YH66_10325 gene modified recombinant strain producing L-isoleucine and construction method and application thereof

文档序号：842703 发布日期：2021-04-02 浏览：20次中文

阅读说明：本技术 一种yh66_10325基因改造的产l-异亮氨酸重组菌株及其构建方法与应用 (YH66_10325 gene modified recombinant strain producing L-isoleucine and construction method and application thereof ) 是由郭小炜赵春光孟刚魏爱英杨立鹏田斌马风勇周晓群苏厚波于 2020-12-28 设计创作，主要内容包括：本发明公开了一种基于YH66-10325基因的产L-异亮氨酸的重组菌株及其构建方法与应用。本发明通过对YH66-10325基因的敲除,发现该基因编码的产物对L-异亮氨酸生产能力产生影响,通过在编码序列引入点突变,或者增加该基因的拷贝数或过表达获得重组菌株,所述点突变是使YH66-10325基因序列第925位碱基由鸟嘌呤(G)突变为腺嘌呤(A),使编码的相应氨基酸序列第309位丙氨酸变为苏氨酸。所获得的菌株与未改造的菌株相比,有利于生产高浓度的L-异亮氨酸。(The invention discloses a YH66_10325 gene-based recombinant strain for producing L-isoleucine, and a construction method and application thereof. The present invention discovers that the product coded by the gene has influence on the L-isoleucine production capacity by knocking out the YH66_10325 gene, and obtains a recombinant strain by introducing a point mutation into the coding sequence, or increasing the copy number or overexpression of the gene, wherein the point mutation is that the 925 th base of the YH66_10325 gene sequence is mutated from guanine (G) to adenine (A), and the 309 th alanine of the coded corresponding amino acid sequence is changed into threonine. The obtained strain is advantageous for producing L-isoleucine at a high concentration as compared with an unmodified strain.)

1. A polynucleotide comprising a polynucleotide encoding an amino acid sequence set forth in SEQ ID No. 3 wherein alanine at position 309 is replaced with a different amino acid; preferably alanine 309 is replaced by threonine;

preferably, the polynucleotide comprises a polynucleotide encoding an amino acid sequence shown in SEQ ID NO. 4;

preferably, the polynucleotide is formed by mutation of 925 th base of the polynucleotide sequence shown in SEQ ID NO. 1; preferably, the mutation is that the 925 site base of the polynucleotide sequence shown in SEQ ID NO. 1 is mutated from guanine (G) to adenine (A);

preferably, the polynucleotide comprises the polynucleotide sequence shown in SEQ ID NO. 2.

2. A protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 4.

3. Recombinant vector, expression cassette, transgenic cell line and/or recombinant bacterium comprising the polynucleotide of claim 1 and/or the protein of claim 2.

4. Use of the polynucleotide of claim 1, the protein of claim 2, the recombinant vector, the expression cassette, the transgenic cell line and/or the recombinant bacterium of claim 3 for the production of L-isoleucine.

5. An L-isoleucine producing bacterium having improved expression of a polynucleotide having an amino acid sequence encoding SEQ ID NO. 3;

preferably, the improved expression is an increased expression of the polynucleotide encoding the amino acid sequence of SEQ ID NO. 3, or the polynucleotide encoding the amino acid sequence of SEQ ID NO. 3 has a point mutation and the expression is increased.

6. The bacterium of claim 5, wherein the polynucleotide encoding the amino acid sequence of SEQ ID NO. 3 is point mutated such that alanine at position 309 of the amino acid sequence of SEQ ID NO. 3 is replaced with a different amino acid; preferably, alanine 309 is replaced by threonine.

7. The bacterium of claim 5 or 6, wherein the polynucleotide encoding the amino acid sequence of SEQ ID NO. 3 comprises the nucleotide sequence of SEQ ID NO. 1.

8. The bacterium of any one of claims 5 to 7, wherein the polynucleotide sequence having the point mutation is formed by mutation of base 925 of the polynucleotide sequence shown in SEQ ID NO. 1;

preferably, the mutation comprises that the 925 site base of the polynucleotide sequence shown in SEQ ID NO. 1 is mutated from guanine (G) to adenine (A);

preferably, the polynucleotide sequence having point mutations comprises the polynucleotide sequence shown in SEQ ID NO. 2.

9. The bacterium according to any one of claims 5 to 8, wherein said bacterium is a bacterium of the genus Corynebacterium, preferably Corynebacterium acetoacidophilum (Corynebacterium acetoacidophilum), Corynebacterium acetoglutamicum (Corynebacterium acetoglutacter), Corynebacterium glutamicum (Corynebacterium glutamicum), Brevibacterium flavum (Brevibacterium flavum), Brevibacterium lactofermentum (Brevibacterium lactofermentum), Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Corynebacterium pekinense (Corynebacterium pekinense), Brevibacterium saccharolyticum (Brevibacterium saccharolyticum), Brevibacterium roseum (Brevibacterium roseum), Brevibacterium thionasum (Brevibacterium thiogeniticum); preferably Corynebacterium glutamicum YPILE001, with the biological collection number CGMCC No.20437, or Corynebacterium glutamicum ATCC 13869.

10. A method of producing L-isoleucine, said method comprising: culturing the bacterium of any one of claims 5-9, and recovering L-isoleucine from the culture.

Technical Field

The invention belongs to the technical field of genetic engineering and microorganisms, and particularly relates to a YH 66-10325 gene modified recombinant strain for producing L-isoleucine, a construction method and application thereof.

