CN117866868A - L-high proline production strain and construction method and application thereof - Google Patents

L-high proline production strain and construction method and application thereof Download PDF

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CN117866868A
CN117866868A CN202410275935.4A CN202410275935A CN117866868A CN 117866868 A CN117866868 A CN 117866868A CN 202410275935 A CN202410275935 A CN 202410275935A CN 117866868 A CN117866868 A CN 117866868A
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gene
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homoproline
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CN117866868B (en
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徐庆阳
王玉博
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Tianjin University of Science and Technology
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Tianjin University of Science and Technology
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Abstract

The invention provides an L-homoproline production strain, a construction method and application thereof, wherein the production strain is a starting strain by utilizing a metabolic engineering meansE.coliFurther modified based on W3110 to E.coliE.coliW3110 is chassis, L-lysine is converted into alpha-aminoadipic semialdehyde by introducing exogenous L-lysine 6-aminotransferase, and then the semialdehyde is spontaneously reacted to produce D-1-piperidine-6-carboxylic acid, which is delta 1 Reduction of pyrroline-5-carboxylate reductase to L-homoproline, production of L-homoproline by fermentation, and use of glucoseAs a substrate, the L-homoproline is directly synthesized from glucose in a fermentation tank, has the advantages of low production cost, no toxic metabolic byproducts, stable heredity, high conversion rate, short fermentation period and the like, can realize the yield of the fermentation 50-h L-homoproline of up to 40.5 g/L, and has very good industrial application value.

Description

L-high proline production strain and construction method and application thereof
Technical Field
The invention relates to the technical production field of genetic engineering and fermentation engineering, in particular to an L-high proline production strain, a construction method and application thereof.
Background
L-homoproline (L-Pipecolic acid), also known as L-Pipecolic acid, is a commercially valuable natural cyclic non-proteinogenic alpha-amino acid found in a variety of animals, plants and microorganisms. L-homoproline is a key component of many natural and synthetic bioactive molecules, such as the immunosuppressant rapamycin, the antineoplastic agent swainsonine, the peptide antibiotic virginiamycin, and the like, and therefore has high market value.
At present, the preparation of the L-homoproline mainly comprises a chemical synthesis method and a biological synthesis method. Traditional chemical synthesis methods have large limitations in production due to the disadvantages of using noble metal catalysts, higher pressures and temperatures, complicated separation and purification steps, lower yields, and unsustainable raw materials.
The microorganism direct fermentation method for synthesizing the L-homoproline has the advantages of safe production raw materials, low cost, simple process, easy industrialization and the like, and becomes the mode for producing the L-homoproline first choice at the present stage. The method of synthesizing L-homoproline by microorganisms mainly catalyzes the decomposition of L-lysine by lysine ring deaminase and lysine-6-dehydrogenase, but researches show that the turnover number of the L-lysine ring deaminase is low (about 0.6-1 s), so that the biocatalytic method requires very high enzyme load; lysine-6-dehydrogenase has a large restriction in its expression due to incompatibility with the intracellular environment.
Disclosure of Invention
The invention aims to provide an L-high proline producing strain.
Another technical problem to be solved by the present invention is to provide a method for constructing the strain for producing L-homoproline.
Another technical problem to be solved by the present invention is to provide an application of the strain for producing L-homoproline.
In order to solve the technical problems, the technical scheme of the invention is as follows:
an L-homoproline producing strain, namely strain PIP11, is prepared from the strain in the way of metabolic engineeringE.coliFurther modified on the basis of W3110, includingcadA、ldcC、ppc、lysC、asd、dapA、 lysA、pntABThe expression intensity of the gene was adjusted tolat，proCThe heterologous expression of the gene is specifically as follows:
knockout on genomecadAThe gene is not expressed by the gene,
knockout on genomeldcCThe gene is not expressed by the gene,
at the position ofycdNPseudogene locus use P trc Promoter controlppcThe gene is over-expressed and the expression of the gene is improved,
at the position ofrphPseudogene locus use P trc Promoter controllysCThe gene is over-expressed and the expression of the gene is improved,
at the position offhiaPseudogene locus use P trc Promoter controlasdThe gene is over-expressed and the expression of the gene is improved,
at the position ofyjiPPseudogene locus use P trc Promoter controldapAThe gene is over-expressed and the expression of the gene is improved,
at the position ofyciQPseudogene locus use P trc Promoter controllysAThe gene is over-expressed and the expression of the gene is improved,
at the position ofyeeLPseudogene locus use P trc Promoters control exogenous sourceslatThe gene is over-expressed and the expression of the gene is improved,
at the position ofmbhAPseudogene locus use P trc Promoters control exogenous sourceslatThe gene is over-expressed and the expression of the gene is improved,
at the position ofilvGPseudogene locus use P trc Promoters control exogenous sourcesproCThe gene is over-expressed and the expression of the gene is improved,
at the position ofygaYPseudogene locus use P trc Promoter controlpntABThe gene is over-expressed.
