32806 strains have at least the following changes in their genomes: BQ9382_C1-2260, 308-310 EKK deletion, hypothetical protein; BQ9382_C1-3800, E129K, 60S ribosomal sub Base assembly/export protein LOC1; BQ9382_C1-5700, I312M, NADH dehydrogenase outside the mitochondria, class II NAD(P)H: quinone oxidoreductase; BQ9382_C2-3950, Q145X, essential protein, with binding partner Psr1p, for complete Activate the general stress response; BQ9382_C3-2220, E188K, hypothetical protein; and BQ9382_C3-4370, W196X, Orotidine 5'-phosphate decarboxylase.

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Abstract

提供了一种表达外源基因的毕赤酵母突变株。具体而言，提供了一株相对于毕赤酵母菌株GS115或CICC32806，其包含以下6个突变中的一个或多个：BQ9382_C1-2260，308-310位EKK缺失，假设蛋白；BQ9382_C1-3800，E129K，60S核糖体亚基组装/输出蛋白LOC1；BQ9382_C1-5700，I312M，线粒体外NADH脱氢酶，II类NAD(P)H:醌氧化还原酶；BQ9382_C2-3950，Q145X，必需蛋白质，具有结合伴侣Psr1p，用于完全激活一般压力反应；BQ9382_C3-2220，E188K，假设蛋白；和BQ9382_C3-4370，W196X，奥罗替丁5\'-磷酸脱羧酶。所提供的毕赤酵母突变株是有效的外源表达通用宿主，可高效表达各种蛋白，尤其是磷脂酶和脂肪酶。

Description

用于表达外源基因的毕赤酵母突变株 技术领域
本发明涉及用于表达外源基因的毕赤酵母突变株。
背景技术
巴斯德毕赤酵母表达系统是20世纪80年代初期发展起来的一种新型的外源蛋白表达系统。由于它既具有原核表达系统操作简易、易于培养、生长速度快、表达量高、成本低等优点，还具有原核生物表达系统所不具有的外源蛋白的翻译后修饰等特点，如糖基化、蛋白磷酸化等。同时它还避免了酿酒酵母(Saccharomyces cerevisiae)分泌效率差、表达菌株不够稳定、表达质粒易丢失等缺陷，所以该表达系统很快成为目前最优秀、应用最为广泛的外源基因表达系统之一。
Koichi Ogata等人于1969年首次发现了某些酵母可以利用甲醇为唯一碳源和能源生长(Ogata,et a1.1969)，此后，用甲醇利用型酵母生产单细胞蛋白作为动物饲料的潜力就引起了广泛关注。1987年，Cregg等人首次报道了用甲醇营养型酵母表达乙型肝炎表面抗原(HbsAg)，随后Philip Petroleum公司与Salk Institute Biotechnology/Industrial Associates(SIBIA)就开始了毕赤酵母表达系统的合作开发。SIBIA的研究人员分离了AOX基因的启动子和宿主菌株，构建了载体，并开发出了相应的毕赤酵母基因操作技术，结合Philip Petroleum公司生产单细胞蛋白的发酵工艺，实现了外源蛋白的高效表达。1993年，Philip Petroleum公司将毕赤酵母表达系统的专利卖给Research Corporation Technologies(RCT)公司。
RCT公司的基础菌株GS115来自化学法诱变原始菌NRRL-Y 11430(ATCC 76273)，使之成为组氨酸营养缺陷型宿主(His-)，便于进行克隆筛选。GS115醇氧化酶基因AOX1完整，已经能够很好地利用甲醇表达大多数外源蛋白，但其本底AOX1依然会高效表达，因而某些基因的产量受到影响。后续毕赤酵母衍生菌株方向之一就是在GS115基础上将AOX1基因敲除，替换成酿酒酵母ARG4基因，得到KM71(his4 arg4 aox1Δ::ARG4)，其AOX2基因保持完整，因此甲醇利用率很低，在甲醇唯一碳源的培养下生长很慢。进一步敲除AOX2基因，得到了无法利用甲醇的宿主MC100-3(his4 arg4 aox1Δ::SARG4aox2Δ::Phis4)。在GS115基础上改造的另一个方向是失活宿主蛋白酶。敲除毕赤酵母液泡蛋白酶B(Proteinase B，prb1)，得到SMD1165(his4 prb1)。或敲除液泡天冬氨酸蛋白酶(PEP4)得到SMD1168(his4 pep4)，该蛋白酶用于激活其他 液泡蛋白酶，包括羧肽酶Y(carboxypeptidase Y)和蛋白酶B。然后在SMD1168基础上进一步敲除液泡蛋白酶B(Proteinase B，prb1)，得到SMD1163(his4 pep4 prb1)。即PEP4蛋白酶比较关键，用于一些蛋白酶的活化，必要时可进一步敲除液泡蛋白酶prb1和羧肽酶。
发明内容
本发明改造毕赤酵母CICC32806，得到可用于高效外源表达各种蛋白，尤其是磷脂酶、脂肪酶的毕赤酵母菌。
本发明的第一方面是提供一种毕赤酵母菌株，相对于毕赤酵母菌株GS115或CICC32806，其包含以下6个突变中的一个或多个：BQ9382_C1-2260，308-310位EKK缺失，假设蛋白；BQ9382_C1-3800，E129K，60S核糖体亚基组装/输出蛋白LOC1；BQ9382_C1-5700，I312M，线粒体外NADH脱氢酶，II类NAD(P)H:醌氧化还原酶；BQ9382_C2-3950，Q145X，必需蛋白质，具有结合伴侣Psr1p，用于完全激活一般压力反应；BQ9382_C3-2220，E188K，假设蛋白；和BQ9382_C3-4370，W196X，奥罗替丁5\'-磷酸脱羧酶。
在一个或多个实施方案中，所述菌株包括如上所述6个突变。
所述毕赤酵母菌株为保藏编号为CGMCC No.16670的巴斯德毕赤酵母(Pichia pastoris)菌株，保藏编号为CGMCC No.16669的巴斯德毕赤酵母(Pichia pastoris)菌株，保藏编号为CGMCC No.19221的巴斯德毕赤酵母(Pichia pastoris)菌株。
在一个或多个实施方案中，本发明提供的毕赤酵母菌株为组氨酸和尿嘧啶双缺陷型菌株。
在一个或多个实施方案中，本发明提供保藏编号为CGMCC No.16670的巴斯德毕赤酵母(Pichia pastoris)菌株。
本发明还提供一种基因工程改造的毕赤酵母菌株，该毕赤酵母菌株为经基因工程改造的保藏编号为CGMCC No.16670巴斯德毕赤酵母菌株，且(a)为组氨酸缺陷型菌株；和/或(b)含有表达生长促进因子的质粒、和/或基因组中整合有生长促进因子的编码序列、和/或表达生长促进因子。
在一个或多个实施方案中，所述基因工程改造的毕赤酵母菌株为保藏编号为CGMCC No.16669的巴斯德毕赤酵母菌株。
