WO2021035793A1 - MviN蛋白突变体、含有该突变体的表达载体和宿主细胞及其应用 - Google Patents

MviN蛋白突变体、含有该突变体的表达载体和宿主细胞及其应用 Download PDF

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WO2021035793A1
WO2021035793A1 PCT/CN2019/104683 CN2019104683W WO2021035793A1 WO 2021035793 A1 WO2021035793 A1 WO 2021035793A1 CN 2019104683 W CN2019104683 W CN 2019104683W WO 2021035793 A1 WO2021035793 A1 WO 2021035793A1
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seq
mvin
host cell
amino acid
mutant
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PCT/CN2019/104683
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孙际宾
李庆刚
周文娟
德莱奥西班乔·泰沃
郑平
马延和
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中国科学院天津工业生物技术研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine

Definitions

  • the present invention belongs to the field of genetic engineering, and specifically relates to mutants of transmembrane lipid II flipping enzyme (MviN) and the application of the mutants in the production of glutamic acid and lysine.
  • McN transmembrane lipid II flipping enzyme
  • Glutamic acid and lysine are important nutrients for humans and animals. They play a very important role in medicine, health, food, animal feed and other industries. They are mainly produced by microbial fermentation.
  • the transformation technology of fermentation strains has been continuously developed. During the synthesis of a compound, people usually transform the pathways directly related to its metabolism (including substrate transportation, product transportation, energy production, etc.).
  • a large number of studies have reported on the modification of modules closely related to the amino acid synthesis pathway, such as removing the product feedback inhibition of key enzymes, weakening the competitive pathway, strengthening substrate or product transportation, etc., and the acid production capacity of industrial production strains has also been greatly improved. improve.
  • MviN is a transmembrane lipid II flipping enzyme that catalyzes the last step of peptidoglycan (PG) synthesis, transports lipid II from the inner cytoplasmic membrane to the periplasmic space, and cross-links the new PG to form a mature PG .
  • PG peptidoglycan
  • JP2010161970A discloses that the mutation of 3 amino acid residues (position 197, position 260, and position 181) in the protein can increase the glutamate of Corynebacterium glutamicum.
  • the yield of glutamic acid is very low. Compared with the original strain, the yield of glutamic acid is only increased by 1-5% (product percentage), and the effect on the conversion rate is not reported.
  • the purpose of the present invention is to provide a new MviN protein mutant, and a method for using the protein mutant to significantly increase the conversion rate of host strains in the production of glutamic acid and lysine.
  • the present invention provides a transmembrane lipid II flipping enzyme (MviN) protein mutant, the amino acid sequence of which includes a group selected from the following:
  • Proline at position 346 of the amino acid sequence corresponding to any one of SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 is replaced by leucine, or by other Aliphatic hydrocarbon side chain amino acids such as isoleucine, valine or alanine replaced by any one; and/or valine at position 599 is replaced by phenylalanine or tryptophan;
  • the amino acid sequence described in a) has the same mutation site, and the amino acid sequence described in a) has more than 90%, for example, more than 95%, more than 98% homology, the amino acid sequence is derived from Corynebacterium glutamicum (Corynebacterium glutamicum), and maintain the activity of maintaining lipid II transport from the cytoplasmic inner membrane to the periplasmic space.
  • Corynebacterium glutamicum Corynebacterium glutamicum
  • the MviN protein mutant further includes or does not include mutations at other sites.
  • the MviN protein mutant includes conservative mutations derived from the above amino acid sequence, and the MviN protein mutant maintains the activity of lipid II transport from the inner cytoplasmic membrane to the periplasmic space.
  • the present invention provides the coding gene of the MviN protein mutant described in the first aspect.
  • the present invention provides an expression vector including the gene encoding the MviN protein mutant described in the second aspect; in one embodiment, the expression vector is constructed by linking the nucleotide sequence of the encoding gene with a plasmid
  • the plasmid is selected from pEC-XK99E.
  • the present invention provides a host cell expressing the MviN protein mutant described in the first aspect.
  • the host cell expressing the mutant comprises the expression vector of the third aspect or the nucleotide sequence of the coding gene of the second aspect is integrated into its genome.
  • the expression vector is introduced into the original host cell to form a host cell containing the gene encoding the mutant.
  • the original host cell is from Escherichia, Corynebacterium, Pantoea, Brevibacterium sp, Bacillus ), Klebsiella, Serratia or Vibrio.
  • Escherichia coli E. Coli
  • Corynebacterium glutamicum Corynebacterium glutamicum
  • Corynebacterium glutamicum Corynebacterium glutamicum
  • the present invention provides the MviN protein mutant described in the first aspect, or the coding gene described in the second aspect, or the expression vector described in the third aspect, or the host cell described in the fourth aspect is used in the production of grains.
  • the present invention provides a method for preparing glutamic acid or lysine, the method comprising the following steps:
  • step b Culturing the host cell constructed in step a to produce a culture medium containing glutamic acid or lysine;
  • step b Optionally separate the produced glutamic acid or lysine from the culture broth of step b.
  • the present invention provides a new mutant of MviN protein, which can increase the production of glutamic acid and lysine and significantly increase the glucose conversion rate by mutating an amino acid at a specific site.
  • the present invention provides a new modified target of MviN protein, which can increase the growth rate of strains and reduce the production cost of glutamic acid and lysine.
  • transmembrane lipid II flippase and “membrane protein”, “murein biosynthesis protein”, “MviN protein”, “protein of the present invention”, and “polypeptide of the present invention” can be used interchangeably and have The meaning generally understood by those of ordinary skill in the art.
  • MviN protein refers to a transmembrane lipid II flipping enzyme that catalyzes the last step of peptidoglycan (PG) synthesis and transports lipid II from the inner cytoplasmic membrane to the periplasmic space.
  • MviN protein can be derived from various species, including but not limited to Escherichia (Escherichia), Corynebacterium (Corynebacterium), Pantoea (Pantoea), Brevibacterium (Brevibacterium), Bacillus (Bacillus), Klebsiella, Serratia or Vibrio.
  • the MviN protein is derived from the genus Corynebacterium, such as the protein corresponding to any amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8; it may also contain SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 is a polypeptide having the activity of transporting lipid II from the inner cytoplasmic membrane to the periplasmic space; It can also have a homology with any amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 greater than 90%, preferably 95%, more preferably 98% or more.