Preferably, the above-mentioned L-homoproline producing strain, the metabolic engineering means is CRISPR-Cas9 gene editing technology.
Preferably, the L-homoproline producing strain described above, the starting strainE.coliW3110 was deposited under ATCC 273250.
Preferably, the L-homoproline producing strain, the P trc The nucleotide sequence of the promoter is shown in a sequence table SEQ ID NO. 1.
Preferably, the above L-homoproline producing strain, saidcadANucleotide sequence of GeneAs shown in SEQ ID NO.2 of the sequence table.
Preferably, the above L-homoproline producing strain, saidldcCThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 3.
Preferably, the above L-homoproline producing strain, saidppcThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 4.
Preferably, the above L-homoproline producing strain, saidlysCThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 5.
Preferably, the above L-homoproline producing strain, saidasdThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 6.
Preferably, the above L-homoproline producing strain, saiddapAThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 7.
Preferably, the above L-homoproline producing strain, saidlysAThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 8.
Preferably, the above L-homoproline producing strain, saidlatThe gene is derived fromFlavobacterium lutescens，Obtained after codon optimization, thelatThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 9.
Preferably, the above L-homoproline producing strain, saidproCThe gene originates from Corynebacterium glutamicum ATCC 13032, whichproCThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 10.
Preferably, the above L-homoproline producing strain, saidpntABThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 11.
The construction method of the L-homoproline production strain is that the strain is a starting strainE.coliThe method for directionally modifying the W3110 comprises the following specific steps:
(1) In the original strainE.coliW3110 genomic knockoutcadAThe gene is obtained into a strain PIP01;
(2) Based on strain PIP01, knockout on genomeldcCThe gene is obtained into a strain PIP02;
(3) Based on strain PIP02, inycdNPseudogene locus use P trc Promoter controlppcThe gene is over-expressed to obtain a strain PIP03;
(4) Based on strain IP03, inrphPseudogene locus use P trc Promoter controllysCThe gene is over-expressed to obtain a strain PIP04;
(5) Based on strain PIP04, infhiaPseudogene locus use P trc Promoter controlasdThe gene is over-expressed to obtain a strain PIP05;
(6) Based on strain PIP05, inyjiPPseudogene locus use P trc Promoter controldapAThe gene is over-expressed to obtain a strain PIP06;
(7) Based on strain PIP06, inyciQPseudogene locus use P trc Promoter controllysAThe gene is over-expressed to obtain a strain PIP07;
(8) Based on strain PIP07, inyeeLPseudogene locus use P trc Promoters control exogenous sourceslatThe gene is over-expressed to obtain a strain PIP08;
(9) Starting from strain PIP08, inmbhAPseudogene locus use P trc Promoters control exogenous sourceslatThe gene is over-expressed to obtain a strain PIP09;
(10) Starting from strain PIP09, inilvGPseudogene locus use P trc Promoters control exogenous sourcesproCThe gene is over-expressed to obtain a strain PIP10;
(11) Starting from strain PIP10, inygaYPseudogene locus use P trc Promoter controlpntABGene overexpression gives strain PIP11.
The L-homoproline producing strain is applied to the fermentation production of L-homoproline.
Preferably, the L-homoproline producing strain is used for producing L-homoproline by fermentation in a mechanically stirred tank, and is synthesized by seed culture and fermentation culture with glucose as a substrate.