前述任一实施方案所述的毕赤酵母菌株可作为基础菌株，经基因工程改造而用于表 达感兴趣的外源基因。因此，本发明还提供一种基因工程改造的毕赤酵母菌株，该菌株为经基因工程改造而包含外源基因或含该外源基因的载体的前述任一实施方案所述的毕赤酵母菌株，包括经基因工程改造而含有外源基因或含该外源基因的载体的CGMCC No.16670的巴斯德毕赤酵母(Pichia pastoris)菌株、所述组氨酸和尿嘧啶双缺陷型菌株、所述组氨酸单缺陷型菌株和/或含有表达生长促进因子的质粒、基因组中整合有生长促进因子的编码序列和/或表达生长促进因子的基因工程改造的毕赤酵母菌株、以及保藏编号为CGMCC No.16669的巴斯德毕赤酵母菌株。应理解的是，此处所述的外源基因不包括编码所述生长促进因子的基因，可通过基因工程改造而在含该生长促进因子的表达基因的基础上进一步含有任何感兴趣的其它外源基因。
在一个或多个实施方案中，所述菌株的基因组中整合了所述外源基因。
在一个或多个实施方案中，所述外源基因为工业、饲料或食品领域中用到的蛋白的编码序列。
在一个或多个实施方案中，所述外源基因为酶的编码序列，优选地，所述酶选自以下酶中的至少一种：脂肪酶、蛋白酶、纤维素酶、淀粉酶、植酸酶、酯酶、果胶酶、半乳糖苷酶和磷脂酶。
本发明还提供一种培养物，所述培养物含有本发明任一实施方案所述的毕赤酵母菌株和任选的培养基。
在一个或多个实施方案中，所述培养基为种子培养基或发酵培养基。
在一个或多个实施方案中，所述培养基为YPD培养基或BMMY培养基。
本发明还提供一种酶制品，所述酶制品含有本发明任一实施方案所述的其外源基因为酶的编码序列的毕赤酵母菌的发酵液或发酵所得细胞的裂解液，或所述发酵液或裂解液的浓缩物。
本发明还提供本发明所述酶制品在酯交换中的应用，其中，该酶制品含有本发明任一实施方案所述的其外源基因为脂肪酶的编码序列的毕赤酵母菌的发酵液或发酵所得细胞的裂解液，或所述发酵液或裂解液的浓缩物。
本发明还提供本发明所述酶制品在油脂脱胶中的应用，其中，该酶制品含有本发明任一实施方案所述的其外源基因为磷脂酶(尤其是磷脂酶C)的编码序列的毕赤酵母菌的发酵液或发酵所得细胞的裂解液，或所述发酵液或裂解液的浓缩物。
在一个或多个实施方案中，所述脂肪酶的氨基酸序列为与SEQ ID NO:7或9具有至少80％、90％、95％、98％或99％同一性的氨基酸序列，更优选地，所述脂肪酶的氨基酸序列如SEQ ID NO:7或9所示。
在一个或多个实施方案中，所述磷脂酶的氨基酸序列为与SEQ ID NO:2具有至少80％、90％、95％、98％或99％同一性的氨基酸序列，更优选地，所述磷脂酶的氨基酸序列如SEQ ID NO:2所示。
本发明提供一种制备组氨酸和尿嘧啶双缺陷型的毕赤酵母菌株的方法，所述方法包括：
(1)对毕赤酵母进行诱变，筛选获得尿嘧啶营养缺陷型突变体；
(2)敲除步骤(1)获得的尿嘧啶营养缺陷型突变体中的HIS4基因，筛选获得组氨酸缺陷型突变体；
(3)在步骤(2)获得的组氨酸缺陷型突变体中敲入外源基因和HIS4基因，筛选获得表达该外源基因的突变体；
(4)对步骤(3)获得的突变体实施诱变，筛选获得步骤(3)中敲入的外源基因未发生突变、但相对而言该外源基因表达量较高或其表达产物活性较高的突变体；和
(5)敲除步骤(4)获得的突变体中步骤(3)敲入的HIS4基因，筛选获得组氨酸缺陷型突变体；和任选的，
(6)敲除步骤(5)获得的组氨酸缺陷型突变体中的URA3基因，筛选获得组氨酸和尿嘧啶双突变型突变体。
本发明还提供一种制备用于外源基因表达的毕赤酵母菌株的方法，所述方法包括：
(1)对毕赤酵母进行诱变，筛选获得尿嘧啶营养缺陷型突变体；
(2)敲除步骤(1)获得的尿嘧啶营养缺陷型突变体中的HIS4基因，筛选获得组氨酸缺陷型突变体；
(3)在步骤(2)获得的组氨酸缺陷型突变体中敲入外源基因和HIS4基因，筛选获得表达该外源基因的突变体；
(4)对步骤(3)获得的突变体实施诱变，筛选获得步骤(3)中敲入的外源基因未发生突变、但相对而言该外源基因表达量较高或其表达产物活性较高的突变体；和
(5)敲除步骤(4)获得的突变体中步骤(3)敲入的HIS4基因，筛选获得组氨酸缺陷型突变体；和任选的，
(6)敲除步骤(5)获得的组氨酸缺陷型突变体中的URA3基因，筛选获得组氨酸和尿嘧啶双突变型突变体；
(7)在步骤(6)获得的双突变体中敲入生长促进基因和URA3基因，筛选获得组氨酸缺陷型突变体；和
(8)在步骤(6)获得的双突变体中敲入液泡蛋白酶A基因和URA3基因，筛选 获得组氨酸缺陷型突变体。
本发明还提供本文所述的巴斯德毕赤酵母菌株在构建表达外源基因的菌株中的应用。
本发明的毕赤酵母突变株是有效的外源表达通用宿主，可高效表达各种蛋白，尤其是磷脂酶和脂肪酶。
附图说明
图1：m314-SPLC表达PLC的PLC酶活对比图。
图2：m314-SPLC表达PLC的对比蛋白电泳图。
图3：m314H表达RML的酶活对比图。
图4：m314H表达RML的对比蛋白电泳图。
图5：m315H表达TL的酶活对比图。
图6：m315H表达TL的对比蛋白电泳图。
图7：m316H表达TL的酶活对比图。
图8：m316H表达TL的对比蛋白电泳图。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
定义：
本发明所使用的术语“基因工程”又称基因拼接技术和DNA重组技术，是以分子遗传学为理论基础，以分子生物学和微生物学的现代方法为手段，将不同来源的基因按预先设计的蓝图，在体外构建杂种DNA分子，然后导入活细胞，以改变生物原有的遗传特性、获得新品种、生产新产品。
本发明所使用的术语“诱变”具有本领域的一般含义，是指人为的措施诱导菌株的遗传基因产生变异，然后在产生变异的菌株中按照需要选育出新的优良品种。诱变育种常用的有物理因素和化学因素，物理因素如各种射线、微波或激光等处理诱变材料，习惯上称之为辐射育种；化学因素是运用能导至遗传物质改变的一些化学药物——诱变剂处理诱变材料促使变异，常称之为化学诱变。
本发明所使用的术语“突变体”是指衍生自酵母菌株CICC32806且在一个或多个位置处包含修饰或改变，即取代、插入和/或缺失的毕赤酵母菌株。可以通过本领域熟知 的各种技术获得毕赤酵母菌株突变体。特别地，用于改变酵母菌株CICC32806序列的技术的实例包括但不限于定点诱变、随机诱变和基因工程。
本发明所使用的术语“同源性”和“同一性”描述了两个或更多个氨基酸序列之间的相似性程度。通过以下方式来确定两个序列之间的“序列同一性”的百分比：在比较窗口上比较两个最佳对齐的序列，使得比较窗口中的序列的部分可以相较于参考序列(其不包含添加或缺失)包含添加或缺失(缺口)以进行两个序列的最佳比对。通过以下方式计算百分比：确定在两个序列中出现相同氨基酸残基的位置的数目，以得到匹配位置的数目，将匹配位置的数目除以比较窗口中的位置总数，并将结果乘以100，得到序列同一性的百分比。与参考序列相比在每个位置相同的序列被认为与参考序列相同，反之亦然。
本发明所使用的术语“基因敲除”是指用含有一定已知序列的DNA片段与受体细胞基因组中序列相同或相近的基因发生同源重组，整合至受体细胞基因组中并得到表达的一种外源DNA导入技术。它针对某个序列已知但功能未知的序列，改变生物的遗传基因，令特定的基因功能丧失作用，从而使部分功能被屏蔽，并可进一步对生物体造成影响，进而推测出该基因的生物学功能。