Background

L-isoleucine is one of eight essential amino acids in human body, and is one of three branched chain amino acids, and has a particularly important position in human life metabolism due to its special structure and function. Therefore, the method is widely applied to industries such as food, animal feed and medicine. The production method of L-isoleucine mainly includes protein hydrolysis method, chemical synthesis method and biological fermentation method. The biological fermentation method is a preferred method for industrial production due to low raw material cost, easy control, energy conservation and environmental protection. At present, the yield of L-isoleucine is not high, and the breeding of high-yield strains becomes an important link for improving the yield of L-isoleucine.

The L-isoleucine-producing strains are mainly Corynebacterium glutamicum (Corynebacterium glutamicum) and Escherichia coli (Escherichia coli). Corynebacterium glutamicum has the characteristics of no endotoxin, less metabolic isozymes, contribution to releasing feedback inhibition or transcription attenuation, strong feedback inhibition resistance of key metabolic enzymes and the like, so most researchers tend to research Corynebacterium glutamicum and subspecies thereof, such as Corynebacterium glutamicum (Brevibacterium flavum), Brevibacterium lactofermentum (Brevibacterium lactofermentum) and the like. The breeding of excellent strains is still the key of high-yield L-isoleucine, and with the development of high-throughput screening technology, the traditional mutagenesis technology is combined, which is helpful for finding out high-yield L-isoleucine mutant strains with unknown characters.

Although some strains have been screened for improved L-isoleucine production, more L-isoleucine-producing mutants still need to be found to meet the increasing demand.

Disclosure of Invention

In view of the disadvantages of the prior art, the present invention provides a polynucleotide and a recombinant strain containing the polynucleotide, and the recombinant strain is used for improving the L-isoleucine-producing ability of bacteria. In order to achieve the above objects, the inventors of the present invention have studied and found that YH66_10325 gene (GenBank: AKF27920.1) in Corynebacterium glutamicum ATCC15168 genome (GenBank: CP011309.1) having L-isoleucine-producing ability can obtain a recombinant strain having an increased L-isoleucine-producing ability as compared with an unmodified wild-type strain by modifying the gene or improving the expression thereof.

The invention is realized by adopting the following technical scheme:

in a first aspect of the present invention, there is provided an L-isoleucine producing bacterium having improved expression of a polynucleotide encoding an amino acid sequence shown as SEQ ID NO. 3. According to the invention, the improved expression is that the expression of the polynucleotide is enhanced, or that the polynucleotide encoding the amino acid sequence of SEQ ID NO. 3 has a point mutation and that the expression is enhanced.

The amino acid sequence of SEQ ID NO. 3 is the protein encoded by gene YH66_ 10325.

The bacterium has an enhanced L-isoleucine-producing ability as compared with an unmodified strain.

In the present invention, the term "bacterium having an L-isoleucine-producing ability" refers to a bacterium having an ability to produce and accumulate L-isoleucine of interest in a medium and/or cells of the bacterium to such an extent that the L-isoleucine can be collected when the bacterium is cultured in the medium. The bacterium having an L-isoleucine-producing ability may be a bacterium capable of accumulating L-isoleucine of interest in a culture medium and/or cells of the bacterium in a larger amount than that obtainable from an unmodified strain.

The term "unmodified strain" refers to a control strain that has not been modified in a manner such that it has particular characteristics. That is, examples of the unmodified strain include a wild-type strain and a parent strain.

In the present invention, the term "L-isoleucine" means L-isoleucine in a free form, a salt thereof or a mixture thereof, unless otherwise specified.

The polynucleotide may encode an amino acid sequence having about 90% or greater, about 92% or greater, about 95% or greater, about 97% or greater, about 98% or greater, or about 99% or greater sequence homology to the amino acid sequence of SEQ ID NO. 3. As used herein, the term "homology" refers to the percent identity between two polynucleotides or two polypeptide modules. Sequence homology between one module and another can be determined by using methods known in the art. Such sequence homology can be determined, for example, by the BLAST algorithm.

Expression of the polynucleotide may be enhanced as follows: expression of regulatory sequences by substitution or mutation, introduction of mutation into polynucleotide sequences, increase in the copy number of polynucleotides by introduction via chromosomal insertion or vectors, or combinations thereof, and the like.

The expression regulatory sequence of the polynucleotide may be modified. Expression regulatory sequences control the expression of the polynucleotide to which they are operably linked and may include, for example, promoters, terminators, enhancers, silencers, and the like. The polynucleotide may have a change in the initiation codon. The polynucleotides may be incorporated into specific sites of the chromosome, thereby increasing copy number. Herein, the specific site may include, for example, a transposon site or an intergenic site. Alternatively, the polynucleotide may be incorporated into an expression vector, which is introduced into a host cell, thereby increasing copy number.

In one embodiment of the invention, the copy number is increased by incorporating the polynucleotide or a polynucleotide having a point mutation into a specific site of the chromosome of the microorganism.

In one embodiment of the invention, the nucleic acid sequence is overexpressed by incorporating a polynucleotide with a promoter sequence or a polynucleotide with a point mutation with a promoter sequence into a specific site of the chromosome of the microorganism.

In one embodiment of the invention, the polynucleotide or the polynucleotide having a point mutation is incorporated into an expression vector, and the expression vector is introduced into a host cell, thereby increasing the copy number.

In one embodiment of the invention, the polynucleotide with a promoter sequence or the polynucleotide with a point mutation with a promoter sequence is incorporated into an expression vector, and the expression vector is introduced into a host cell, thereby overexpressing the amino acid sequence.

In one embodiment of the invention, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO. 1.

In one embodiment of the invention, the polynucleotide encoding the amino acid sequence of SEQ ID NO. 3 has a point mutation such that alanine at position 309 of the amino acid sequence of SEQ ID NO. 3 is replaced by a different amino acid.