  • the "MviN protein mutant" of the present invention refers to the amino acid sequence obtained by mutating any one of the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8. Specifically, the MviN protein mutant of the present invention has an amino acid sequence at position 346 corresponding to any one of the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8. Proline is replaced by leucine, or valine at position 599 corresponding to any of the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8.
  • Phenylalanine substitution; or the MviN protein mutant of the present invention is a fragment of the amino acid sequence including the above-mentioned mutation site.
  • it may also contain any amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 and have the ability to transport lipid II from the inner cytoplasmic membrane to A polypeptide with periplasmic space activity; it may also include a sequence homology with any amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 greater than 90%, preferably 95 %, most preferably 98% or more of a polypeptide derived from Corynebacterium glutamicum and having the activity of transporting lipid II from the inner cytoplasmic membrane to the pericytoplasmic space, as long as it corresponds to SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8, the proline at
  • the NCBI database https://www.ncbi.nlm.nih.gov/ has published the "transmembrane lipid II flipase" derived from Corynebacterium glutamicum (part of the sequence in the database is annotated as hypothetical protein Or membrane protein) has 22 amino acid sequences in total, of which only one sequence has 93% homology with the sequence shown in SEQ ID NO: 2 of the present invention, and the remaining 21 sequences are shown in SEQ ID NO: 2 of the present invention. The sequence homology is greater than 98%; and all 22 sequences are proline at position 346 corresponding to the sequence shown in SEQ ID NO: 2, and the sequence corresponding to the sequence shown in SEQ ID NO: 2 is proline.
  • the 599th position is all valine. It can be seen that in Corynebacterium glutamicum, the transmembrane lipid II flippase is highly conserved, and it is also highly conserved at position 346 or 599 corresponding to the sequence shown in SEQ ID NO: 2. Furthermore, the present invention modified four "transmembrane lipid II flippase" sequences derived from Corynebacterium glutamicum, and the results showed that the 346th proline of any of these four sequences was mutated to Leucine, or the mutation of valine at position 599 to phenylalanine, can increase the production of glutamic acid and lysine and significantly increase the glucose conversion rate.
  • polypeptides that have greater than 98% homology with the sequence shown in SEQ ID NO: 2 and are derived from Corynebacterium glutamicum, as long as they are at position 346 corresponding to the sequence shown in SEQ ID NO: 2
  • the replacement of proline by leucine or the replacement of valine by phenylalanine at position 599 of the sequence corresponding to SEQ ID NO: 2 is within the protection scope of the present invention.
  • the term "host cell” as used herein has the meaning commonly understood by those of ordinary skill in the art, that is, a cell containing the MviN protein of the present invention or a mutant thereof.
  • the present invention can use any host cell, as long as the cell contains the MviN protein of the present invention or a mutant thereof and is capable of producing amino acids.
  • the host cell can be from Escherichia (Escherichia), Corynebacterium (Corynebacterium), Pantoea (Pantoea), Brevibacterium (Brevibacterium sp), Bacillus (Bacillus), Klebsiella (Klebsiella), Serratia or Vibrio.
  • Escherichia coli E.
  • Coli or Corynebacterium glutamicum (Corynebacterium glutamicum) is preferred, and Corynebacterium glutamicum (Corynebacterium glutamicum) is most preferred.
  • the host described in the present invention refers to a strain capable of producing glutamic acid and lysine.
  • containing the MviN of the present invention has the meaning conventionally understood by those skilled in the art, and can be implemented by methods known in the art, including but not limited to, for example, a polynucleus containing a polynucleotide sequence encoding a protein
  • the nucleotide is inserted into the chromosome, or the polynucleotide is cloned into a vector and introduced into the microorganism, or the copy of the polynucleotide is directly added to the chromosome. It can also include, without limitation, any known protein that can be introduced. Active method.
  • the term "has the activity of MviN protein” has the same or similar meaning as conventionally understood by those skilled in the art, and both mean that the amino acid sequence of a certain fragment is a part of the amino acid sequence of a complete protein or polypeptide, and has the same meaning as that of the complete protein or polypeptide.
  • the same or similar function or activity means any amino acid fragment obtained from the MviN protein of the present invention that has the same function as the MviN protein.
  • Those of ordinary skill in the art should understand that changing a few amino acid residues in certain regions of the polypeptide, such as non-important regions, will not substantially change the biological activity. For example, the sequence obtained by appropriately replacing certain amino acids will not affect its activity.
  • MviN of the present invention is further mutated to obtain further mutants that still have the function of MviN.
  • the present invention should include conservative mutants of MviN of the present invention. These conservative mutants can be produced based on, for example, amino acid substitutions as shown in the table below.
  • the present invention also provides polynucleotides encoding the polypeptides of the present invention.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the gene of the MviN protein can be a probe prepared under stringent conditions with any of the nucleotide sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7, for example, SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 is a DNA that hybridizes with a complementary sequence of part or the entire sequence, as long as it maintains its original function .
  • the "stringent conditions" refer to conditions under which so-called specific hybridization can be formed without forming non-specific hybridization.
  • DNAs with high homology hybridize with each other
  • DNAs with a homology of less than 90% do not hybridize with each other, or the cleaning conditions of the usual Southern hybridization, that is, under comparable conditions. Wash once at 60°C, 1*SSC, 0.1% SDS, preferably 60°C, 0.1*SSC, 0.1% SDS, more preferably 68°C, 0.1*SSC, 0.1% SDS salt concentration and temperature, preferably 2-3 Second condition.
  • any codon in the gene of the MviN protein can be replaced with a corresponding equivalent codon, that is, the gene of the MviN protein can be due to the degeneracy of the genetic code.
  • the MviN protein gene may be a modified gene so that it has the best codon according to the codon frequency in the host to be used.
  • the MviN protein gene may be DNA homologous to any amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 Or the sequence identity is 90% or more, preferably 95% or more, and more preferably 99% DNA.
  • Such active fragments can be the mutation site of the present invention (corresponding to SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 (the 346th and 599th positions) are mutated into active fragments obtained by other amino acid residues, which can also be the active fragments described in the present invention.
  • the mutation site (corresponding to SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 346 and 599 of SEQ ID NO: 8) is obtained by mutating adjacent or similar sites Active fragments, such active fragments should be regarded as equivalent rights of the present invention and fall within the protection scope of the present invention.