本发明通过对毕赤酵母进行诱变，然后筛选出营养缺陷型突变体，从而制备得到用于外源基因表达的毕赤酵母菌株。具体而言，本发明以购自中国工业微生物菌种保藏管理中心(简称CICC)的CICC32806为初始菌株，经过紫外诱变后，筛选获得尿嘧啶营养缺陷型菌株U7。再通过基因敲除的方式，将U7菌株中的HIS4基因进行失活，获得了组氨酸营养缺陷型菌株7H3。然后在7H3中转入PLC编码序列，筛选获得重组毕赤酵母7H3-SPLC。本发明进一步对7H3-SPLC实施诱变，筛选得到突变体m314-SPLC。通过敲除掉m314-SPLC中的PLC基因和HIS4基因，获得了m314H菌株。进一步地，可构建组氨酸敲除载体，转入本发明的菌株m314H中，在含有YNB和组氨酸的培养基、含有YNB和尿嘧啶的培养基以及含有YNB、组氨酸和尿嘧啶的培养基中进行筛选，筛选到只能在含YNB、组氨酸和尿嘧啶的培养基中生长的菌株，即为组氨酸和尿嘧啶双营养缺陷型菌株，命名为菌株m314HU。进一步地，本发明还在m314HU菌株中过表达3种生长促进基因并敲入URA3基因，构建得到菌株m315H。另外，本发明在m314HU菌株中过表达液泡蛋白酶A基因并敲入URA3基因，构建得到菌株m316H。
经苏州金维智生物科技有限公司进行全基因测序。金维智提取菌株基因组后，打断并补平，3’末端加A后与含Index序列的接头连接，按照此策略构建的测序文库，桥式结合到测序芯片上后，进行Illumina HiSeq测序。重测序下机数据经过处理后获得 原始数据，经过过滤去接头、去污染、然后与参考基因组比对。通过比对结果，去除出每个文库中由于PCR扩增引起的重复序列，然后计算出相对于参考基因组的测序深度和覆盖度、单核苷酸位点变异(SNV)、插入/缺失(InDels)等。对于有参考基因集、SNV数据集，还可以进行突变注释和功能富集等一些标准生物信息分析。覆盖率99.99％及以上，平均深度400×及以上且其中深度分布200×以上占99.95％以上。通过测序，在M314H、M315H、M316H中，相对于GS115或CICC32806，至少包含以下6个位点差别，上述位点所在基因编码序列及基因功能注释如下：
BQ9382_C1-2260，308EKKdel，假设蛋白。其中，BQ9382_C1-2260为GENEBANK给出的编号，其中，C1：第1个染色体，2260是基因序号，其中308-310位的EKK缺失。
BQ9382_C1-3800，E129K，60S核糖体亚基组装/输出蛋白LOC1。BQ9382_C1-3800为GENEBANK给出的编号，其中，C1：第1个染色体，3800是基因序号，129位的E突变为K。
BQ9382_C1-5700，I312M，线粒体外NADH脱氢酶,II类NAD(P)H:醌氧化还原酶。BQ9382_C1-5700为GENEBANK给出的编号，其中，C1：第1个染色体，5700是基因序号，312位的I突变为M。
BQ9382_C2-3950,Q145X,必需蛋白质，具有结合伴侣Psr1p，用于完全激活一般压力反应。BQ9382_C2-3950为GENEBANK给出的编号，其中，C2：第2个染色体，3950是基因序号，145位的Q变成终止密码子。
BQ9382_C3-2220,E188K,假设蛋白。BQ9382_C3-2220为GENEBANK给出的编号，其中，C3：第3个染色体，2220是基因序号。
BQ9382_C3-4370,W196x,奥罗替丁5\'-磷酸脱羧酶。BQ9382_C3-4370为GENEBANK给出的编号，其中，C3：第3个染色体，4370是基因序号,196位W突变为终止密码子。本领域普通技术人员可通过包括但不限于同源重组、基因敲除及回补、锌指核酸酶、TALE核酸酶、Crisp/Cas9等策略，将本发明所公开的上述6个突变的一个或多个，引入到感兴趣的毕赤酵母菌株中。
本发明中，诱变可以是物理诱变或化学诱变。物理诱变包括紫外诱变，如将毕赤酵母菌株置于紫外光下一段时间，例如60～120秒。化学诱变包括使毕赤酵母菌株与化学诱变剂如亚硝基胍接触一段时间，例如15～60分钟。
本发明中，可利用含有尿嘧啶和5-氟乳清酸(5-FOA)的培养基，如含YNB(无氨基酵母氮源)的培养基培养诱变的菌株，可筛选获得尿嘧啶缺陷型菌株。例如，在某些实施方案中，可将诱变的菌株培养于含有YNB、甘油、琼脂糖、尿嘧啶和5-氟乳清酸的培养基上，25-33℃避光培养3-8天，然后挑出在该培养基中长出来的单菌落，转接到 含YNB和尿嘧啶的培养基(如含有YNB、甘油、琼脂糖和尿嘧啶)上，挑取只能在此培养基上生长的菌株，即可获得尿嘧啶缺陷型菌株。这些培养基中，YNB的浓度可为10-20g/L，甘油含量可为0.5-2％，琼脂糖含量可为1-3％，尿嘧啶浓度可为30-100μg/mL；含有时，5-FOA浓度可为0.5-1.2mg/mL。
在某些实施方案中，本发明以保藏编号为CICC32806的毕赤酵母为初始菌株进行诱变。因此，在这些实施方案中，所述尿嘧啶缺陷型菌株是保藏编号为CICC32806的毕赤酵母经诱变和尿嘧啶营养缺陷型筛选后获得的菌株。
获得的尿嘧啶缺陷型菌株可经基因敲除法敲除其组氨酸脱氢酶基因(HIS4)，并在含有组氨酸的培养基中进行筛选，筛选出能在含组氨酸的培养基中生长的菌株，即为组氨酸缺陷型菌株。通常，使转入了组氨酸敲除载体的尿嘧啶缺陷型菌株在含有组氨酸(如10-50微克/mL)的MDS筛选平板中培养，将获得的单菌落分别接种到含YNB的培养基和含YNB及组氨酸的培养基中，通过对比可找出在含组氨酸的培养基中能生长但在不含组氨酸的培养基中不能生长的菌株，由此可筛选获得组氨酸缺陷型菌株。
在本发明的某些实施方案中，经诱变和组氨酸营养缺陷型筛选后获得的菌株，转入了表达PLC的表达载体，并筛选得到磷脂平板上培养时水解圈较大、且转入的用于表达PLC的核酸序列(如AOX启动子、信号肽、PLC基因、转录终止子等)未发生突变的突变子7H3-SPLC。通常，在转入PLC的表达载体时，如同时也转入了HIS4基因，由此构建得到的菌株不是组氨酸营养缺陷型菌株。
在进一步的实施方案中，可对该突变子实施进一步的物理诱变，如紫外照射，筛选出磷脂平板培养中水解圈较大、且转入该用于表达PLC的核酸序列(如AOX启动子、信号肽、PLC基因、转录终止子等)未发生突变的突变子m314-SPLC。
可利用常规的技术敲除该突变子m314-SPLC中先前转入的外源基因，包括PLC基因和标记基因如HIS4基因。例如，构建AOX-His基因片段，电转突变子m314-SPLC，在含组氨酸的磷脂酶筛选平板上培养，挑选出没有水解圈的转化子，分别在不含组氨酸和含有组氨酸的平板上划线，选择表型正确(在含有组氨酸的平板上生长，而在不含组氨酸的平板上不生长)的转化子，可获得本发明的菌株m314H。应理解的是，在构建此菌株过程中所获得的各种突变子，如前文所述的突变子也包括在本发明的保护范围之内。
本发明的菌株m314H已于2018年10月31日保藏于中国普通微生物菌种保藏管理中心(CGMCC，北京市朝阳区北辰西路1号院3号，100101)，分类命名为巴斯德毕赤酵母(Pichia pastoris)，保藏编号为CGMCC No.16670。
在某些实施方案中，本发明还包括组氨酸和尿嘧啶双营养缺陷型菌株。具体而言， 可构建组氨酸敲除载体，转入本发明的菌株m314H中，在含有YNB和组氨酸的培养基、含有YNB和尿嘧啶的培养基以及含有YNB、组氨酸和尿嘧啶的培养基中进行筛选，筛选到只能在含YNB、组氨酸和尿嘧啶的培养基中生长的菌株，即为组氨酸和尿嘧啶双营养缺陷型菌株，命名为菌株m314HU。