According to the invention, it is preferred that alanine 309 is replaced by threonine.

According to the present invention, the amino acid sequence shown in SEQ ID NO. 3, wherein the amino acid sequence in which alanine at position 309 is replaced with threonine is shown in SEQ ID NO. 4.

In one embodiment of the present invention, the polynucleotide sequence having a point mutation is formed by mutation at the 925 nd base of the polynucleotide sequence shown in SEQ ID NO. 1.

According to the invention, the mutation comprises that base 925 of the polynucleotide sequence shown in SEQ ID NO. 1 is mutated from guanine (G) to adenine (A).

In one embodiment of the present invention, the polynucleotide sequence having a point mutation comprises the polynucleotide sequence shown in SEQ ID NO. 2.

As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a polynucleotide sequence, whereby the regulatory sequence controls transcription and/or translation of the polynucleotide sequence. The regulatory sequence may be a strong promoter capable of increasing the expression level of the polynucleotide. The regulatory sequence may be a promoter derived from a microorganism belonging to the genus Corynebacterium or may be a promoter derived from other microorganisms. For example, the promoter may be trc promoter, gap promoter, tac promoter, T7 promoter, lac promoter, trp promoter, araBAD promoter, or cj7 promoter.

In one embodiment of the present invention, the promoter is the promoter of the polynucleotide (YH 66-10325) encoding the amino acid sequence of SEQ ID NO. 3.

As used herein, the term "vector" refers to a polynucleotide construct containing the regulatory sequences of a gene and the gene sequences and configured to express a target gene in a suitable host cell. Alternatively, a vector may in turn be a polynucleotide construct containing sequences useful for homologous recombination, so that the regulatory sequences of an endogenous gene in the genome of a host cell may be altered or a target gene which may be expressed may be inserted into a specific site in the genome of a host cell as a result of the vector introduced into the host cell. In this regard, the vector used in the present invention may further comprise a selection marker to determine the introduction of the vector into the host cell or the insertion of the vector into the chromosome of the host cell. The selection marker may comprise a marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to a cytotoxic agent, or expression of a surface protein. In the context of treatment with such a selection agent, transformed cells may be selected as cells that only express the selection marker may survive or display a different phenotypic trait.

In some embodiments of the invention, the vector used is the pK18mobsacB plasmid, the pXMJ19 plasmid.

As used herein, the term "transformation" refers to the introduction of a polynucleotide into a host cell such that the polynucleotide can replicate as an extra-genomic element or as an insert into the genome of the host cell. The method of transforming the vector used in the present invention may include a method of introducing a nucleic acid into a cell. In addition, as disclosed in the related art, the electric pulse method may be performed depending on the host cell.

According to the present invention, the bacterium may be a microorganism belonging to the genus Corynebacterium, such as Corynebacterium acetoacidophilum (Corynebacterium acetoacidophilum), Corynebacterium acetoglutamicum (Corynebacterium acetobacter), Corynebacterium melaleucum (Corynebacterium callunae), Corynebacterium glutamicum (Corynebacterium glutamicum), Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Corynebacterium beijerinckii (Corynebacterium pekinense), Brevibacterium saccharolyticum (Brevibacterium saccharolyticum), Brevibacterium roseum (Brevibacterium roseum), Brevibacterium thiogenes (Brevibacterium thiogenes), and the like.

In one embodiment of the present invention, the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum ATCC 15168.

In one embodiment of the present invention, the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum YPILE001, which has been deposited at the general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at 17.08.2020 at the following deposition address: west road No. 1, north chen, chaoyang district, beijing, zip code: 100101, preservation organization abbreviation: CGMCC, with the biological preservation number of CGMCC No. 20437.

In a second aspect of the invention, there is provided a polynucleotide sequence, an amino acid sequence encoded by the polynucleotide sequence, a recombinant vector comprising the polynucleotide sequence, a recombinant strain comprising the polynucleotide sequence.

According to the invention, the polynucleotide sequence comprises a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 3, in which alanine at position 309 is replaced by a different amino acid.

According to the invention, it is preferred that alanine 309 is replaced by threonine. .

According to the present invention, it is preferred that the polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 3 comprises the polynucleotide sequence shown in SEQ ID NO. 1.

In one embodiment of the present invention, the polynucleotide sequence is formed by mutation at base 925 of the polynucleotide sequence shown in SEQ ID NO. 1.

According to the present invention, the mutation refers to a change in the base/nucleotide at the site, and the mutation method may be at least one selected from the group consisting of mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination. In the present invention, PCR site-directed mutagenesis and/or homologous recombination are preferably used.

According to the invention, the mutation comprises a mutation of guanine (G) to adenine (A) at position 925 of the polynucleotide sequence shown in SEQ ID NO. 1.

In one embodiment of the invention, the polynucleotide sequence comprises the polynucleotide sequence shown in SEQ ID NO. 2.

According to the invention, the amino acid sequence comprises the amino acid sequence shown as SEQ ID NO. 4.

According to the invention, the recombinant vector is constructed by introducing the polynucleotide sequence into a plasmid.

In one embodiment of the invention, the plasmid is a pK18mobsacB plasmid.

In another embodiment of the invention, the plasmid is the pXMJ19 plasmid.

In particular, the polynucleotide sequence and the plasmid may be constructed into a recombinant vector by a NEBuider recombination system.

According to the invention, said recombinant strain contains said polynucleotide sequence.

As an embodiment of the invention, the germination bacteria of the recombinant strain is Corynebacterium glutamicum YPILE001, and the biological preservation number is CGMCC No. 20437.

As an embodiment of the present invention, the recombinant strain has an outgrowth of ATCC 15168.