  • corresponding to has the meaning commonly understood by those of ordinary skill in the art. Specifically, “corresponding to” means that after two sequences are aligned for homology or sequence identity, one sequence corresponds to a specified position in the other sequence. Therefore, for example, in terms of "corresponding to the amino acid residue at position 40 of any amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8", if Add a 6 ⁇ His tag to one end of any amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8, then the resulting mutant corresponds to SEQ ID NO: 2 or SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, the 40th position may be the 46th position.
  • the homology or sequence identity may be 90% or more, preferably 95% or more, and more preferably 98% homology and derived from the genus Corynebacterium.
  • the preferred method for determining identity is to obtain the largest match between the tested sequences.
  • the method for determining identity is compiled in a publicly available computer program.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to: GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990).
  • the public can obtain the BLASTX program from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • MviN (named MviNZ, nucleoside derived from Corynebacterium glutamicum Z188)
  • the acid and amino acid sequences are SEQ ID NO: 1 and SEQ ID NO: 2), derived from the MviN of Corynebacterium glutamicum B253 (named MviNB, and the nucleotide and amino acid sequences are SEQ ID NO: 3 and SEQ ID, respectively) NO: 4
  • MviN named MviN9, the nucleotide and amino acid sequences are SEQ ID NO: 5 and SEQ ID NO: 6) derived from Corynebacterium glutamicum ATCC 13869 (GeneBank accession number CP016335), derived from Corynebacterium glutamicum ATCC 13032 (Gene
  • PCR amplification was performed using primer MF (SEQ ID NO: 9CCTCGAATTCATGAATGGTCAACAAGTGAGTTCTTC) and primer MR1 (SEQ ID NO: 10TCTACTGCAGTTACCAACCAACGAGTTGTACTTCAG), respectively using Z188, B253, ATCC 13869 genome as templates; primer MF and primer MR1 (SEQ ID NO: 10TCTACTGCAGTTACCAACCAACGAGTTGTACTTCAG) were used for PCR amplification; primer MF and primer MR1 (SEQ ID NO: 10TCTACTGCAGTTACCAACCAACGAGTTGTACTTCAG) were used as templates; ), using the ATCC 13032 genome as a template for PCR amplification.
  • PCR amplification was performed using primers ATGCTGCGCCGTTTATTTAT (SEQ ID NO: 12) and ATAAATAAACGGCGCAGCAT (SEQ ID NO: 13), using pK18-MviNZ, pK18-MviN9 and pK18-MviN2 as templates, respectively; using primers TGATGCTGCACCGTTTATTT (SEQ ID NO: 14) And AAATAAACGGTGCAGCATCA (SEQ ID NO: 15), using pK18-MviNB as a template, PCR amplification.
  • the ClonExpress II One Step Cloning kit (The ClonExpress II One Step Cloning kit, Nanjing Novazin Biotechnology Co., Ltd., China) to self-ligate the PCR products to obtain the P346L mutation of MviN on the corresponding plasmid, and name the mutant plasmids respectively They are pK18-Mvi NZ P346L , pK18-MviNB P346L , pK18-MviN9 P346L , and pK18-MviN2 P346L .
  • the PCR products were self-ligated to obtain the V599F mutation of MviN on the corresponding plasmid, and the mutant plasmids were named separately They are pK18-MviNZ V599F , pK18-MviNB V599F , pK18-MviN9 V599F , and pK18-MviN2 V599F .
  • Plasmid pK18-MviNZ, pK18-MviNB, pK18-MviN9, pK18-MviN2, pK18-MviNZ P346L, pK18-MviNB P346L, pK18-MviN9 P346L, pK18-MviN2 P346L, pK18-MviNZ V599F, pK18-MviNB V 599F, pK18 -MviN9 V599F and pK18-MviN2 V599F were introduced into Z188 and B253 competent cells respectively (where pK18-MviNZ does not need to be introduced into Z188, pK18-MviNB does not need to be introduced into B253), using LBG medium (glucose 10g/L, yeast powder 5g) /L, peptone 10g/L, sodium chloride 10g/L) After overnight culture, kanamycin-resistant clo
  • the cloned genes amplified by MF and MR1 were used to select clones in which the original MviN gene on the Z188 or B253 genome was completely replaced by the corresponding MviN and its mutants from different sources by sequencing to obtain the strains Z188-MviNB, Z188-MviN9, Z188-MviN2, Z188-MviNZ P346L , Z188-MviNB P346L, Z188-MviN9 P 346L, Z188-MviN2 P346L, Z188-MviNZ V599F, Z188-MviNB V599F, Z188-MviN9 V599F Z188-Mvi N2 V599F, and strain B253-MviNZ , B253-MviN9, B253-MviN2, B253-MviNZ P346L , B253-MviNB P346L , B253-MviN9 P346L , B253
  • Example 2 Using plasmids to overexpress different MviN gene mutants in Corynebacterium glutamicum strains Z188 and B253
  • the plasmids pEC, pEC-MviNB P346L and pEC-MviNB V599F were transformed into B253, respectively, to obtain strains B253 (pEC), B253 (pEC-MviNB P346L ) and B253 (pEC-MviNB V599F ).
  • the seed culture medium is: glucose 50g/L, phosphoric acid 0.7g/L, magnesium sulfate heptahydrate 0.8g/L, ammonium sulfate 10g/L, 3-(N-maleinide) propanesulfonic acid 84g/L, corn steep liquor 3g/L, urea 10g/L, peptone 1g/L, yeast powder 0.5g/L, adjust the pH to 7.0 with sodium hydroxide.
  • the difference between the fermentation medium and the seed medium is that peptone and yeast powder are not added to the fermentation medium.
  • the rotating speed of the orifice plate shaker was 800 rpm, and the glutamic acid production and the conversion rate from glucose to glutamic acid were measured after 33 hours of cultivation. Part of the results are shown in Table 1 and Table 2. It can be seen from Table 1 and Table 2 that by introducing MviN from different sources with P346L or V599F mutation sites into the genome, and by overexpression of MviN with P346L or V599F mutation sites on the plasmid, the strain yield can be improved. The level of amino acid, the relative increase in conversion rate of 15.77-22.29%.
  • Example 4 Test of lysine production by fermentation of Corynebacterium glutamicum B253 and its MviN gene mutant strain
  • the fermentation medium components are: glucose 80g/L; yeast powder 8g/L; urea 9g/L; K 2 HPO 4 1.5g/L; MnSO 4 0.01g/L; MgSO 4 0.6g/ L; FeSO 4 0.01g/L; MOPS 42g/L.