Preferably, the application of the strain for producing L-homoproline comprises the following specific steps:
(1) Seed culture: the culture temperature is 35 ℃, the pH of the culture is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 40% by adjusting the stirring rotation speed or ventilation, and the OD is obtained 600nm Meet the inoculation requirement when the number is 20;
(2) Fermentation culture: the inoculation amount is 30%, the culture temperature is 35 ℃, the pH is controlled to be 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained to be 40% by adjusting the stirring rotation speed or ventilation amount, the glucose concentration in the tank is controlled to be less than or equal to 0.5g/L by feeding 80% (mass volume fraction) glucose solution, and the fermentation period is less than or equal to 50h.
Preferably, the application of the L-homoproline production strain comprises inoculating a storage strain at-80deg.C on an activating inclined plane without resistance, culturing at 37deg.C for 12 hr, and passaging for 2 times; eluting the activated thalli on the inclined plane by sterilized distilled water, and transferring the thalli into a mechanical stirring type fermentation tank to start seed culture.
Preferably, the use of the above-mentioned L-homoproline producing strain, the seed culture medium used in the seed culture: 30g/L glucose, 5g/L yeast powder, 2g/L peptone, (NH) 4 ) 2 SO 4 1g/L，KH 2 PO 4 3g/L，MgSO 4 ·7H 2 0.8g/L of O, 1g/L of monosodium glutamate and the balance of water.
Preferably, the use of the above-mentioned L-homoproline producing strain, the fermentation medium used in the fermentation culture: 30g/L glucose, 5g/L yeast powder, 2g/L peptone, (NH) 4 ) 2 SO 4 1g/L，KH 2 PO 4 4g/L，MgSO 4 ·7H 2 O1.5 g/L, methionine 0.3g/L, isoleucine 0.5g/L, feSO 4 ·7H 2 O20 mg/L, and the balance of water.
The above culture medium can be prepared by standard method.
Preferably, when the L-homoproline producing strain is used for fermentation culture, 9mg of pyridoxal phosphate (PLP) and 2.25g of choline chloride (namely, pyridoxal phosphate (PLP) 6mg/L of sugar solution and 1.5g/L of choline chloride) are added to 1.5L of 80% (mass volume fraction) glucose solution.
The beneficial effects are that:
the L-homoproline producing strain is a starting strain by using metabolic engineering meansE.coliFurther engineering the obtained strain PIP11 on the basis of W3110 to E.coliE.coliW3110 is chassis by introduction of exogenous L-lysine 6-aminotransferaselatThe gene transfers L-lysine into alpha-aminoadipic semialdehyde, then spontaneously reacts to produce D-1-piperidine-6-carboxylic acid, and the D-1-piperidine-6-carboxylic acid is reacted with the semialdehyde to form the L-lysine-semialdehyde 1 Pyrroline-5-carboxylic acid reductaseproCThe gene is reduced into L-homoproline, the strain PIP11 is used for producing the L-homoproline by a fermentation method, glucose is used as a substrate, the L-homoproline is directly synthesized from the glucose in a fermentation tank, the production cost is low, toxic metabolic byproducts are not generated, the genetic stability is realized, the conversion rate is high, the fermentation period is short, and the like, and the yield of the fermented 50h L-homoproline can reach 40.5 g/L, so that the method has very good industrial application value.
Drawings
FIG. 1 is a diagram showing the process of the genetic engineering of L-homoproline producing strain from the head synthesis pathway.
Detailed Description
In order to enable those skilled in the art to better understand the technical scheme of the present invention, the technical scheme of the present invention will be further described in detail below with reference to the specific embodiments.
The percentage "%" referred to in the examples is the mass percentage, the percentage of the solution is the gram of the solute contained in 100mL, and the percentage between the liquids is the volume ratio of the solution at 25 ℃.
The starting strain used in the examples is wild-typeE.coliW3110, accession number ATCC 273250, the corresponding promoters and genes, etc. are shown in the sequence listing.