In a third aspect of the present invention, there are provided the above polynucleotide sequence, an amino acid sequence encoded by the polynucleotide sequence, a recombinant vector comprising the polynucleotide sequence, and the use of a recombinant strain comprising the polynucleotide sequence for the production of L-isoleucine.

In the fourth aspect of the invention, a method for constructing a recombinant strain which can generate L-isoleucine is also provided.

According to the invention, the construction method comprises the following steps:

the polynucleotide sequence of the wild type YH66_10325 gene shown as SEQ ID NO. 1 in the host strain is transformed, so that the 925 th base of the wild type YH66_10325 gene is mutated, and the recombinant strain containing the mutant YH66_10325 encoding gene is obtained.

According to the construction method of the invention, the modification comprises at least one of mutagenesis, PCR site-directed mutagenesis, homologous recombination and the like.

According to the construction method of the invention, the mutation is that the 925 th base of SEQ ID NO. 1 is mutated from guanine (G) to adenine (A); specifically, the polynucleotide sequence of the gene encoding mutant YH66_10325 is shown as SEQ ID NO. 2.

Further, the construction method comprises the following steps:

(1) modifying the nucleotide sequence of a wild YH66_10325 gene shown as SEQ ID NO. 1 to make the 925 th base generate mutation, and obtaining the mutant YH66_10325 gene polynucleotide sequence;

(2) connecting the mutated polynucleotide sequence with a plasmid to construct a recombinant vector;

(3) and (3) introducing the recombinant vector into a host strain to obtain the recombinant strain containing the mutant YH66_10325 encoding gene.

According to the construction method of the present invention, the step (1) includes: construction of point-mutated YH66_10325 Gene: synthesizing two pairs of primers P1 and P2 and P3 and P4 for amplifying YH66_10325 gene fragment according to genome sequence of unmodified strain, introducing point mutation in SEQ ID NO:1 of wild-type YH66_10325 gene by PCR (polymerase chain reaction) site-directed mutagenesis method to obtain the nucleotide sequence SEQ ID NO:2 of point-mutated YH66_10325 gene, which is marked as YH66_10325^G925A。

In one embodiment of the present invention, the genome of the unmodified strain may be derived from ATCC15168 strain, whose genome sequence GenBank: CP011309.1 may be obtained from the NCBI website.

In one embodiment of the present invention, in the step (1), the primers are:

P1:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTCTGAAGGCAGCT CAATGC 3'(SEQ ID NO:5)

P2:5'GGTGGCTTCTCCTTCGAAGGCACATCTCCTCGTCAC 3'(SEQ ID NO:6)

P3:5'GTGACGAGGAGATGTGCCTTCGAAGGAGAAGCCACC 3'(SEQ ID NO: 7)

P4:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTCTGTAGATG ACGTGATC 3'(SEQ ID NO:8)

in one embodiment of the invention, the PCR amplification is performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 40s (30 cycles), and over-extension at 72 ℃ for 10 min.

In one embodiment of the invention, the overlapping PCR amplification is performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 90s (30 cycles), and over-extension at 72 ℃ for 10 min.

According to the construction method of the present invention, the step (2) comprises construction of a recombinant plasmid comprising: separating and purifying YH66_10325^G925AAnd pK18mobsacB plasmid, and assembling through a NEBuider recombination system to obtain a recombinant plasmid.

According to the construction method of the present invention, the step (3) includes constructing a recombinant strain, and transforming the recombinant plasmid into a host strain to obtain the recombinant strain.

In one embodiment of the present invention, the conversion of step (3) is an electrical conversion process.

In one embodiment of the present invention, the host strain is ATCC 15168.

In one embodiment of the invention, the host strain is Corynebacterium glutamicum YPILE001 with the biological collection number of CGMCC No. 20437.

In one embodiment of the invention, the recombination is effected by homologous recombination.

In the fifth aspect of the present invention, there is also provided a method for constructing a recombinant strain that produces L-isoleucine.

According to the invention, the construction method comprises the following steps:

amplifying upstream and downstream homologous arm fragments of YH66_10325, coding region of YH66_10325 gene and promoter region sequence thereof, and introducing YH66_10325 or YH66_10325 into genome of host strain by homologous recombination^G925AGenes to achieve overexpression of YH66_10325 or YH66_10325 by the strain^G925AA gene.

In one embodiment of the invention, the primers for amplifying the upstream homology arm fragments are:

P7:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTGTGTGCCAGG ATGTCGGC 3'(SEQ ID NO:11)

P8:5'CATCAAAGGAACTGTTCTAATTAATAGGTGGGCGCGAGC 3'(SEQ ID NO:12)

in one embodiment of the invention, the primers for amplifying the downstream homology arm fragments are:

P11:5'GATGCGGTTCTGGCTCATGGAGTATGGTGGCCTTAC 3'(SEQ ID NO: 15)

P12:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCACCACGCCG TCAAGGTAATC 3'(SEQ ID NO:16)

in one embodiment of the present invention, the primers for amplifying the coding region of the gene and the sequence of its promoter region are:

P9:5'GCTCGCGCCCACCTATTAATTAGAACAGTTCCTTTGATG 3'(SEQ ID NO:13)

P10:5'GTAAGGCCACCATACTCCATGAGCCAGAACCGCATC 3'(SEQ ID N O:14)

in one embodiment of the present invention, the integrated homologous arm fragment is obtained by amplifying the three amplified fragments of the upstream homologous arm fragment, the downstream homologous arm fragment, the gene coding region and the promoter region sequence fragment thereof by using the P7/P12 as primers.