  • B253 (pEC), B253 (pEC-MviNB P346L ) and B253 (pEC-MviNB V599F ) were cultured with IPTG at a final concentration of 0.1 mM and kanamycin at a final concentration of 25 ⁇ g/L.
  • the strain was inoculated into LBG medium for overnight culture, and the culture was inoculated as seeds into a 96-well deep-well plate containing 150 ⁇ L of fermentation medium per well.
  • the inoculum amount was 5%.
  • the plate shaker speed At 800 rpm, 3 parallel experiments were performed for each strain. After the fermentation, the lysine production and the conversion rate from glucose to lysine were measured.
  • the test results of the plasmid overexpressing MviNB mutant strain are shown in Table 3 and Table 4.

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Abstract

提供一种MviN蛋白突变体，所述突变体通过在MviN蛋白的氨基酸序列的特定位点引入氨基酸突变，改善了棒杆菌产谷氨酸、赖氨酸的能力。

Description

MviN蛋白突变体、含有该突变体的表达载体和宿主细胞及其应用
本申请要求2019年8月26日向中国国家知识产权局提交的专利申请号为201910787948.9，发明名称为“MviN蛋白突变体、含有该突变体的表达载体和宿主细胞及其应用”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于基因工程领域，具体地涉及跨膜脂质II翻转酶(MviN)的突变体，以及突变体在生产谷氨酸、赖氨酸方面的应用。
背景技术
谷氨酸、赖氨酸是人类和动物的重要营养物质，在医药、健康、食品、动物饲料等行业中有着十分重要的地位，主要采用微生物发酵法来生产。近年来，作为发酵工业的核心，发酵菌种的改造技术不断开发，在某一化合物的合成过程中人们通常对其代谢直接相关途径(包括底物运输、产物运输、能量产生等)进行改造。已有大量研究报道了与氨基酸合成途径密切相关的模块改造，例如解除关键酶的产物反馈抑制、弱化竞争性途径、加强底物或产物运输等，工业生产菌株的产酸能力也因而得到了大幅提高。尽管当前谷氨酸、赖氨酸工业菌株已达到高水平，然而进一步提高菌株产酸水平，特别是提高转化率(原料转化为产物的百分比)，降低生产成本的需求仍然很迫切。可是在高水平的谷氨酸、赖氨酸生产菌株上进一步提升，甚至是小幅提升都已经十分困难。
目前针对与氨基酸合成不明显相关的基因的改造比较少。其原因在于，众所周知细胞是一个复杂的代谢网络集合体，对不相关基因的改造对菌株造成的影响是不可预知的；另外从众多的基因中挖掘出与谷氨酸、赖氨酸生产可能相关的改造靶点的难度巨大。
MviN，是一种跨膜脂质II翻转酶，催化肽聚糖(PG)合成的最后一步，将脂质II从胞质内膜转运到细胞周质间隙，使新生PG交联形成成熟的PG。目前对于棒状杆菌属来源的MviN研究较少，仅有一篇专利(JP2010161970A)公开了该蛋白中的3个氨基酸残基(197位、260位、181位)突变可以提高谷氨酸棒杆菌的谷氨酸产量，但其谷氨酸产量提高幅度很低，与出发菌株相比仅仅提高了1-5％(产物百分比)，而对转化率的影响则没有报道。
发明内容
本发明的目的在于提供新的MviN蛋白突变体，以及利用该蛋白突变体显著提高宿主菌株生产谷氨酸、赖氨酸的转化率的方法。
第一方面，本发明提供了一种跨膜脂质II翻转酶(MviN)蛋白突变体，其氨基酸序列包括选自以下的组：
a)在对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8任一所示氨基酸序列的第346位脯氨酸被亮氨酸取代，或被其他脂肪烃侧链氨基酸例如异亮氨酸、缬氨酸或丙氨酸中的任一种所取代；和/或者第599位缬氨酸被苯丙氨酸或色氨酸取代；
b)与a)所述的氨基酸序列具有相同的突变位点，并且具有与a)所述的氨基酸序列90％以上，例如95％以上、98％以上的同源性，所述氨基酸序列来源于谷氨酸棒状杆菌(Corynebacterium glutamicum)，并保持保持脂质II从胞质内膜转运到细胞周质间隙的活性。
在一个实施方式中，所述MviN蛋白突变体进一步包括或不包括其他位点的突变。例如，所述MviN蛋白突变体包括来自于上述氨基酸序列的保守性突变，并且所述MviN蛋白突变体保持脂质II从胞质内膜转运到细胞周质间隙的活性。
第二方面，本发明提供了第一方面所述的MviN蛋白突变体的编码基因。
第三方面，本发明提供了包括第二方面所述的MviN蛋白突变体编码基因的表达载体；在一个实施方式中，所述表达载体是将编码基因的核苷酸序列与质粒相连构建而成，例如所述质粒选自pEC-XK99E。
第四方面，本发明提供了一种表达第一方面所述的MviN蛋白突变体的宿主细胞。
在优选的实施方式中，表达所述突变体的宿主细胞包含第三方面所述的表达载体或在其基因组中整合有第二方面所述的编码基因的核苷酸序列。在一个实施方式中，将所述表达载体导入原始宿主细胞中，形成含有所述突变体编码基因的宿主细胞。
在进一步优选的实施方式中，所述原始宿主细胞来自埃希氏菌属(Escherichia)、棒状杆菌属(Corynebacterium)、泛菌属(Pantoea)、短杆菌属(Brevibacterium sp)、芽孢杆菌属(Bacillus)、克雷伯氏菌属(Klebsiella)、沙雷氏菌属(Serratia)或弧菌属(Vibrio)。优选大肠杆菌(E.Coli)或谷氨酸棒状杆菌(Corynebacterium glutamicum)，更优选谷氨酸棒状杆菌(Corynebacterium glutamicum)。
第五方面，本发明提供第一方面所述的MviN蛋白突变体、或第二方面所述的编码基因、或第三方面所述的表达载体、或第四方面所述的宿主细胞在生产谷氨酸、赖氨酸中的应用。
在第六方面，本发明提供一种制备谷氨酸或赖氨酸的方法，所述方法包括以下步骤：
a.构建第四方面所述的宿主细胞；
b.培养步骤a构建的宿主细胞，使之产生含谷氨酸或赖氨酸的培养液；和
c.任选从步骤b的培养液中分离产生的谷氨酸或赖氨酸。
本发明的有益效果
1.本发明提供了新的MviN蛋白突变体，所述突变体经特定位点的氨基酸突变，能提高谷氨酸、赖氨酸的产量并显著提高葡萄糖转化率。