As shown in FIG. 1, an L-homoproline producing strain was constructed by the following method: in Escherichia coliE.coliBased on W3110 (ATCC 273250), lysine decarboxylase was knocked outcadAAndldcCgenes which are not expressed inycdNPseudogene locus use P trc Start-upSub-controlled phosphoenolpyruvate carboxylaseppcGene overexpression inrphPseudogene locus use P trc Promoter controls aspartokinaselysCGene overexpression infhiaPseudogene locus use P trc Promoter controls aspartate semialdehyde dehydrogenaseasdGene overexpression inyjiPPseudogene locus use P trc Promoters controlling dihydropyridine dicarboxylic acid synthasedapAThe gene is over-expressed. At the position ofyciQPseudogene locus use P trc Promoters control diaminopimelate decarboxylaselysAGene overexpression inyeeLPseudogene locus use P trc Promoter controls exogenous L-lysine-6-aminotransferaselatGene overexpression inmbhAPseudogene locus use P trc Promoter controls exogenous L-lysine-6-aminotransferaselatGene overexpression inilvGPseudogene locus use P trc Promoter controls exogenous events 1 Pyrroline-5-carboxylic acid reductaseproCGene overexpression inygaYPseudogene locus use P trc Promoter controls membrane border inducer enzymespntABThe gene is over-expressed.
Example 1
1. Method for gene editing
Gene editing methods employed are described in the literature (Li Y, lin Z, huang C, et al Metabolic engineering of Escherichia coli using CRISPR-Cas9 mediated genome coding Metabolic Engineering,2015, 31:13-21.). Engineering plasmids pREDCas9 and pGRB related by the method, wherein pREDCas9 carries an elimination system of a gRNA expression plasmid pGRB, a Red recombination system of lambda phage, a Cas9 protein expression system and the resistance of Qamycin (working concentration: 100 mg/L); pGRB takes pUC18 as a backbone, and comprises the promoter J23100, the gRNA-Cas9 binding region sequence and the terminator sequence, and ampicillin resistance (working concentration: 100 mg/L). The terminology involved in the following embodiments is explained in this article.
2. The primers used in the strain construction are shown in Table 1.
TABLE 1 primers involved in the construction of strains
Primer name Primer sequence (5 '-3') Sequence number
cadA-pGRB-S AGTCCTAGGTATAATACTAGTAAGAAACACCAAACGCAACCGTTTTAGAGCTAGAA SEQ ID NO.12
cadA-pGRB-A TTCTAGCTCTAAAACGGTTGCGTTTGGTGTTTCTTACTAGTATTATACCTAGGACT SEQ ID NO.13
cadA-U-S ACTGGGTTGCGTGTTCTGC SEQ ID NO.14
cadA-U-A GTACGGAAGGATCATATTGGCGTCAGTCAAAAATAACGCCGCACA SEQ ID NO.15
cadA-D-S TGTGCGGCGTTATTTTTGACTGACGCCAATATGATCCTTCCGTAC SEQ ID NO.16
cadA-D-A AAACCAGAGAAGCATATGCGCT SEQ ID NO.17
ldcC-pGRB-S AGTCCTAGGTATAATACTAGTTGTACGCCGGGGCATATGGGGTTTTAGAGCTAGAA SEQ ID NO.18
ldcC-pGRB-A TTCTAGCTCTAAAACCCCATATGCCCCGGCGTACAACTAGTATTATACCTAGGACT SEQ ID NO.19
ldcC-U-S TGGGTATCATTGCTCCGCG SEQ ID NO.20
ldcC-U-A GATAAGGCAGGATCATATTTGCCGCAAAAATCACGCCGCAAATTCG SEQ ID NO.21
ldcC-D-S CGAATTTGCGGCGTGATTTTTGCGGCAAATATGATCCTGCCTTATC SEQ ID NO.22
ldcC-D-A CGGCGGGAACGGAAATGAGAA SEQ ID NO.23
ycdN-pGRB-U AGTCCTAGGTATAATACTAGTGCGTGGAAATCATCATGGCTGTTTTAGAGCTAGAA SEQ ID NO.24
ycdN-pGRB-D TTCTAGCTCTAAAACAGCCATGATGATTTCCACGCACTAGTATTATACCTAGGACT SEQ ID NO.25
ycdN-U-S GATTTTGACGCCACCAACACC SEQ ID NO.26