可在菌株m314HU中转入生长促进基因并敲入URA3基因，构建表达生长促进基因的菌株。本文中，生长促进基因优选为来源于酵母自身的生长促进基因，包括但不限于Protein required general stress response(Genbank no:XM_002491428.1)、Mitochondrial external NADH脱氢酶(Genbank no:XM_002490375.1)、液泡蛋白酶A(Genbank no:XM_002493288.1)。可转入一种或多种所述生长促进基因。本发明也包括成功转入生长促进基因的单组氨酸缺陷型菌株。在某些实施方案中，本发明获得的所述单组氨酸缺陷型菌株在本文中命名为菌株m315H。
本发明的菌株m315H已于2018年10月31日保藏于中国普通微生物菌种保藏管理中心(CGMCC，北京市朝阳区北辰西路1号院3号，100101)，分类命名为巴斯德毕赤酵母(Pichia pastoris)，保藏编号为CGMCC No.16669。
在菌株m314HU中转入液泡蛋白酶A(Genbank no:XM_002493288.1)并敲入URA3基因，构建过表达液泡蛋白酶A基因的单组氨酸缺陷型菌株。在某些实施方案中，本发明获得的所述单组氨酸缺陷型菌株在本文中命名为菌株m316H。
本发明的菌株m316H已于2019年月日保藏于中国普通微生物菌种保藏管理中心(CGMCC，北京市朝阳区北辰西路1号院3号，100101)，分类命名为巴斯德毕赤酵母(Pichia pastoris)，保藏编号为CGMCC No.19221。
本发明的营养缺陷型毕赤酵母，尤其是菌株m314H、m314HU、m315H和m316H，可作为基础菌株，用做构建表达外源基因的宿主菌株。本文中，外源基因指从外部转入宿主菌株的具有感兴趣功能的基因，不论该基因是来自其它物种还是本身存在于该宿主基因组中。外源基因可以是编码任何感兴趣蛋白的基因。所述感兴趣蛋白包括但不限于工业、饲料或食品等领域中用到的各种蛋白，包括但不限于各种脂肪酶、蛋白酶、纤维素酶、淀粉酶、植酸酶、酯酶、果胶酶、半乳糖苷酶和磷脂酶。尤其是，在某些实施方案中，可利用本文所述的组氨酸营养缺陷型毕赤酵母构建表达外源磷脂酶C的菌株。优选地，所述磷脂酶的氨基酸序列为与SEQ ID NO:2具有至少80％、90％、95％、98％或99％同一性的氨基酸序列；或者为在高严格条件下与SEQ ID NO:1、其cDNA序列或其全长互补体杂交的多核苷酸编码的氨基酸。在某些实施方案中，所述磷脂酶C的氨基酸序列如SEQ ID NO:2所示，优选地，其编码序列如SEQ ID NO:1所示。在某些实施方 案中，可利用本文所述的组氨酸营养缺陷型毕赤酵母构建表达外源脂肪酶的菌株。优选地，所述脂肪酶的氨基酸序列为与SEQ ID NO:7或9具有至少80％、90％、95％、98％或99％同一性的氨基酸序列；或者为在高严格条件下与SEQ ID NO:6或8、其cDNA序列或其全长互补体杂交的多核苷酸编码的氨基酸序列。术语“高严格条件”指在该条件下形成所谓的特异性杂交，并且不形成非特异性杂交。高严格条件的例子包括典型的Southern杂交的洗涤条件，即，在对应于1x SSC,0.1％SDS在60℃，优选0.1x SSC,0.1％SDS在60℃，更优选0.1x SSC,0.1％SDS在68℃的盐浓度和温度下洗涤一次，优选两次或三次。在某些实施方案中，所述脂肪酶的氨基酸序列如SEQ ID NO:7或9所述，其编码序列优选如SEQ ID NO:6或8所示。或者，在某些实施方案中，可用本发明的菌株表达其氨基酸序列与SEQ ID NO:2、7或9相比具有一个或多个(例如10个以内)氨基酸残基突变的磷脂酶C或脂肪酶，包括一个或者多个氨基酸残基的取代、插入或缺失。优选地，上述一个或多个氨基酸残疾的取代、插入或缺失为是保持蛋白质正常功能(即所述突变的磷脂酶C或者脂肪酶的活性基本不发生变化)的保守突变。保守突变的典型例子是保守取代，其中如果取代位点是芳香族氨基酸，取代在Phe,Trp,和Tyr之间相互发生；如果其是疏水性氨基酸，取代发生在Leu,Ile,和Val之间；如果其是极性氨基酸，取代发生在Gln和Asn之间；如果其是碱性氨基酸，发生在Lys,Arg,和His之间；如果其是酸性氨基酸，发生在Asp和Glu之间；以及如果其是具有羟基的氨基酸，取代发生在Ser和Thr之间。认为是保守取代的取代的实例特别包括用Ser或Thr取代Ala、用Gln，His或Lys取代Arg、用Glu，Gln，Lys，His或Asp取代Asn、用Asn，Glu或Gln取代Asp、用Ser或Ala取代Cys、用Asn，Glu，Lys，His，Asp或Arg取代Gln、用Gly，Asn，Gln，Lys或Asp取代Glu、用Pro取代Gly、Asn，Lys，Gln，Arg或Tyr取代His、用Leu，Met，Val或Phe取代Ile、用Ile，Met，Val或Phe取代Leu、用Asn，Glu，Gln，His或Arg取代Lys、用Ile，Leu，Val或Phe取代Met、用Trp，Tyr，Met，Ile或Leu取代Phe、用Thr或Ala取代Ser、用Ser或Ala取代Thr、用Phe或Tyr取代Trp、用His，Phe或Trp取代Tyr，以及用Met,Ile,或Leu取代Met。此外，上述此类氨基酸残基取代，缺失，插入或添加包括由于个体差异，或基因来源于的细菌的物种差异所带来的天然存在的突变(突变体或变体)。
可利用本领域常规的用于在毕赤酵母中表达外源基因的骨架载体构建适用于本发明的表达载体，用于转化本文所述的组氨酸营养缺陷型毕赤酵母。这类骨架载体包括但不限于pPIC3、pPIC9、pPIC9k、pHIL-D1、pAO804、pAO815和pPSC3K等。典型的毕赤酵母表达载体包含醇氧化酶-1(AOX1)基因的启动子和转录终止子(5'AOX1和 AOXTT)，它们被多克隆位点(MCS)分开，外源基因可以在此插入。这类载体还可包含组氨醇脱氢酶基因(HIS4)选择标记及3'AOX1区。当将这类载体转化毕赤酵母时，载体的5'AOX1、AOXTT、3'AOX1以及HIS4能单独或一起与染色体上的同源基因重组，从而使整个载体连同待表达的外源基因插入到受体菌染色体上，外源基因在5'AOX1启动子控制下表达。本领域研究者熟知的，AOX1启动子可被替代，合适的启动子包括但不限于诱导型、组成型启动子。
载体的构建方法为本领域所熟知。例如，PCR扩增获得目的基因后，对PCR产物以及骨架载体进行相应的限制性内切酶酶切，利用DNA连接酶将PCR产物的酶切片段与载体的酶切片段连接，将连接物转入大肠杆菌中，在合适的培养基中培养后，利用市售的质粒提取试剂盒提取获得用于转化本文所述组氨酸营养缺陷型毕赤酵母的质粒。
毕赤酵母的转化方法也为本领域所熟知。例如，用限制性内切酶酶切构建得到的表达载体，获得线性化的载体。然后，可按照标准的转化方法(Shixuan Wu&Geoffrey J Letchworth，2004)，电击转化毕赤酵母的感受态细胞，然后涂布到合适的平板(如MDS筛选平板)上培养数日。之后再挑取转化子至合适的平板上，根据其所表达的外源蛋白的生物学活性来挑选所需的重组菌株。例如，在某些实施方案中，所述外源蛋白是磷脂酶(如磷脂酶C)，则将在磷脂平板上培养转化子。通常，磷脂平板含有1～3％YNB、1～3％磷脂和1～3％琼脂。由于磷脂酶能水解磷脂，因此，可依据水解圈的大小确定转化子表达的磷脂酶的活性。挑取水解圈相对较大的转化子，即可获得性能优异的组氨酸营养缺陷型毕赤酵母。
因此，在某些实施方案中，本发明还包括包含待表达的外源基因的组氨酸营养缺陷型毕赤酵母。该外源基因通常整合到该毕赤酵母的基因组中。含有该外源基因的组氨酸营养缺陷型毕赤酵母能稳定地表达所述外源基因。在某些实施方案中，所述外源基因是磷脂酶或脂肪酶的编码序列。在某些实施方案中，所述外源基因是磷脂酶C、RML脂肪酶或TL脂肪酶的编码序列。在某些实施方案中，所述组氨酸营养缺陷型毕赤酵母转入了利用pPIC9构建的含外源基因的表达载体。在某些实施方案中，利用pPIC9构建的含外源基因的表达载体含有SEQ ID NO:1、6或8所示的核苷酸序列。