2.本发明提供了新的MviN蛋白的改造靶点，能提高菌株的生长速度，降低谷氨酸、赖氨酸的生产成本。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照设备制造厂商所建议的条件。
定义与说明：
本文所用的术语“跨膜脂质II翻转酶”和“膜蛋白”、“murein biosynthesis protein”、“MviN蛋白”、“本发明的蛋白”、“本发明的多肽”可互换使用，并具有本领域普通技术人员通常理解的含义。
本发明的“MviN蛋白”是指一种跨膜脂质II翻转酶，催化肽聚糖(PG)合成的最后一步，将脂质II从胞质内膜转运到细胞周质间隙。MviN蛋白可以来源于各种物种，包括但不限于埃希氏菌属(Escherichia)、棒状杆菌属(Corynebacterium)、泛菌属(Pantoea)、短杆菌属(Brevibacterium)、芽孢杆菌属(Bacillus)、克雷伯氏菌属(Klebsiella)、沙雷氏菌属(Serratia)或弧菌属(Vibrio)。关于本发明，MviN蛋白来源于棒状杆菌属，如SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列对应的蛋白；也可以是含有SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列且具有将脂质II从胞质内膜转运到细胞周质间隙活性的多肽；还可以是与SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列同源性大于 90％，优选95％，更优选98％以上来源于棒状杆菌属且具有将脂质II从胞质内膜转运到细胞周质间隙活性的多肽。
本发明的“MviN蛋白突变体”是指通过对氨基酸序列如SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列进行突变获得。具体地说，本发明的MviN蛋白突变体是氨基酸序列在对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列的第346位脯氨酸被亮氨酸取代，或者在对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列的第599位缬氨酸被苯丙氨酸取代；或者本发明的MviN蛋白突变体是包括上述突变位点的氨基酸序列的片段。关于本发明，也可以是含有SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列且具有将脂质II从胞质内膜转运到细胞周质间隙活性的多肽；还可以包括与SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列同源性大于90％，优选95％，最优选98％以上来源于谷氨酸棒杆菌且具有将脂质II从胞质内膜转运到胞质周间隙活性的多肽，只要其在对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列的第346位脯氨酸被亮氨酸取代，或在对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列的第599位缬氨酸被苯丙氨酸取代，也在本发明的范围内。
目前，NCBI数据库(https://www.ncbi.nlm.nih.gov/)中已经公布的来源于谷氨酸棒杆菌的“跨膜脂质II翻转酶”(数据库中部分序列注释为假定蛋白或者膜蛋白)的氨基酸序列共有22条，其中仅一条序列与本发明的SEQ ID NO:2所示序列的同源性为93％，其余21条序列与本发明的SEQ ID NO:2所示序列的同源性均大于98％；并且，全部的22条序列在对应于SEQ ID NO:2所示序列的第346位均为脯氨酸，在对应于SEQ ID NO:2所述序列的第599位均为缬氨酸。可见，在谷氨酸棒杆菌中，跨膜脂质II翻转酶是高度保守的，且在对应于SEQ ID NO:2所示序列的第346位或第599位也是高度保守的。进一步，本发明对来源于谷氨酸棒杆菌的4条“跨膜脂质II翻转酶”序列进行了改造，结果显示，将这4条中的任一条序列的第346位脯氨酸突变为亮氨酸，或将第599位缬氨酸突变为苯丙氨酸，均能够提高谷氨酸、赖氨酸的产量并显著提高葡萄糖转化率。因此，基于以上原因，其他与SEQ ID NO:2所示序列同源性大于98％且来源于谷氨酸棒杆菌的多肽，只要其在对应于SEQ ID NO:2所示序列的第346位的脯氨酸被亮氨酸取代，或者在对应于SEQ ID NO:2所示序列的第599位的缬氨酸被苯丙氨酸取代，均在本发明的保护范围内。
本文所用的术语“宿主细胞”是具有本领域普通技术人员通常理解的含义，即，含有本 发明所述MviN蛋白或其突变体的细胞。换言之，本发明可以利用任何宿主细胞，只要所述细胞中含有本发明所述的MviN蛋白或其突变体且能够生产氨基酸的细胞。所述宿主细胞可以来自埃希氏菌属(Escherichia)、棒状杆菌属(Corynebacterium)、泛菌属(Pantoea)、短杆菌属(Brevibacterium sp)、芽孢杆菌属(Bacillus)、克雷伯氏菌属(Klebsiella)、沙雷氏菌属(Serratia)或弧菌属(Vibrio)。优选大肠杆菌(E.Coli)或谷氨酸棒状杆菌(Corynebacterium glutamicum)，最优选谷氨酸棒状杆菌(Corynebacterium glutamicum)。具体地，本发明所述的宿主是指能够生产谷氨酸、赖氨酸的菌株。
本文所用的术语“含有本发明的MviN”具有本领域技术人员常规理解的含义，并且可以通过本领域已知的方法实施，包括但不限于，如：将包含编码蛋白的多核苷酸序列的多核苷酸插入到染色体上，或将多核苷酸克隆到载体上引入微生物，或在染色体上直接增加该多核苷酸的拷贝等方法来实现，也可以非限制性地包括任何已知的可以引入蛋白活性的方法。
本文所用的术语“具有MviN蛋白的活性”与本领域技术人员常规理解的含义相同或相似，均是指某一片段的氨基酸序列是完整蛋白或多肽的氨基酸序列的一部分，具备与完整蛋白或多肽相同或相似的功能或活性。具体地说，在本发明中，表示从本发明的MviN蛋白获得的具有MviN蛋白具有相同功能的任意氨基酸片段。本领域普通技术人员应当理解，在多肽的某些区域，例如非重要区域改变少数氨基酸残基基本上不会改变生物活性，例如，适当替换某些氨基酸得到的序列并不会影响其活性(可参见Watson等，Molecular Biology of The Gene，第四版，1987，The Benjamin/Cummings Pub.Co.P224)。因此，本领域普通技术人员能够实施这种替换并且确保所得分子仍具有所需生物活性。因此，对本发明的MviN作进一步突变而得到仍具备MviN功能的进一步突变体是显而易见的。例如，本领域技术人员公知在多肽的任一端增加或减少数个氨基酸残基，例如优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基不会影响得到的突变体的功能。例如，为便于纯化，技术人员往往在得到的蛋白的任一端带上6×His标签，而这种蛋白与不具备6×His标签的蛋白具有相同的功能。此外，本发明应包括本发明的MviN的保守性突变体。这些保守性突变体可以根据，例如下表所示进行氨基酸替换而产生。