ycdN-U-A GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAAT CCACATCACACAATCCATC SEQ ID NO.27
ycdN-D-S CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAA TGAAGGGATTTTTGGCTATCAGG SEQ ID NO.28
ycdN-D-A GTATTCGCCAGGCTGTAAATTC SEQ ID NO.29
ycdN-ppc-S CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGAC CATGAACGAACAATATTCCGCATTGC SEQ ID NO.30
ycdN-ppc-A TCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTT TTATTTGTTAGCCGGTATTACGCATACCTGC SEQ ID NO.31
rph-pGRB-S AGTCCTAGGTATAATACTAGTTGCGACGTGCTTCAGGCTGAGTTTTAGAGCTAGAA SEQ ID NO.32
rph-pGRB-A TTCTAGCTCTAAAACTCAGCCTGAAGCACGTCGCAACTAGTATTATACCTAGGACT SEQ ID NO.33
rph-U-S ATAGCGCAGGGTACATTCCACT SEQ ID NO.34
rph-U-A GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCA ACCTTCTTCAATAGAGGCGGTACA SEQ ID NO.35
rph-D-S TGCCGCAGAGACCGACATCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGG TGAACGCTCTCCTGAGTAGGACAAAT SEQ ID NO.36
rph-D-A ACAGCGGTTGTGGTGGCA SEQ ID NO.37
rph-lysC-S CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGAC CATGTCTGAAATTGTTGTCTCCAAATTT SEQ ID NO.38
rph-lysC-A GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATT TGTTACTCAAACAAATTACTATGCAGTT SEQ ID NO.39
fhia-pGRB-S AGTCCTAGGTATAATACTAGTTGACGTGCGTAACCAGCTGCGTTTTAGAGCTAGAA SEQ ID NO.40
fhia-pGRB-A TTCTAGCTCTAAAACGCAGCTGGTTACGCACGTCAACTAGTATTATACCTAGGACT SEQ ID NO.41
fhia-U-S GGGCAATGGTGTTGATACTGG SEQ ID NO.42
fhia-U-A AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAA TCGCCAGAATCATCATCCC SEQ ID NO.43
fhia-D-S AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAG GACAAAT CAAGCAGGAGCTGACGGTGT SEQ ID NO.44
fhia-D-A TGCACCAATGCTGGATACTTACA SEQ ID NO.45
fhia-asd-S ATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAA ATGTTGGTTTTATCGGCTG SEQ ID NO.46
fhia-asd-A CGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTAT TTGTTACGCCAGTTGACGAAGCATC SEQ ID NO.47
yjiP-pGRB-S AGTCCTAGGTATAATACTAGTTGGAAAGCGCCTCGGGGAATGTTTTAGAGCTAGAA SEQ ID NO.48
yjiP-pGRB-A TTCTAGCTCTAAAACATTCCCCGAGGCGCTTTCCAACTAGTATTATACCTAGGACT SEQ ID NO.49
yjiP-U-S GCCATACCGCCAGCAAGAT SEQ ID NO.50
yjiP-U-A AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAG CAGATATTCCCCTTTCCACC SEQ ID NO.51
yjiP-D-S AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGG ACAAATGACGGATGACAAACGCAAAGC SEQ ID NO.52
yjiP-D-A AAAGGCGGATTTTTACTGTGGA SEQ ID NO.53
yjiP-dapA-S CTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCAT GTTCACGGGAAGTATTGTCGC SEQ ID NO.54
yjiP-dapA-A ACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTA CAGCAAACCGGCATGCTTAA SEQ ID NO.55
yciQ-pGRB-S AGTCCTAGGTATAATACTAGTAAACAACGTTTCTTGCCTCAGTTTTAGAGCTAGAA SEQ ID NO.56
yciQ-pGRB-A TTCTAGCTCTAAAACTGAGGCAAGAAACGTTGTTTACTAGTATTATACCTAGGACT SEQ ID NO.57
yciQ-U-S TTACTTGAAGCATTGGGCGAAC SEQ ID NO.58
yciQ-U-A AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAC CAGTCAAGATGCCAGGGTTC SEQ ID NO.59
yciQ-D-S AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGG ACAAATGTCTGACAAGAACCAGCAAATCCT SEQ ID NO.60
yciQ-D-A ATAGCTTCACCGTGGGCATAAC SEQ ID NO.61
yciQ-lysA-S CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGAC CATGCCACATTCACTGTTCAGCA SEQ ID NO.62
yciQ-lysA-A GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATT TGTTAAAGCAATTCCAGCGCCAGT SEQ ID NO.63
yeeL-pGRB-S AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA SEQ ID NO.64
yeeL-pGRB-A TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT SEQ ID NO.65