In one embodiment of the present invention, a PCR system is used: 10 XEx Taq Buffer 5. mu.L, dNTP mix (2.5 mM each) 4. mu.L, Mg²⁺4. mu.L (25mM), 2. mu.L each of primers (10. mu.M), 0.25. mu.L of Ex Taq (5U/. mu.L), and a total volume of 50. mu.L; PCR amplification was performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 180s (30 cycles), and over-extension at 72 ℃ for 10 min.

In one embodiment of the present invention, shuttle plasmid PK18mobsacB and the integration homology arm fragment were assembled using the NEBuider recombination system to obtain an integrated plasmid.

In one embodiment of the present invention, the integration plasmid is transfected into the host strain, and YH66_10325 or YH66_10325 is introduced into the genome of the host strain by homologous recombination^G925AA gene.

In one embodiment of the invention, the host strain is Corynebacterium glutamicum YPILE001 with the biological collection number CGMCC No. 20437.

In one embodiment of the present invention, the host strain is ATCC 15168.

In one embodiment of the present invention, the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO. 2.

In the sixth aspect of the invention, a method for constructing a recombinant strain for producing L-isoleucine is also provided.

According to the invention, the construction method comprises the following steps:

amplification of coding region and promoter region sequences of YH66_10325 Gene, or YH66_10325^G925AGene coding region and promoter region sequence, constructing over-expression plasmid vector, transferring the vector intoIn a host strain to achieve overexpression of YH66_10325 or YH66_10325^G925AA gene.

In one embodiment of the present invention, the primers for amplifying the coding region of the gene and the sequence of its promoter region are:

P17:5'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTTAGAACAGTTC CTTTGATG 3'(SEQ ID NO:21)

P18:5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACATGAGCCAGAACC GCATC 3'(SEQ ID NO:22)。

in one embodiment of the present invention, the PCR system: 10 XEx Taq Buffer 5. mu.L, dNTP mix (2.5 mM each) 4. mu.L, Mg²⁺4. mu.L (25mM), 2. mu.L each of primers (10. mu.M), 0.25. mu.L of Ex Taq (5U/. mu.L), and a total volume of 50. mu.L; the PCR amplification was performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 120s (30 cycles), and over-extension at 72 ℃ for 10 min.

In one embodiment of the present invention, the shuttle plasmid pXMJ19 and YH66_10325 or YH66_10325 with its own promoter were ligated using the NEBuider recombination system^G925AAssembling the fragments to obtain an overexpression plasmid.

In one embodiment of the invention, the host strain is Corynebacterium glutamicum YPILE001 with the biological collection number CGMCC No. 20437.

In one embodiment of the present invention, the host strain is ATCC 15168.

In one embodiment of the present invention, the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO. 2.

The recombinant strain obtained by the invention can be independently applied to the fermentation production of L-isoleucine, and can also be mixed with other L-isoleucine-producing bacteria for fermentation production of L-isoleucine.

Another aspect of the present invention provides a method for producing L-isoleucine, which comprises culturing said bacterium; and obtaining L-isoleucine from the culture.

The cultivation of the bacteria may be carried out in a suitable medium under culture conditions known in the art. The culture medium may comprise: carbon sources, nitrogen sources, trace elements, and combinations thereof. In the culture, the pH of the culture may be adjusted. Further, prevention of bubble generation, for example, by using an antifoaming agent, may be included in the culture. In addition, the culturing may include injecting a gas into the culture. The gas may include any gas capable of maintaining aerobic conditions of the culture. In the culture, the temperature of the culture may be 20 to 45 ℃. The produced L-isoleucine can be recovered from the culture by treating the culture with sulfuric acid or hydrochloric acid or the like, followed by a combination of methods such as anion exchange chromatography, concentration, crystallization and isoelectric precipitation.

The invention has the advantages of

According to the invention, through knocking out YH66_10325 gene, the product coded by the gene is found to affect L-isoleucine production capacity, and through introducing point mutation into coding sequence, or increasing copy number or overexpression of the gene, a recombinant strain is obtained, and compared with an unmodified strain, the obtained strain is favorable for producing high-concentration L-isoleucine.

Biological preservation Instructions

Corynebacterium glutamicum (Corynebacterium glutamicum), which is deposited in the China general microbiological culture Collection center, with the deposition address: west road No. 1, north chen, chaoyang district, beijing, zip code: 100101, preservation organization abbreviation: CGMCC, the preservation date is 2020, 08 and 17 days, the biological preservation number is CGMCC number 20437, and the strain is named: YPILE 001.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Unless defined otherwise or clearly indicated by the background, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The basic culture medium used for culturing the strain in the following examples has the same composition, and correspondingly required sucrose, kanamycin or chloramphenicol and the like are added to the basic culture medium, and the basic culture medium has the following composition:

TABLE 1 composition of solid media

Composition (I)	Formulation of
		Glucose	5g/L
Polypeptone	10g/L
		Beef extract	10g/L
Yeast powder	5g/L
		Sodium chloride	55g/L
Agar powder	20g/L
		pH (NaOH adjusted)	7.0
Culture conditions	31℃

Example 1 construction of transformation vector pK18-YH66_10325 containing the coding region of the Point-mutated YH66_10325 Gene^G925A