初始残基 代表性的取代残基 优选的取代残基
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码本发明多肽的多核苷酸。术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
MviN蛋白的基因可以是在严格的条件下与SEQ ID NO:1或SEQ ID NO:3或SEQ ID NO:5或SEQ ID NO:7所示任一核苷酸序列制备的探针，例如与SEQ ID NO:1或SEQ ID NO:3或SEQ ID NO:5或SEQ ID NO:7所示任一核苷酸序列的部分或者整个序列互补的序列杂交的DNA，只要保持其初始功能即可。所述“严格的条件”是指可形成所谓的特异性杂交且未形成非特异性杂交的条件。例如，同源性较高的DNA，例如具有90％以上同源性的DNA彼此杂交，同源性低于90％的DNA彼此不杂交的条件，或者通常的Southern杂交的清洗条件，即在相当于60℃、1*SSC、0.1％SDS、优选60℃、0.1*SSC、0.1％SDS，更优选68℃、0.1*SSC、0.1％SDS的盐浓度及温度下清洗1次、优选2-3次的条件。
此外，由于密码子的简并性因宿主而异，MviN蛋白的基因中的任意密码子可以用相应的等价密码子替换，也就是说，MviN蛋白基因可以是由于遗传密码的简并性而在上面例如的任何MviN蛋白基因的突变。例如，MviN蛋白基因可以是经修饰的基因，使得其具有根据要使用的宿主中密码子频率的最佳密码子。
在具体的实施方式中，所述MviN蛋白基因可以是与编码SEQ ID NO:2或SEQ ID NO: 4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列的DNA同源性或序列相同性在90％以上，优选95％以上，更优选99％的DNA。
因此，基于本发明的教导和本发明具体得到的MviN蛋白，本领域技术人员不难得到具有相同或相似活性或功能的活性片段，这样的活性片段可以是将本发明的突变位点(对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8的第346位、第599位)突变成为其他氨基酸残基得到的活性片段，也可以是对本发明所述突变位点(对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8的第346位、第599位)的相邻或相近位点进行突变而得到的活性片段，这样的活性片段应该视为本发明的等同权利而落入本发明的保护范围内。
本文所用的术语“对应于”具有本领域普通技术人员通常理解的意义。具体地说，“对应于”表示两条序列经同源性或序列相同性比对后，一条序列与另一条序列中的指定位置相对应的位置。因此，例如，就“对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列的第40位的氨基酸残基”而言，如果在SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列的一端加上6×His标签，那么所得突变体中对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示任一氨基酸序列的第40位就可能是第46位。
在具体的实施方式中，所述同源性或序列相同性可以是90％以上，优选95％以上，更优选98％的同源性且来源于棒状杆菌属。
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987；序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991；和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手 册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。
实施例：
实施例1、在谷氨酸棒杆菌菌株Z188和B253基因组中引入MviN基因突变
在对产谷氨酸的谷氨酸棒杆菌Z188(GenBank Accession number：NZ_AKXP00000000)进行诱变筛选时，我们获得两株突变菌，基因组测序分析发现，一株突变株对应于Z188的MviN基因核苷酸序列第1037位点的C突变为T(对应的氨基酸在第346位点的P突变为L)，另一株突变株对应于其MviN基因核苷酸序列第1795位点的G突变为T(对应的氨基酸在第599位点的V突变为F)。为了测试不同来源MviN的P346L和V599F突变对Z188产谷氨酸和B253(GeneBank accession number CP010451)产赖氨酸的影响，选择了来源于谷氨酸棒杆菌Z188的MviN(命名为MviNZ，核苷酸和氨基酸序列分别为SEQ ID NO:1和SEQ ID NO:2)，来源于谷氨酸棒杆菌B253的MviN(命名为MviNB，核苷酸和氨基酸序列分别为SEQ ID NO:3和SEQ ID NO:4)，来源于谷氨酸棒杆菌ATCC 13869(GeneBank accession number CP016335)的MviN(命名为MviN9，核苷酸和氨基酸序列分别为SEQ ID NO:5和SEQ ID NO:6)，来源于谷氨酸棒杆菌ATCC 13032(GeneBank accession number CP025533)的MviN(命名为MviN2，核苷酸和氨基酸序列分别为SEQ ID NO:7和SEQ ID NO:8)。
利用同源重组的方法对Z188和B253的MviN基因进行突变。利用引物MF(SEQ ID NO:9CCTCGAATTCATGAATGGTCAACAAGTGAGTTCTTC)和引物MR1(SEQ I D NO:10TCTACTGCAGTTACCAACCAACGAGTTGTACTTCAG)，分别以Z188，B253，ATCC 13869基因组为模板，进行PCR扩增；利用引物MF和引物MR2(SEQ ID NO:11TCTACTGCAGTTACCAACCAACAAGTTGTACTTCAG)，以ATCC 13032基因组为模板，进行PCR扩增。利用限制性内切酶EcoRI和PstI对PCR片段分别进行双酶切后，分别连接到同样双酶切的质粒pK18mobsacB(GenBank Accession number：FJ437239)上，获得相应质粒pK18-MviNZ，pK18-MviNB，pK18-MviN9，pK18-MviN2。利用引物ATGCTGCGCCGTTTATTTAT(SEQ ID NO:12)和ATAAATAAACGGCGCAGCAT(SEQ ID NO:13)，分别以pK18-MviNZ，pK18-MviN9和pK18-MviN2为模板，PCR扩增；利用引物TGATGCTGCACCGTTTATTT(SEQ ID NO:14)和AAATAAACGGTGCAGCATCA(SEQ ID NO:15)，以pK18-MviNB为模板，PCR扩增。