yeeL-U-S TTCATCGGGACGAGTGGAGA SEQ ID NO.66
yeeL-U-A TCCACACATTATACGAGCCGGATGATTAATTGTCAACCATAGCATCGCCAATCTGAT CGGG SEQ ID NO.67
yeeL-D-S AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGG ACAAATACCCAAAGGTGAAGATA SEQ ID NO.68
yeeL-D-A CATTCCCTCTACAGAACTAG SEQ ID NO.69
yeeL-lat-S CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGAC CATGAGCTTATTAGCGCCACTGG SEQ ID NO.70
yeeL-lat-A TCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTT TTATTTGTTACGCGCGGCGCGGG SEQ ID NO.71
mbhA-pGRB-S TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAAT TGTCAACACGGTGGCAGGTTTTGG SEQ ID NO.72
mbhA-pGRB-A AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGG ACAAATGACCAAAAGTGCGTCCGATAC SEQ ID NO.73
mbhA-U-S GCCAGCACGAACATAATCCC SEQ ID NO.74
mbhA-U-A GGTCTGTTTCCTGCTAGCACTATACCTAGGACTGAGCTAGCCGTAAACACGGTGGCA GGTTTTGG SEQ ID NO.75
mbhA-D-S AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGG ACAAATGACCAAAAGTGCGTCCGATAC SEQ ID NO.76
mbhA-D-A CGGCGTAATCACAAACTGGC SEQ ID NO.77
ilvG-pGRB-S AGTCCTAGGTATAATACTAGTTATCGGCACTGACGCATTTCGTTTTAGAGCTAGAA SEQ ID NO.78
ilvG-pGRB-A AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA SEQ ID NO.79
ilvG-U-S ACCGAGGAGCAGACAATGAATAA SEQ ID NO.80
ilvG-U-A AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAG GTGATGGCAACAACAGGGA SEQ ID NO.81
ilvG-D-S CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAA TCTATCTACGCGCCGTTGTTGTT SEQ ID NO.82
ilvG-D-A GCGCTGGCTAACATGAGGAA SEQ ID NO.83
ilvG-proC-S CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGAC CATGACAACAATTGCTGTAATCGGC SEQ ID NO.84
ilvG-proC-A CAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTG CTAGCGCTTTCCGAGTTCTTCAG SEQ ID NO.85
ygaY-pGRB-S AGTCCTAGGTATAATACTAGTCACTGATGGCGCTGGCATTAGTTTTAGAGCTAGAA SEQ ID NO.86
ygaY-pGRB-A TTCTAGCTCTAAAACTAATGCCAGCGCCATCAGTGACTAGTATTATACCTAGGACT SEQ ID NO.87
ygaY-U-S CCTACAAACCACATCGCACATT SEQ ID NO.88
ygaY-U-A TCCACACATTATACGAGCCGGATGATTAATTGTCAAACACCGAAGCAACCCAAAAGA CGGT SEQ ID NO.89
ygaY-D-S AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGG ACAAATTTGCTTGCCGCTCCACC SEQ ID NO.90
ygaY-D-A GGAGTAGGGCTTTCCATAGAGTGT SEQ ID NO.91
ygaY-pntAB-S CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGA CCATGCGAATTGGCATACCAAGAG SEQ ID NO.92
ygaY-pntAB-A CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAC AGAGCTTTCAGGATTGCATCCAC SEQ ID NO.93
Example 2
This example is intended to illustrate the knockout of a genomecadAThe gene comprises the following steps:
(1) the E.coli W3110 genome was used as a template, respectivelycadA-U-S、cadA-U-A andcadA-D-S、cadAD-A is used as a primer, an upstream homology arm and a downstream homology arm are obtained through PCR amplification, and delta is obtained through overlap PCR by using the same as a templatecadAA gene knockout fragment consisting ofcadAUpstream homology armcadADownstream homology arms.
(2) To be used forcadApGRB-ScadApGRB-A as primer, construction of pGRB by PCR annealing procedurecadAThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-cadA；
(3) Delta obtained in the steps (1) and (2) is reducedcadAGene knockout fragment and pGRBcadAPlasmid electrotransformation intoE.coliIn the W3110 strain, positive transformants were obtained by selection, and pGRB-cadAThe plasmid was designated PIP01.