可采用本领域常规的培养基和方法培养本文的组氨酸营养缺陷型毕赤酵母。例如，培养基可以是常规的BMGY培养基。可在28～32℃、180～300rpm条件下培养。诱导外源基因表达时，可在培养液中加入一定量的甲醇，用于诱导表达。诱导表达结束后，离心发酵液，过滤上清液，即可获得含有外源基因表达的目的蛋白的发酵液。可采用常规的方法进一步浓缩该发酵液。
在某些实施方案中，发酵时，上罐初始培养基可以是基础发酵培养基，其含有硫酸钙、磷酸二氢钾、无水硫酸镁、硫酸铵、乳化硅油消泡剂和甘油，并添加有PTM，即五水硫酸铜、碘化钠、一水硫酸锰、二水钼酸钠、六水氯化钴、五水氯化锌、七水硫酸亚铁、硼酸、浓硫酸和生物素。基础发酵培养基中的成分浓度或含量为本领域所熟知。例如，硫酸钙的浓度可以是0.5～1.5g/L，磷酸二氢钾的浓度可以是30～40g/L，无水硫酸镁的浓度可以是10～13g/L，硫酸铵的浓度可以是6～12g/L，乳化硅油消泡剂的浓度可以是0.1～0.5ml/L，甘油的浓度可以是30～70g/L。PTM中，五水硫酸铜的浓度可以是5～6.5g/L，碘化钠的浓度可以是60～100mg/L，一水硫酸锰的浓度可以是2.0～4.0g/L，二水钼酸钠的浓度可以是0.2～0.4g/L，六水氯化钴的浓度可以是0.4～0.6g/L，五水氯化锌的浓度可以是18～22g/L，七水硫酸亚铁的浓度可以是60～70g/L，硼酸的浓度可以是0.01～0.03g/L，浓硫酸的浓度可以是19.0～19.5ml/L，生物素的浓度可以是0.3～0.5g/L。
菌体生长阶段流加甘油、PTM、消泡剂和氨水。当发酵至一定阶段时，例如菌体生长湿重达200～220g/L时，停止甘油补料并经过一段时间的饥饿期后，加入甲醇发酵。加入甲醇发酵诱导外源基因表达可根据需要，调控发酵过程的pH、温度、溶氧和甲醇流加速率等参数。本领域研究者熟知的，毕赤酵母还可以使用组成型启动子，如GAP启动子，TEF启动子等，使用相应的启动子并使用对应的发酵条件。
可对发酵液进行离心处理或板框过滤处理去除菌体，上清进行微滤、超滤处理，并使用缓冲液置换并浓缩。进一步的，可加入适当的保护剂待完全溶解后作为酶制剂置于4℃保存。
因此，本申请也提供一种酶制品，所述酶制品含有本文任一实施方案所述的含有编码酶的外源基因的毕赤酵母菌株的发酵液或发酵所得细胞的裂解液，或所述发酵液或裂解液的浓缩物。在某些优选的实施方案中，所述毕赤酵母菌株含有利用pPIC9构建得到的含所述酶的编码序列的表达载体。在某些实施方案中，酶制剂还可含有甘油和防腐剂。防腐剂可以是常规的防腐剂，如山梨酸钾。甘油和防腐剂的量可为本领域的常规用量。例如，可加入占该酶制品重量40-70％的甘油和0.1-0.8％的山梨酸钾。在某些实施方案中，所述酶制品含有磷脂酶C或脂肪酶。
本文所述的含磷脂酶C的酶制剂可用于油脂脱胶。脱胶可采用常规的方法实施，包括将待脱胶油脂与本文所述的含磷脂酶C的酶制剂接触的步骤。例如，取毛油加热至一定的温度(如50±5℃)，加入一定量的纯水和酶液，高速剪切(如10000r/min)，然后在一定温度下搅拌(如750r/min)，反应1～5小时。最后可将反应混合物升温至80－90℃并维持一段时间，将酶灭活，离心即可获得脱胶油。
因此，本申请还提供一种脱胶方法，该方法包括将待脱胶油脂与本文所述的含磷脂酶C的酶制剂接触的步骤；以及本文所述的含磷脂酶C的酶制剂在油脂脱胶中的应用。
在某些实施方案中，本申请还涉及本文所述的含有脂肪酶的酶制品在酯交换中的应用。例如，本文提供一种酯交换方法，所述方法包括将反应底物与本发明的含脂肪酶的酶制品接触的步骤。
本申请还提供选自Genbank登陆号为XM_002491428.1所示的序列、Genbank登陆号为XM_002490375.1的所示的序列、Genbank登陆号为XM_002493288.1所示的序列以及与XM_002491428.1、XM_002490375.1或XM_002493288.1具有至少80％、优选至少90％、优选至少95％、优选至少98％、优选至少99％的序列中的任一种或多种或其表达载体在提高外源基因在宿主细胞中的表达中的应用，或者促进含外源基因的宿主细胞的生长中的应用，或在制备外源基因表达能力提高或生长能力提高的宿主细胞中的应用。优选地，所述具有一定序列同一性的序列也来自酵母，更优选来自毕赤酵母。优选地，所述宿主细胞为酵母，更优选为毕赤酵母，更优选为本文所述的m314H菌株或其组氨酸和尿嘧啶双缺陷型突变体、组氨酸单缺陷型突变体。可采用本领域周知的工具计算两条或多条序列之间的序列同一性，这些工具可来自NCBI提供的各种在线工具。
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非限制本发明的保护范围。实施例中所用到的方法和材料，除非另有说明，否则均为本领域常规的方法和材料。
实施例1：尿嘧啶营养缺陷型毕赤酵母CICC32806-U7菌株的获得
将10uL CICC-32806菌种接种至10mL的YPD(2％蛋白胨，1％酵母粉，1％甘油)培养基中，30℃，240rpm培养过夜，检测菌液OD600数值，吸取200OD培养液，室温4000rpm离心后去除上清，使用无菌水洗涤菌体2次，最后重悬菌液浓度至20OD/mL，取2mL菌液均匀分散在培养皿表层，置于超净工作台紫外灯下照射90s，取100μL涂布于YNB-Uracil-FOA(13.4g/L YNB，1％甘油，2％琼脂糖，50μg/mL尿嘧啶(Uracil)，0.75mg/mL5-氟乳清酸(5-FOA))固体培养基上，30℃避光培养(全过程在红光下操作，防止回复突变)7天。将上一步在YNB-Uracil-FOA固体培养基上长出来的单菌落转接到YNB固体培养基、YNB-Uracil固体培养基上，挑取只能在YNB-Uracil固体培养基上生长的菌株，获得尿嘧啶代谢缺陷型毕赤酵母CICC32806-U7菌株。
实施例2：组氨酸缺陷型毕赤酵母CICC32806-7H3菌株的获得
以CICC32806的基因组为模板，HIS-A-F/R扩增HIS-A片段，HIS-B-F/R扩增HIS-B片段，URA3-1F/2R扩增URA3片段，依次连接入pSP72质粒，从而构建好组氨酸敲除载体pHISA-URA3-HISB，引物序列如下：
HIS-A-F:5'CCGCTCGAGTCACCTCAGCCAGATCAAAGT 3'(SEQ ID NO:10)；
HIS-A-R:5'ACATGCATGCCTTTGGACAACTCTTTCTGCC 3'(SEQ ID NO:11)；
HIS-B-F:5'CGGGGTACCCCTGGTTGATAAAGTTGCAT 3'(SEQ ID NO:12)；
HIS-B-R:5'GGCGAGCTCAGGTGTCTTCAAAGCGACTC 3'(SEQ ID NO:13)；
URA3-1F:ACATGCATGCCTGCAGAAATGGGGAGATAACCACC(SEQ ID NO:14)；
URA3-2R:CGGGGTACCACTAGTGGTTTTCTGGGGGTATTTGCTG(SEQ ID NO:15)。