利用一步法定向克隆试剂盒(The ClonExpress II One Step Cloning kit，南京诺唯赞生物科技有限公司，中国)，将PCR 产物分别自连，获得相应质粒上MviN的P346L突变，突变后的质粒分别命名为pK18-Mvi NZ P346L，pK18-MviNB P346L，pK18-MviN9 P346L，pK18-MviN2 P346L。利用引物TCTGCCAGAGGTCCAAAACT(SEQ ID NO:16)和AGTTTTGGACCTCTGGCAGA(SEQ ID NO:17)，分别以pK18-MviNZ，pK18-MviNB和pK18-MviN9为模板，PCR扩增；利用引物TTTGCCGGAGGTCCAAAACT(SEQ ID NO:18)和AGTTTTGGACCTCCGGCAAA(SEQ ID NO:19)，以pK18-MviN2为模板，PCR扩增。利用一步法定向克隆试剂盒(The ClonExpress II One Step Cloning kit，南京诺唯赞生物科技有限公司，中国)，将PCR产物分别自连，获得相应质粒上MviN的V599F突变，突变后的质粒分别命名为pK18-MviNZ V599F，pK18-MviNB V599F，pK18-MviN9 V599F，pK18-MviN2 V599F。
将质粒pK18-MviNZ，pK18-MviNB，pK18-MviN9，pK18-MviN2，pK18-MviNZ P346L，pK18-MviNB P346L，pK18-MviN9 P346L，pK18-MviN2 P346L，pK18-MviNZ V599F，pK18-MviNB V 599F，pK18-MviN9 V599F，pK18-MviN2 V599F分别导入Z188和B253感受态细胞中(其中，pK18-MviNZ无需导入Z188中，pK18-MviNB无需导入B253中)，利用LBG培养基(葡萄糖10g/L，酵母粉5g/L，蛋白胨10g/L，氯化钠10g/L)过夜培养后，在含有卡那霉素(25mg/L)的LBG固体平板上挑选具有卡那霉素抗性的克隆。挑取克隆后，在含有10g/L蔗糖的LBG液体培养基中过夜培养，涂布含有10g/L蔗糖的LBG固体平板，挑选能够生长的克隆，并利用含有卡那霉素(25mg/L)的LBG固体平板验证其丢失了卡那霉素抗性基因从而无法生长。利用MF和MR1扩增所获克隆的基因，通过测序选择Z188或B253基因组上原有的MviN基因被相应不同来源的MviN及其突变体完全替换的克隆，从而获得菌株Z188-MviNB，Z188-MviN9，Z188-MviN2，Z188-MviNZ P346L，Z188-MviNB P346L，Z188-MviN9 P 346L，Z188-MviN2 P346L，Z188-MviNZ V599F，Z188-MviNB V599F，Z188-MviN9 V599F Z188-Mvi N2 V599F，以及菌株B253-MviNZ，B253-MviN9，B253-MviN2，B253-MviNZ P346L，B253-Mv iNB P346L，B253-MviN9 P346L，B253-MviN2 P346L，B253-MviNZ V599F，B253-MviNB V599F，B253-MviN9 V599F，B253-MviN2 V599F。
实施例2、在谷氨酸棒杆菌菌株Z188和B253中利用质粒过表达不同MviN基因突变体
首先从不同菌株中克隆MviN基因及其突变体。利用引物MV-F(SEQ ID NO:20CTCGATGGAATTC CGTTAAGGCGCGCACTCGTACATGAATGGTC)和MV-R(SEQ ID NO:21CGCGGGATCCTTACCAACCAACGAGTTGTACTTCAGCGATC)，分别以Z188-MviNZ P346L，Z188-MviNZ V599F，B253-MviNB P346L，B253-MviNB V599F基因组为模板， PCR扩增，扩增产物分别用EcoRI和BamHI双酶切，然后分别连接到同样用EcoRI和BamHI双酶切的质粒pEC-XK99E【Kirchner O,Tauch A.J Biotechnol.2003,104(1-3):287-299】上，分别获得pEC-MviNZ P346L和pEC-MviNZ V599F，以及pEC-MviNB P346L和pEC-MviNB V599F。另外，将EcoRI和BamHI双酶切的质粒pEC-XK99E末端补平后自连，获得不含MviN基因的对照质粒，命名为pEC。将质粒pEC，pEC-MviNZ P346L和pEC-MviNZ V599F分别转化到Z188中，获得菌株Z188(pEC)，Z188(pEC-MviNZ P346L)和Z188(pEC-MviNZ V599F)。将质粒pEC，pEC-MviNB P346L和pEC-MviNB V599F分别转化到B253中，分别获得菌株B253(pEC)，B253(pEC-MviNB P346L)和B253(pEC-MviNB V599F)。
实施例3、谷氨酸棒杆菌Z188及其MviN基因突变菌株发酵产谷氨酸测试
利用96孔板测试Z188菌株，基因组上MviN突变的菌株Z188-MviNB，Z188-MviN9，Z188-MviN2，Z188-MviNZ P346L，Z188-MviNB P346L，Z188-MviN9 P346L，Z188-MviN2 P346L，Z188-MviNZ V599F，Z188-MviNB V599F，Z188-MviN9 V599F，Z188-MviN2 V599F，以及利用质粒过表达MviNZ突变体的菌株Z188(pEC-MviNZ P346L)和Z188(pEC-MviNZ V599F)和其对照菌株Z188(pEC)，发酵产谷氨酸水平。种子培养基为：葡萄糖50g/L，磷酸0.7g/L，七水硫酸镁0.8g/L，硫酸铵10g/L，3-(N-玛琳代)丙磺酸84g/L，玉米浆粉3g/L，尿素10g/L，蛋白胨1g/L，酵母粉0.5g/L，用氢氧化钠调pH为7.0。发酵培养基与种子培养基的区别为发酵培养基中不添加蛋白胨和酵母粉。培养Z188(pEC)，Z188(pEC-MviNZ P346L)和Z188(pEC-MviNZ V599F)时，加入终浓度为0.1mM的异丙基硫代半乳糖苷(IPTG)，以及终浓度为25μg/L的卡那霉素。菌株在种子培养基中过夜培养后，以10％的接种量接种到发酵培养基中，96孔板的每个孔中含有150μL发酵培养基，每个菌株3个平行实验，培养温度为30摄氏度，孔板摇床转速为800rpm，培养33小时后检测谷氨酸产量和从葡萄糖到谷氨酸的转化率，部分结果如表1和表2所示。由表1和表2可以看出，通过在基因组上引入带有P346L或V599F突变位点的不同来源MviN，以及通过质粒过表达带有P346L或V599F突变位点的MviN，都能够提高菌株产谷氨酸的水平，转化率相对提高15.77-22.29％。另外，相对于Z188-MviNB，Z188-MviN9以及Z188-MviN2，在基因组上引入含有P346L或V599F点突变的菌株，其产谷氨酸的转化率相对提高9.06-22.18％。