According to the genome (GenBank: CP011309.1) sequence of Corynebacterium glutamicum ATCC15168 published by NCBI, two pairs of primers for amplifying the sequence of coding region of YH66_10325 gene (GenBank: AKF27920.1) were designed and synthesized, point mutations were introduced by allelic replacement in strain ATCC15168 and Corynebacterium glutamicum YPILE001 (organism accession No. CGMCC No.20437) of high yield of L-isoleucine obtained from the right of ATCC15168, the nucleotide sequence corresponding to the encoded protein was SEQ ID NO:3, and guanine (G) at position 925 of YH66_10325 gene was changed to adenine (A) (SEQ ID NO: 2: YH66_10325 gene) at position 925 (A)^G925A) Alanine (a) to threonine (T) at position 309 of the amino acid sequence corresponding to the encoded protein (SEQ ID NO: 4: YH66_10325^A309T). The primers were designed as follows (synthesized by Shanghai Invitrogen corporation):

P1:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTCTGAAGGCAGC TCAATGC 3'(SEQ ID NO:5)

P2:5'GGTGGCTTCTCCTTCGAAGGCACATCTCCTCGTCAC 3'(SEQ ID NO:6)

P3:5'GTGACGAGGAGATGTGCCTTCGAAGGAGAAGCCACC 3'(SEQ ID NO: 7)

P4:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTCTGTAGATG ACGTGATC 3'(SEQ ID NO:8)

the construction method comprises the following steps: PCR amplification was carried out using Corynebacterium glutamicum ATCC15168 as a template and primers P1 and P2, and P3 and P4, respectively.

And (3) PCR system: 10 XEx5 μ L of Taq Buffer, 4 μ L of dNTP mix (2.5 mM each), Mg²⁺mu.L (25mM), 2. mu.L each of primers (10pM), 0.25. mu.L of Ex Taq (5U/. mu.L), and a total volume of 50. mu.L.

The PCR amplification was performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 40s (30 cycles), and over-extension at 72 ℃ for 10min to obtain two DNA fragments (YH66_10325) with the sizes of about 770bp and 848bp respectively and containing YH66_10325 gene coding region^G925AUp and YH66_10325^G925A-Down)。

YH66_10325^G925AUp and YH66_10325^G925A-Down after separation and purification by agarose gel electrophoresis; then using the above two DNA fragments as templates and P1 and P4 as primers, amplifying YH66_10325 with length of 1628bp by overlap PCR^G925A-Up-Down fragment.

And (3) PCR system: 10 XEx Taq Buffer 5. mu.L, dNTP mix (2.5 mM each) 4. mu.L, Mg²⁺mu.L (25mM), 2. mu.L each of primers (10pM), 0.25. mu.L of Ex Taq (5U/. mu.L), and a total volume of 50. mu.L.

The PCR amplification was performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 90s (30 cycles), and over-extension at 72 ℃ for 10 min.

This DNA fragment resulted in the conversion of guanine (G) at position 925 of the coding region of YH66_10325 gene of the mutagenized strain Corynebacterium glutamicum YPILE001 (accession number CGMCC No.20437) of ATCC15168 to adenine (A) and finally in the conversion of amino acid 309 of the encoded protein from alanine (A) to threonine (T).

The pK18mobsacB plasmid (from Addgene) was digested with XbaI and purified by agarose gel electrophoresis to isolate YH 66-10325^G925Athe-Up-Down and the linearized pK18mobsacB plasmid are assembled by a NEBuider recombination system to obtain a vector pK18-YH66_10325^G925AThe plasmid contains a kanamycin resistance marker. And the vector p K18-YH66_10325^G925ASequencing and identifying by a sequencing company, and carrying out sequencing identification on the vector pK18-YH66_10325 containing the correct point mutation (C-T)^G925AAnd (5) storing for later use.

Example 2 construction of YH66_10325 comprising a Point mutation^G925AOf (4) an engineered strain

The construction method comprises the following steps: the allele substitution plasmid pK18-YH66_10325^G925ATransforming into Corynebacterium glutamicum YPILE001 (biological preservation number is CGMCC No.20437) with high isoleucine yield by electric shock; the single colony produced by the culture is respectively identified by the primer P1 and the universal primer M13R, and the strain which can amplify a band with the size of about 1628bp is a positive strain. Culturing the positive strain on a culture medium containing 15% of sucrose; the strains that were cultured and produced single colonies were cultured on kanamycin-containing and kanamycin-free media, respectively, and grown on kanamycin-free media, but not grown on kanamycin-containing media were further subjected to PCR identification using the following primers (synthesized by Shanghai invitrogen Co.):

P5:5'TGCGATCTGGGAATGCAG 3'(SEQ ID NO:9)

P6:5'CGCATCGATGCCATCAAC 3'(SEQ ID NO:10)

the PCR amplification product was subjected to sscp electrophoresis (plasmid pK18-YH 66-10325) after high temperature denaturation and ice-bath^G925AThe amplified fragment is a positive control, the amplified fragment of ATCC15168 is a negative control, and water is used as a blank control). due to the different fragment structures and different electrophoresis positions, the strain with the fragment electrophoresis position inconsistent with the position of the negative control fragment and consistent with the position of the positive control fragment is a strain with successful allelic replacement. Carrying out allelic replacement on the target fragment of the successful strain by using primers P5 and P6 through PCR amplification again, connecting the target fragment to PMD19-T vector sequencing, and determining whether the replacement is successful or not by aligning base sequences; and a mutant strain obtained by successfully replacing Corynebacterium glutamicum YPILE001 (biological preservation number is CGMCC No.20437) which is derived from high-yield L-isoleucine is named YPI 013.

TABLE 2 preparation of PAGE by sscp electrophoresis

Example 3 construction of genome overexpression of YH66_10325 or YH66_10325^G925AEngineered strains of genes

Corynebacterium glutamicum ATCC15168 genome published under NCBI(GenBank: CP011309.1) sequence, designing and synthesizing three pairs of primers for amplifying upstream and downstream homologous arm fragments and YH66_10325 gene coding region and promoter region sequences, and introducing YH66_10325 or YH66_10325 into Corynebacterium glutamicum YPILE001 (biological collection number CGMCC number 20437) strain by homologous recombination^G925AA gene.