用XhoI和SacI线性化敲除载体，通过电击法转化入CICC32806-U7中，涂布在含有组氨酸(20μg/mL)的MDS筛选平板，将长出来的单菌落，分别转接到YNB固体培养基、YNB-HIS固体培养基上。组氨酸营养缺陷型突变子不能在YNB固体培养基上生长，但是可以在YNB-HIS固体培养基上生长。将表型正确的菌株再次在YNB固体培养基、YNB-HIS固体培养基上进行单菌落划，挑取只能在YNB-HIS固体培养基上生长的单菌落菌株。最终获得组氨酸缺陷型毕赤酵母CICC32806-7H3菌株。
实施例3：产磷脂酶菌株7H3-PLC的构建
经过密码子优化并由生工生物工程(上海)股份有限公司合成的磷脂酶PLC核苷酸序列为：
Figure PCTCN2019129359-appb-000001
Figure PCTCN2019129359-appb-000002
该PLC的氨基酸序列为：
Figure PCTCN2019129359-appb-000003
使用引物PLC_F：TACGTATGGTCAGCTGAGGACAAGC(SEQ ID NO:3)及PLC_R：CCTAGGTTACCTGTCACCGTAAGTGTCGAAC(SEQ ID NO:4)，扩增PLC的成熟肽部分，PCR使用Takara公司PrimeSTAR HS(DRR010A)DNA聚合酶进行，反应体系为：水33μL，5×PrimeSTAR缓冲液10μL，dNTP混合物(各2.5mM)4μL，引物各1μL，质粒模板0.5μL，PrimeSTAR酶0.5μL。PCR反应程序为30个循环的98℃10秒，68℃1min。
PCR产物经Axygen公司PCR产物纯化试剂盒纯化(AP-PCR-50)，使用NEB公司SnaBI和AvrII限制性内切酶酶切，并再次经过Axygen公司PCR产物纯化试剂盒纯化。同时使用相同的限制性内切酶酶切pPIC9K质粒，酶切物同样进行纯化。使用Fermentas公司T4 DNA连接酶，按照产品说明，将PCR产物酶切片段和pPIC9k载体酶切片段进行连接，连接物热激法转入大肠杆菌DH5α中，在含氨苄的LB平板上过夜培养。次日挑取单克隆在LB液体培养基中培养，使用Axygen公司质粒提取试剂盒提取质粒并送交上海生工测序。
将测序正确的重组表达载体用Bgl II限制性内切酶酶切线性化后，按照毕赤酵母的标准转化方法(Shixuan Wu&Geoffrey J Letchworth,2004)，电击转化毕赤酵母 CICC32806-7H3、SMD1168感受态细胞，涂布到选择培养基MDS筛选平板，于28℃培养三天。挑取转化子至磷脂平板(1％YNB，2％磷脂，2％琼脂糖，1％甘油)上，30℃培养2天，挑取具有水解圈且磷脂酶基因为单拷贝的转化子，编号7H3-SPLC和SMD1168-SPLC重组菌株。
实施例4：紫外诱变法获得m314-SPLC菌株
挑取7H3-SPLC菌落至5mL YPD培养基中，于30℃，240rpm摇床震荡培养过夜，检测菌液OD600数值；吸取重量200OD的菌液，室温4000rpm离心后去除上清，使用无菌水洗涤细胞2次，最后重悬菌液浓度至20OD/mL，取2mL菌液均匀分散在培养皿表层，置于超净工作台紫外灯下照射90s，取100μL涂布于磷脂平板上，30℃培养箱避光培养(全过程在红光下操作，防止回复突变)4天。挑取水解圈变大的突变子，使用KOD-FX酶和毕赤酵母表达测序通用5’AOX及3’AOX引物，对该突变子菌落进行PCR扩增，按照该酶的使用说明进行PCR，PCR产物送上海生工进行测序，发现人为转入酵母中的核酸部分：AOX启动子、信号肽、PLC基因、转录终止子等都未发生突变，因此该突变子的改变为菌株自身的突变，命名为m314-SPLC。
实施例5：7H3-SPLC、m314-SPLC和SMD1168-SPLC摇瓶发酵
根据实施例2和实施例3可知，7H3-SPLC、m314-SPLC和SMD1168-SPLC三个菌中所含PLC基因的氨基酸序列完全一样且拷贝数均为单拷贝。接种这三个菌至50mL的BMGY培养基中，30℃、240rpm培养过夜，取200OD菌体离心收集。用无菌水重悬洗涤菌体2次，然后用BMMY培养基重悬菌体。向BMMY培养基中添加2％的甲醇，30℃、240rpm诱导表达。每隔12h向50mL培养基中补加0.5mL甲醇。诱导3天后，8000rpm、4℃离心发酵液，取发酵液上清进行酶活测定及蛋白电泳检测。
pNPPC法磷脂酶测定方法：
pNPPC法磷脂酶酶活单位的定义：在温度为37℃，pH值7.6的条件下，1min催化底物释放出1μmol磷酸胆碱的酶量为1个磷脂酶活力单位(U)。
反应缓冲液的配制：0.1M硼酸-硼酸钠缓冲液(pH7.6)，20mM pNPPC，1％Triton-X-100，1mM CaCl 2。
测定具体步骤：取两支干净的离心管，其中一个做样品管，一个做空白对照管。每个离心管中加入600μL反应缓冲液，样品管加入25μL的待测酶液，空白管不加，两管同时放在37℃恒温水浴锅中反应15min，立即加入500μL 0.5M的氢氧化钠溶液终止反 应，空白管加入25μL待测酶液，405nm测定吸光度，用空白管校正零点。
酶活测定结果如图1所示。结果显示，m314-SPLC磷脂酶酶活比出发菌株7H3-SPLC提高了127％，比SMD1168-SPLC提高了152％。
聚丙烯酰胺凝胶电泳分析：使用0.22μm滤膜过滤处理上清液，将等量上清液使用Milipore 10KDa超滤浓缩罐浓缩到相同体积后，取相同体积的浓缩酶液进行聚丙烯酰胺凝胶电泳分析。
电泳结果如图2所示。结果显示，3个泳道都有明显的目标条带，位于35-40KDa条带之间。各泳道的目标蛋白含量与测定的磷脂酶活性高低一致。
因此，经过紫外诱变获得的m314-SPLC菌株的蛋白表达能力比诱变前提高了127％。
实施例6：m314-SPLC菌株中PLC基因和His基因的敲除
用于敲除PLC基因的核苷酸序列(AOX-His)如下所示：
Figure PCTCN2019129359-appb-000004
Figure PCTCN2019129359-appb-000005
PCR法扩增出AOX-His基因片段，电击转化m314-SPLC菌株，涂布在含有组氨酸的磷脂酶筛选平板上。挑选出没有水解圈的转化子分别在不含组氨酸和含有组氨酸的平板上划线，选择表型正确(在含有组氨酸的平板上生长，而在不含组氨酸的平板上不生长)的转化子，命名为m314H。菌株m314H已于2018年10月31日保藏于中国普通微生物菌种保藏管理中心(CGMCC，北京市朝阳区北辰西路1号院3号，100101)，分类命名为巴斯德毕赤酵母(Pichia pastoris)，保藏编号为CGMCC No.16670。
实施例7：以RML基因为报告基因评估m314H菌株
通过RML脂肪酶基因来评估m314H菌株对其它蛋白表达水平的增强作用，所用RML基因经过密码子优化并由生工生物工程(上海)股份有限公司合成，核苷酸序列为：
Figure PCTCN2019129359-appb-000006
Figure PCTCN2019129359-appb-000007
RML氨基酸序列为：
Figure PCTCN2019129359-appb-000008
根据合成的RML基因序列设计引物，构建pPIC9K-RML表达载体，经限制性内切酶BglII线性化后分别电击转化7H3和m314H菌株，经脂肪酶平板筛选后选择具有水解圈且RML基因为单拷贝的转化子，编号7H3-SRML和m314-SRML，将选择出来的转化子进行摇瓶发酵(发酵条件如实施例5所述)。发酵液经超滤浓缩后取用相同体积的浓缩酶液进行脂肪酶酶活测定和聚丙烯酰胺凝胶电泳分析。
pNPP法脂肪酶测定方法：