表1 Z188基因组不同突变体的发酵产谷氨酸水平
Figure PCTCN2019104683-appb-000001
表2菌株Z188(pEC)，Z188(pEC-MviNZ P346L)和Z188(pEC-MviNZ V599F)发酵产谷氨酸水平
Figure PCTCN2019104683-appb-000002
实施例4、谷氨酸棒杆菌B253及其MviN基因突变菌株发酵产赖氨酸测试
利用96孔板测试B253菌株，基因组上MviN突变的菌株B253-MviNZ，B253-MviN9，B253-MviN2，B253-MviNZ P346L，B253-MviNB P346L，B253-MviN9 P346L，B253-MviN2 P346L，B253-MviNZ V599F，B253-MviNB V599F，B253-MviN9 V599F，B253-MviN2 V599F，以及利用质粒过表达MviNB突变体的菌株B253(pEC-MviNB P346L)、B253(pEC-MviNB V599F)和其对照菌株B253(pEC)发酵产赖氨酸水平。利用LBG作为种子培养基，发酵培养基成份为：葡萄糖80g/L；酵母粉8g/L；尿素9g/L；K 2HPO 4 1.5g/L；MnSO 4 0.01g/L；MgSO 4 0.6g/L；FeSO 40.01g/L；MOPS 42g/L。培养时B253(pEC)，B253(pEC-MviNB P346L)和B253(pEC-MviNB V599F)时加入终浓度为0.1mM的IPTG，以及终浓度为25μg/L的卡那霉素。首 先将菌株接种到LBG培养基中过夜培养，培养物作为种子接种到每孔含有150μL发酵培养基的96孔深孔板中，接种量为5％，30℃培养48小时，孔板摇床转速为800rpm，每个菌株3个平行实验，发酵结束后检测赖氨酸产量和从葡萄糖到赖氨酸的转化率。质粒过表达MviNB突变体菌株测试结果如表3和表4所示。可以看出，通过质粒过表达带有P346L或V599F突变位点的MviN，能够提高菌株产赖氨酸的水平，转化率相对提高7.48％。相对于B253，B253-MviNZ，B253-MviN9以及B253-MviN2，在基因组上引入含有P346L或V599F点突变的菌株，其产赖氨酸的转化率相对提高5.23-8.22％。
表3 B253(pEC)，B253(pEC-MviNB P346L)和B253(pEC-MviNB V599F)发酵产赖氨酸水平
Figure PCTCN2019104683-appb-000003
表4 B253基因组不同突变体的发酵产赖氨酸水平
Figure PCTCN2019104683-appb-000004
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 一种跨膜脂质II翻转酶(MviN)蛋白突变体，其特征在于，所述蛋白突变体的氨基酸序列选自如下的组：
    a)在对应于SEQ ID NO:2或SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8任一所示氨基酸序列的第346位脯氨酸被亮氨酸取代；和/或者第599位缬氨酸被苯丙氨酸取代；或者
    b)与a)所述的氨基酸序列具有相同的突变位点，并且具有与a)所述的氨基酸序列90％以上，例如95％以上、98％以上的同源性，所述氨基酸序列来源于谷氨酸棒状杆菌(Corynebacterium glutamicum)，并保持脂质II从胞质内膜转运到细胞周质间隙的活性。
  2. 如权利要求1所述MviN蛋白突变体的编码基因。
  3. 包含权利要求2所述的编码基因的表达载体。
  4. 一种宿主细胞，其特征在于，所述宿主细胞表达权利要求1所述的MviN蛋白突变体，或者含有权利要求2所述的编码基因。
  5. 根据权利要求4所述的宿主细胞，其特征在于，所述宿主细胞含有所述MviN蛋白突变体编码基因的表达载体,或者在所述宿主细胞的基因组中整合有编码所述MviN蛋白突变体的基因。
  6. 根据权利要求4或5所述的宿主细胞，其特征在于，所述宿主细胞来源于棒状杆菌属。
  7. 如权利要求1所述的MviN蛋白突变体、权利要求2所述的编码基因、权利要求3所述的表达载体、权利要求4或权利要求5所述的宿主细胞在生产谷氨酸或赖氨酸中的应用。
  8. 一种制备谷氨酸或赖氨酸的方法，其特征在于，包括以下步骤：
    a.将MviN蛋白突变体的编码基因导入到产谷氨酸的谷氨酸棒杆菌中，或者导入到产赖氨酸的谷氨酸棒杆菌中，构建权利要求4-6任一所述的宿主细胞；
    b.培养步骤a构建的宿主细胞，使之产生含谷氨酸或赖氨酸的培养液；和
    c.任选从步骤b的培养液中分离产生的谷氨酸或赖氨酸。
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RUDNICK PAUL A., ARCONDÉGUY TANIA, KENNEDY CHRISTINA K., KAHN DANIEL: "glnD and mviN Are Genes of an Essential Operon in Sinorhizobium meliloti", JOURNAL OF BACTERIOLOGY, vol. 183, no. 8, 15 April 2001 (2001-04-15), pages 2682 - 2685, XP055788340, ISSN: 0021-9193, DOI: 10.1128/JB.183.8.2682-2685.2001 *
ULLAND TYLER K., BUCHAN BLAKE W., KETTERER MARGARET R., FERNANDES-ALNEMRI TERESA, MEYERHOLZ DAVID K., APICELLA MICHAEL A., ALNEMRI: "Cutting Edge: Mutation of Francisella tularensis mviN Leads to Increased Macrophage Absent in Melanoma 2 Inflammasome Activation and a Loss of Virulence", THE JOURNAL OF IMMUNOLOGY, WILLIAMS & WILKINS CO., US, vol. 185, no. 5, 1 September 2010 (2010-09-01), US, pages 2670 - 2674, XP055788343, ISSN: 0022-1767, DOI: 10.4049/jimmunol.1001610 *

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