The primers were designed as follows (synthesized by Shanghai Invitrogen corporation):

P7:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTGTGTGCCAGGA TGTCGGC3'

P8:5'CATCAAAGGAACTGTTCTAATTAATAGGTGGGCGCGAGC3'

P9:5'GCTCGCGCCCACCTATTAATTAGAACAGTTCCTTTGATG3'

P10:5'GTAAGGCCACCATACTCCATGAGCCAGAACCGCATC 3'

P11:5'GATGCGGTTCTGGCTCATGGAGTATGGTGGCCTTAC3'

P12:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCACCACGCCG TCAAGGTAATC 3'

the construction method comprises the following steps: PCR amplification was performed using Corynebacterium glutamicum ATCC15168 or YPI013 as template and primers P7/P8, P9/P10, P11/P12, respectively, to obtain upstream homology arm fragments of about 864bp, YH66_10325 or YH66_10325^G925AThe gene fragment is about 1232bp and the downstream homologous arm fragment is about 785bp, and then the integrated homologous arm fragment is obtained by taking P7/P12 as a primer and mixing the three amplified fragments as a template for amplification. After the PCR reaction is finished, carrying out electrophoretic recovery on the amplified product, recovering a required DNA fragment of about 2881bp by using a column type DNA gel recovery kit, and connecting a NEBuider recombination system with a shuttle plasmid pk18mobsacB recovered by Xba I enzyme digestion to obtain an integrated plasmid pk18mobsacB-YH66_10325 or pk18mobsacB-YH66_10325^G925AThe plasmid contains a kanamycin resistance marker, and recombinants with the plasmid integrated on the genome can be obtained by kanamycin screening.

And (3) PCR system: 10 XEx Taq Buffer 5. mu.L, dNTP mix (2.5 mM each) 4. mu.L, Mg²⁺mu.L (25mM), 2. mu.L each of primers (10pM), 0.25. mu.L of Ex Taq (5U/. mu.L), and a total volume of 50. mu.L.

The PCR amplification was performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 180s (30 cycles), and over-extension at 72 ℃ for 10 min.

The 2 integration plasmids are respectively transformed into Corynebacterium glutamicum YPILE001 (biological preservation number is CGMCC number 20437), a single colony generated by culture is identified by PCR through a P13/P14 primer, a positive strain containing a fragment with the size of about 1056bp is amplified by PCR, and a raw strain containing the fragment which can not be amplified is obtained. The positive strains are screened by 15% sucrose, then are respectively cultured on a culture medium containing kanamycin and a culture medium not containing kanamycin, the strains grow on the culture medium not containing kanamycin, the strains which do not grow on the culture medium containing kanamycin are further subjected to PCR identification by adopting a P15/P16 primer, and the strains with the amplified size of about 845bp are YH66_10325 or YH66_10325^G925AThe strain with gene integrated into the genome of the strain Corynebacterium glutamicum YPILE001 (biological preservation number CGMCC No.20437) was named YPI-014 (without mutation point) and YPI-015 (with mutation point).

P13:5'GATTGATCCGGTGAACGCC 3'

P14:5'GGCCCTGAGAACGTCATTG 3'

P15:5'TTGATGTTTGCATCAAGTAG 3'

P16:5'TTGTGAGGTTTGCGTGCCAAG 3'

Example 4 construction of plasmids overexpressing YH66_10325 or YH66_10325^G925AEngineered strains of genes

A pair of primers for amplifying the sequences of the coding region and the promoter region of YH66_10325 gene were designed and synthesized based on the sequence of Corynebacterium glutamicum ATCC15168 genome (GenBank: CP011309.1) published by NCBI, and the primers were designed as follows (synthesized by Shanghai invitrogen corporation):

P17:5'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTTAGAACAGTTC CTTTGATG 3'

P18:5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACATGAGCCAGAACCG CATC 3'

the construction method comprises the following steps: PCR amplification with ATCC15168 or YPI013 and primers P17/P18 to obtain YH66_10325 or YH66_10325^G925AGene fragment is about 1278bp, the amplified product is recovered by electrophoresis, the column type DNA gel recovery kit is adopted to recover the needed DNA fragment of 1278bp, and the DNA fragment is collectedConnecting the recombinant NEBuider system with the EcoRI digested and recovered shuttle plasmid pXMJ19 to obtain an over-expression plasmid pXMJ19-YH66_10325 or pXMJ19-YH66_10325^G925A. The plasmid contains a chloramphenicol resistance marker, and the plasmid obtained by chloramphenicol screening can be transformed into a strain.

And (3) PCR system: 10 XEx Taq Buffer 5. mu.L, dNTP mix (2.5 mM each) 4. mu.L, Mg²⁺mu.L (25mM), 2. mu.L each of primers (10pM), 0.25. mu.L of Ex Taq (5U/. mu.L), and a total volume of 50. mu.L.

The PCR amplification was performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 120s (30 cycles), and over-extension at 72 ℃ for 10 min.

The plasmids are respectively electrically transformed into Corynebacterium glutamicum YPILE001 (biological preservation number is CGMCC No.20437), single colonies generated by culture are identified by PCR through M13R (-48) and P18 primers, and the single colonies amplified by PCR contain a fragment with the size of about 920bp and are taken as transfer strains which are named YPI-016 (without point mutation) and YPI-017 (with point mutation).