pNPP法脂肪酶酶活单位的定义：在温度为40℃，pH值8.0的条件下，样品水解底物棕榈酸对硝基苯脂，每分钟释放出1μmol对硝基苯酚(pNP)所需的酶量为1个酶活力单位(U)。
底物缓冲液：取5.3mL 0.2mol/L NaH 2PO 4溶液和94.7mL 0.2mol/L Na 2HPO 4溶液， 混合后加入约280mL水，加入0.92g脱氧胆酸钠、0.44g阿拉伯树胶粉，搅拌溶解，用H 3PO 4或NaOH调节pH值至8.0，定容至400mL，4℃保存。
底物pNPP溶液(0.0795mol/L，3mg/L)：称取棕榈酸对硝基苯脂(pNPP)0.03g，加入10mL异丙醇搅拌溶解，4℃保存。
测定具体步骤：取1mL pNPP溶液和9mL底物缓冲溶液，混合。取两支干净的离心管，其中一个做样品管，一个做空白对照管。每个离心管中加入600μL反应缓冲液，样品管加入25μL的待测酶液，空白管不加，两管同时放在40℃恒温水浴锅中反应15min，立即加入500μL无水乙醇终止反应，空白管加入25μL待测酶液，12000rpm离心2min。405nm测定吸光度，用空白管校正零点。
酶活测定结果如图3所示。结果显示，m314-SRML表达的RML脂肪酶酶活比出发菌株7H3-SRML提高了92％。
聚丙烯酰胺凝胶电泳分析：使用0.22μm滤膜过滤处理上清液，将等量上清液使用Milipore 10KDa超滤浓缩罐浓缩到相同体积后，取相同体积的浓缩酶液进行聚丙烯酰胺凝胶电泳分析。
电泳结果如图4所示。2个泳道都有明显的目标条带，位于35-40KDa条带之间。各泳道的目标蛋白含量与测定的磷脂酶活性高低一致。
总结实施例5及实施例7的结果，经过紫外诱变获得的m314H菌株用来表达PLC和RML时，其蛋白表达能力比出发菌株7H3提高了127％和92％。
实施例8：在m314H菌株中过表达生长促进基因
以CICC32806的基因组为模板，HIS-A-F/R扩增HIS-A片段，HIS-B-F/R扩增HIS-B片段，依次连接入pSP72质粒，从而构建好组氨酸敲除载体pHISA-HISB。用XhoI和SacI线性化敲除载体，通过电击法转化入m314H中，涂布在含有组氨酸、Uracil和5-FOA的MDS筛选平板，将长出来的单菌落，分别转接到YNB-HIS固体培养基、YNB-Uracil和YNB-Uracil-HIS固体培养基上。组氨酸和尿嘧啶双营养缺陷型转化子只能在YNB-Uracil-HIS固体培养基上生长的单菌落菌株。由此最终获得组氨酸和尿嘧啶双营养缺陷型毕赤酵母m314HU菌株。
以CICC32806的基因组为模板，用引物URA3-1F/2R扩增URA3片段。经生工生物工程(上海)股份有限公司合成Protein required general stress response(Genbank no:XM_002491428.1)、Mitochondrial external NADH dehydrogenase(Genbank no:XM_002490375.1)、Proteinase A(Genbank no:XM_002493288.1)这三个生长促进基因片 段，再将此三个基因片段分别与URA3片段依次连接入pSP72质粒，从而构建好过表达载体pURA3-Stress、pURA3-NADH和pURA3-ProA。用XhoI和BglII线性化敲除载体，通过电击法将三个生长促进基因过表达载体片段共转化入m314HU中，涂布在含有组氨酸的MDS筛选平板，通过PCR验证保证所有过表达基因成功转入，从而获得m315H菌株。菌株m315H已于2018年10月31日保藏于中国普通微生物菌种保藏管理中心(CGMCC，北京市朝阳区北辰西路1号院3号，100101)，分类命名为巴斯德毕赤酵母(Pichia pastoris)，保藏编号为CGMCC No.16669。
实施例9：以TL基因为报告基因评估m315H菌株
通过TL脂肪酶基因来评估m315H菌株对其它蛋白表达水平的增强作用，所用TL基因经过密码子优化并由生工生物工程(上海)股份有限公司合成，核苷酸序列如下：
Figure PCTCN2019129359-appb-000009
TL氨基酸序列为：
Figure PCTCN2019129359-appb-000010
Figure PCTCN2019129359-appb-000011
根据合成的TL基因序列设计引物，构建pPIC9K-TL表达载体，经限制性内切酶BglII线性化后分别电击转化7H3、m314H和m315H菌株，经脂肪酶平板筛选后选择具有水解圈且TL基因为单拷贝的转化子，编号7H3-STL、m314-STL和m315-STL，将选择出来的转化子进行摇瓶发酵。发酵液经超滤浓缩后取用相同体积的浓缩酶液，用pNPP法脂肪酶测定方法进行脂肪酶酶活测定和聚丙烯酰胺凝胶电泳分析。
酶活测定结果如图5所示。结果显示，m315-STL表达的TL脂肪酶酶活比m314-STL提高了39％。
聚丙烯酰胺凝胶电泳分析：使用0.22μm滤膜过滤处理上清液，将等量上清液使用Milipore 10KDa超滤浓缩罐浓缩到相同体积后，取相同体积的浓缩酶液进行聚丙烯酰胺凝胶电泳分析。
电泳结果如图6所示。3个泳道都有明显的目标条带，位于35-40KDa条带之间。各泳道的目标蛋白含量与测定的TL脂肪酶酶活性高低一致。
总结实施例5、实施例7及实施例9的结果，将菌株7H3通过紫外诱变获得的m314H，用m314H来表达PLC、RML和TL时，其蛋白表达能力比出发菌株7H3分别提高了127％、92％和86％，证明菌株m314H的外源表达蛋白的能力较出发菌株7H3显著提升，具有优良的特性；再在m314H中过表达了3种生长促进基因，构建了菌株m315H，发现表达TL时，比m314H的蛋白表达水平再提高了39％，菌株蛋白表达水平再次获得了提高。
实施例10：在m314H菌株中过表达液泡蛋白酶A
以CICC32806的基因组为模板，用引物URA3-1F/2R扩增URA3片段；经生工生物工程(上海)股份有限公司合成液泡蛋白酶A(Genbank no:XM_002493288.1)基因片段，用引物proAF：CGAGTTTCTCCGTATCTAAT和proAR：TCCTCATCTATACCCCAGG扩增Proteinase A基因片段。通过电击法将扩展获得的URA3片段和Proteinase A片段共转化入实施例8中获得的m314HU中。电转物涂布在含有组氨酸的MDS筛选平板，通过PCR验证保证所有转化基因成功转入，从而获得m316H菌株。菌株m316H已于2019年月日保藏于中国普通微生物菌种保藏管理中心(CGMCC，北京市朝阳区北辰西路1号院3号，100101)，分类命名为巴斯德毕赤酵母(Pichia pastoris)，保藏编号为CGMCC No.19221。
经基因组测序，m316H菌株中Proteinase A有3个拷贝，2个位于基因组原Proteinase  A位置，另一个拷贝插入在BQ9382_C2-3700基因Subunit of the Anaphase-Promoting Complex/Cyclosome编码区域。
实施例11：以TL基因为报告基因评估m316H菌株
将实施例9中构建pPIC9K-TL表达载体，经限制性内切酶BglII线性化后分别电击转化m316H菌株，经脂肪酶平板筛选后选择具有水解圈且TL基因为单拷贝的转化子，编号m316-STL，将选择出来的转化子与实施例9中获得的7H3-STL、m314-STL和m315-STL一起进行摇瓶发酵比较。发酵液经超滤浓缩后取用相同体积的浓缩酶液，用pNPP法脂肪酶测定方法进行脂肪酶酶活测定和聚丙烯酰胺凝胶电泳分析。