Example 5 construction of engineered Strain with deletion of YH66_10325 Gene on genome

Two pairs of primers for amplifying fragments at both ends of the coding region of YH66_10325 gene were synthesized as upstream and downstream homology arm fragments based on the genomic sequence of Corynebacterium glutamicum ATCC15168 (GenBank: CP011309.1) published by NCBI. The primers were designed as follows (synthesized by shanghai handsome corporation):

P19:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAACTCCTTGCTAA GGATGG 3'

P20:5'CTTTTAGATTGGATTTTCAGCTAGACAACGAGGGTTGCTAG 3'

P21:5'CTAGCAACCCTCGTTGTCTAGCTGAAAATCCAATCTAAAAG 3'

P22:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAGCGAGAAAT GCTGGGTCTG 3'

the upstream homology arm fragment 868bp and the downstream homology arm fragment 780bp are obtained by PCR amplification with Corynebacterium glutamicum ATCC15168 as a template and primers P19/P20 and P21/P22, respectively. Then OVER PCR was performed with primers P19/P22 to obtain the entire homology arm fragment of 1648 bp. And after the PCR reaction is finished, carrying out electrophoretic recovery on the amplified product, recovering the required 1648bp DNA fragment by using a column type DNA gel recovery kit, and connecting the recovered shuttle plasmid pk18mobsacB plasmid with Xba I enzyme digestion through a NEBuider recombination system to obtain a knocked-out plasmid. The plasmid contains a kanamycin resistance marker.

The knockout plasmid is electrically transformed into Corynebacterium glutamicum YPILE001 (biological preservation number is CGMCC No.20437), and the single colony generated by culture is respectively identified by PCR through the following primers (synthesized by Shanghai Yingjun company):

P23:5'GCGCTGACTATGCAGATTC 3'

P24:5'AAACTCGACC TTCACGTC 3'

the strain with the band of about 1648bp and about 2819bp amplified by the PCR is a positive strain, and the strain with the band of only 2819bp amplified is a spawn. The positive strains are screened on a 15% sucrose culture medium, then are respectively cultured on a culture medium containing kanamycin and a culture medium not containing kanamycin, and grow on the culture medium not containing kanamycin, and the strains which do not grow on the culture medium containing kanamycin are further subjected to PCR identification by adopting a P23/P24 primer, so that the strain with an 1648bp band is a genetically engineered strain with a YH66_10325 gene coding region knocked out, and the strain is named YPI-018.

Example 6L-isoleucine fermentation experiment

The strains constructed in examples 2 to 5 and Corynebacterium glutamicum YPILE001 (organism accession number CGMCC No.20437) were subjected to fermentation experiments in a BLBIO-5GC-4-H type fermenter (available from Bailan Biotech Co., Ltd., Shanghai) using the media shown in Table 3 and the fermentation control process shown in Table 4. Each strain was replicated three times, and the results are shown in Table 5.

TABLE 3 fermentation Medium formulation

TABLE 4 fermentation control Process

TABLE 5 results of L-isoleucine fermentation experiments

As shown in Table 5, the coding region of YH66_10325 gene was point-mutated into YH66_10325 in Corynebacterium glutamicum YPILE001 (accession number: CGMCC No.20437), an engineered bacterium producing isoleucine^G925AAll contribute to the improvement of L-isoleucine production; overexpression of YH66_10325 and YH66_10325^G925AIs helpful to improve the yield of L-isoleucine, and the YH66_10325 gene knockout is not beneficial to the accumulation of L-isoleucine.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Ningxia Yipin Biotechnology Ltd

<120> YH66_10325 gene modified recombinant strain producing L-isoleucine, construction method and application thereof

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atgagccaga accgcatcag gaccactcac gttggttcct tgccccgtac cccagagcta 60

cttgatgcaa acatcaagcg ttctaacggt gagattgggg aggaggaatt cttccagatt 120

ctgcagtctt ctgtagatga cgtgatcaag cgccaggttg acctgggtat cgacatcctt 180

aacgagggcg aatacggcca cgtcacctcc ggtgcagttg acttcggtgc atggtggaac 240

tactccttca cccgcctggg cggactgacc atgaccgata ccgaccgttg ggcaagccag 300

gaagcagtgc gttccacccc tggcaacatc aagctgacca gcttctctga tcgtcgcgac 360

cgcgcattgt tcagcgaagc atacgaggat ccagtatctg gcatcttcac cggccgcgct 420

tctgtgggca acccagagtt caccggacct attacctaca ttggccagga agaaactcag 480

acggatgttg atctgctgaa gaagggcatg aacgcagcgg gagctaccga cggcttcgtt 540

gcagcactat ccccaggatc tgcagctcga ttgaccaaca agttctacga cactgatgaa 600

gaagtcgtcg cagcatgtgc cgatgcgctt tcccaggaat acaagatcat caccgatgca 660

ggtctgaccg ttcagctcga cgcaccggac ttggcagaag catgggatca gatcaaccca 720

gagccaagcg tgaaggatta cttggactgg atcggtacac gcatcgatgc catcaacagt 780

gcagtgaagg gccttccaaa ggaacagacc cgcctgcaca tctgctgggg ctcttggcac 840

ggaccacacg tcactgacat cccattcggt gacatcattg gtgagatcct gcgcgcagag 900

gtcggtggct tctccttcga aggcgcatct cctcgtcacg cacacgagtg gcgtgtatgg 960

gaagaaaaca agcttcctga aggatctgtt atctaccctg gtgttgtgtc tcactccatc 1020

aacgctgtgg agcacccacg cctggttgct gatcgtatcg ttcagttcgc caagcttgtt 1080

ggccctgaga acgtcattgc gtccactgac tgtggtctgg gcggacgtct gcattcccag 1140

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