酶活测定结果如图7所示。结果显示，m316-STL表达的TL脂肪酶酶活与m315-STL类似比m314-STL提高了45％。
聚丙烯酰胺凝胶电泳分析：使用0.22μm滤膜过滤处理上清液，将等量上清液使用Milipore 10KDa超滤浓缩罐浓缩到相同体积后，取相同体积的浓缩酶液进行聚丙烯酰胺凝胶电泳分析。
电泳结果如图8所示。4个泳道都有明显的目标条带，位于35-40KDa条带之间。各泳道的目标蛋白含量与测定的TL脂肪酶酶活性高低一致。
实施例12：本发明毕赤酵母菌株的基因组测序及与已有毕赤酵母基因组比较
将本发明提到的31806、m314、m315和m316菌种提交苏州金维智生物科技有限公司进行基因组测序。金维智提取菌株基因组后，打断并补平，3’末端加A后与含Index序列的接头连接，按照此策略构建的测序文库，桥式结合到测序芯片上后，进行Illumina HiSeq测序。重测序下机数据经过处理后获得原始数据，经过过滤去接头、去污染、然后与参考基因组比对。通过比对结果，去除出每个文库中由于PCR扩增引起的重复序列，然后计算出相对于参考基因组的测序深度和覆盖度、单核苷酸位点变异(SNV)、插入/缺失(InDels)等。对于有参考基因集、SNV数据集，还可以进行突变注释和功能富集等一些标准生物信息分析。覆盖率99.99％及以上，平均深度400×及以上且其中深度分布200×以上占99.95％以上。
经过基因组测序及与已有毕赤酵母基因组比较，本发明出发菌株CICC32806与GS115在基因组有1125个SNV事件及830个INDEL事件报告，显然本发明出发菌株与GS115有显著区别。经过比较，本发明m314H、m315H及m316H菌株与GS115或CICC32806菌株的基因组有至少如下位点发生改变：BQ9382_C1-2260，308-310位EKK 缺失，假设蛋白；BQ9382_C1-3800，E129K，60S核糖体亚基组装/输出蛋白LOC1；BQ9382_C1-5700，I312M，线粒体外NADH脱氢酶，II类NAD(P)H:醌氧化还原酶；BQ9382_C2-3950，Q145X，必需蛋白质，具有结合伴侣Psr1p，用于完全激活一般压力反应；BQ9382_C3-2220，E188K，假设蛋白；和BQ9382_C3-4370，W196X，奥罗替丁5\'-磷酸脱羧酶。

Claims (11)

  1. 一种毕赤酵母菌株，其特征在于，相对于毕赤酵母菌株GS115或CICC32806，其包含以下6个突变中的一个或多个：BQ9382_C1-2260，308-310位EKK缺失，假设蛋白；BQ9382_C1-3800，E129K，60S核糖体亚基组装/输出蛋白LOC1；BQ9382_C1-5700，I312M，线粒体外NADH脱氢酶，II类NAD(P)H:醌氧化还原酶；BQ9382_C2-3950，Q145X，必需蛋白质，具有结合伴侣Psr1p，用于完全激活一般压力反应；BQ9382_C3-2220，E188K，假设蛋白；和BQ9382_C3-4370，W196X，奥罗替丁5\'-磷酸脱羧酶；优选地，所述毕赤酵母菌株包含上述6个突变。
  2. 如权利要求1所述的毕赤酵母菌株，其特征在于，所述毕赤酵母菌株为保藏编号为CGMCC No.16670的巴斯德毕赤酵母(Pichia pastoris)菌株，保藏编号为CGMCC No.16669的巴斯德毕赤酵母(Pichia pastoris)菌株，或者保藏编号为CGMCC No.19221的巴斯德毕赤酵母(Pichia pastoris)菌株。
  3. 保藏编号为CGMCC No.16670的巴斯德毕赤酵母(Pichia pastoris)菌株。
  4. 一种毕赤酵母菌株，其特征在于，所述毕赤酵母菌株为权利要求3所述的巴斯德毕赤酵母菌株经基因工程改造或诱变获得，且(a)为组氨酸缺陷型菌株；和/或(b)含有表达生长促进因子的质粒、和/或基因组中整合有生长促进因子的编码序列、和/或表达生长促进因子，所述生长促进因子优选为来源于酵母自身的生长促进基因，包括但不限于Protein required general stress response(Genbank no:XM_002491428.1)、Mitochondrial external NADH脱氢酶(Genbank no:XM_002490375.1)、液泡蛋白酶A(Genbank no:XM_002493288.1)，所述毕赤酵母菌株优选为保藏编号为CGMCC No.16669的巴斯德毕赤酵母菌株。
  5. 一种毕赤酵母菌株，其特征在于，所述毕赤酵母菌株为权利要求3所述的巴斯德毕赤酵母菌株经基因工程改造或诱变获得，且(a)为组氨酸缺陷型菌株；和/或(b)含有表达液泡蛋白酶A基因的质粒、和/或基因组中整合有液泡蛋白酶A基因的编码序列、和/或表达液泡蛋白酶A基因，所述毕赤酵母菌株优选为保藏编号为CGMCC No.19221的巴斯德毕赤酵母菌株。
  6. 一种基因工程改造或诱变获得的毕赤酵母菌株，其特征在于，所述菌株 为权利要求1-5中任一项所述的毕赤酵母菌株经基因工程改造或诱变获得，其中，所述基因工程改造使得所述毕赤酵母菌株含有外源基因或含该外源基因的载体；优选地，所述菌株的基因组中整合了所述外源基因，所述外源基因优选为工业、饲料或食品领域中用到的蛋白的编码序列；优选地，所述外源基因为酶的编码序列，所述酶选自以下酶中的至少一种：脂肪酶、蛋白酶、纤维素酶、淀粉酶、植酸酶、酯酶、果胶酶、半乳糖苷酶和磷脂酶。
  7. 如权利要求6所述的毕赤酵母菌株，其特征在于，所述酶选自脂肪酶或磷脂酶，优选地，所述脂肪酶的氨基酸序列如为与SEQ ID NO:7或9所示具有至少80％、90％、95％、98％或99％同一性的氨基酸序列，优选所述脂肪酶的氨基酸序列如SEQ ID NO:7或9所示；所述磷脂酶的氨基酸序列如为与SEQ ID NO:2所示具有至少80％、90％、95％、98％或99％同一性的氨基酸序列，优选所述磷脂酶的氨基酸序列如SEQ ID NO:2所示。
  8. 一种培养物，所述培养物含有权利要求1-7中任一项所述的毕赤酵母菌株和任选的培养基；
    优选地，所述培养基为种子培养基或发酵培养基；更优选地，所述培养基为YPD培养基或BMMY培养基。
  9. 一种酶制品，其特征在于，所述酶制品含有权利要求6所述的含有编码酶的外源基因的毕赤酵母菌株的发酵液或发酵所得细胞的裂解液，或所述发酵液或裂解液的浓缩物，优选地，所述毕赤酵母菌含有以pPIC9K质粒为骨架载体构建的含酶的编码序列的表达载体。
  10. 权利要求9所述的酶制品在酯交换或油脂脱胶中的应用，其中，所述酶制品含有脂肪酶或磷脂酶；优选地，所述脂肪酶的氨基酸序列为与SEQ ID NO:7或9具有至少80％、90％、95％、98％或99％同一性的氨基酸序列，更优选地，所述脂肪酶的氨基酸序列如SEQ ID NO:7或9所示；优选地，所述磷脂酶的氨基酸序列为与SEQ ID NO:2具有至少80％、90％、95％、98％或99％同一性的氨基酸序列，更优选地，所述磷脂酶的氨基酸序列如SEQ ID NO:2所示。
  11. 权利要求1-7中任一项所述的巴斯德毕赤酵母菌株在构建表达外源基因的菌株中的应用。
PCT/CN2019/129359 2018-12-28 2019-12-27 用于表达外源基因的毕赤酵母突变株 WO2020135763A1 (zh)

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