Example 3
This example is intended to illustrate the knockout of a genomeldcCThe gene comprises the following steps:
(1) the E.coli W3110 genome was used as a template, respectivelyldcC-U-S、ldcC-U-A andldcC-D-S、ldcCD-A is used as a primer, an upstream homology arm and a downstream homology arm are obtained through PCR amplification, and delta is obtained through overlap PCR by using the same as a templateldcCA gene knockout fragment consisting ofldcCUpstream homology armldcCDownstream homology arms.
(2) To be used forldcCpGRB-SldcCpGRB-A as primer, construction of pGRB by PCR annealing procedureldcCThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-ldcC；
(3) Delta obtained in the steps (1) and (2) is reducedldcCGene knockout fragment and pGRBldcCThe plasmid is electrotransferred into PIPO1 strain, and positive transformant is obtained by screening, pGRB-ldcCThe plasmid was designated PIP02.
Example 4
The present embodiment is intended to be described inycdNPseudogene locus use P trc Promoter controlppcThe method comprises the following steps of gene overexpression:
(1) the E.coli W3110 genome was used as a template, respectivelyycdN-U-S、ycdN-U-A、ycdN-D-S、ycdN-D-A andycdN-ppc-S、ycdN-ppc-A is a primer, and upstream homology is obtained by PCR amplificationThe source arm, the downstream homologous arm and the target gene fragment are taken as templates, and P is obtained by overlapping PCR trc -ppc（ycdN) A gene integration fragment consisting ofycdNUpstream homology arm, P trc -ppcGenes of interestycdNDownstream homology arms.
(2) To be used forycdNpGRB-SycdNpGRB-A as primer, construction of pGRB by PCR annealing procedureycdNThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-ycdN。
(3) The P obtained in the steps (1) and (2) is reacted with trc -ppc（ycdN) Gene integration fragment and pGRBycdNThe plasmid is electrotransferred into PIP02 strain, and positive transformant is obtained by screening, pGRB-ycdNThe plasmid was designated PIP03.
Example 5
The present embodiment is intended to be described inrphPseudogene locus use P trc Promoter controllysCThe method comprises the following steps of gene overexpression:
(1) the E.coli W3110 genome was used as a template, respectivelyrph-U-S、rph-U-A、rph-D-S、rph-D-A andrph-lysC-S、rph-lysCthe-A is used as a primer, the upstream homology arm, the downstream homology arm and the target gene fragment are obtained through PCR amplification, and then the P is obtained through overlap PCR by using the fragment as a template trc -lysC（rph) A gene integration fragment consisting ofrphUpstream homology arm, P trc -lysCGenes of interestrphDownstream homology arms.
(2) To be used forrphpGRB-SrphpGRB-A as primer, construction of pGRB by PCR annealing procedurerphThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-rph。
(3) The P obtained in the steps (1) and (2) is reacted with trc -lysC（rph) Gene integration fragment and pGRBrphPlasmid electrotransformation into PIP03 StrainIn the step (2), positive transformants are obtained through screening, and pGRB (beta-cell growth promoting) is eliminatedrphThe plasmid was designated PIP04.
Example 6
The present embodiment is intended to be described infhiaPseudogene locus use P trc Promoter controlasdThe method comprises the following steps of gene overexpression:
(1) the E.coli W3110 genome was used as a template, respectivelyfhia-U-S、fhia-U-A、fhia-D-S、fhia-D-A andfhia-asd-S、fhia-asdthe-A is used as a primer, the upstream homology arm, the downstream homology arm and the target gene fragment are obtained through PCR amplification, and then the P is obtained through overlap PCR by using the fragment as a template trc -asd（fhia) A gene integration fragment consisting offhiaUpstream homology arm, P trc -asdGenes of interestfhiaDownstream homology arms.
(2) To be used forfhiapGRB-SfhiapGRB-A as primer, construction of pGRB by PCR annealing procedurefhiaThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-fhia。
(3) The P obtained in the steps (1) and (2) is reacted with trc -asd（fhia) Gene integration fragment and pGRBfhiaThe plasmid is electrotransferred into PIP04 strain, and positive transformant is obtained by screening, pGRB-fhiaThe plasmid was designated PIP05.
Example 7
The present embodiment is intended to be described inyjiPPseudogene locus use P trc Promoter controldapAThe method comprises the following steps of gene overexpression:
(1) the E.coli W3110 genome was used as a template, respectivelyyjiP-U-S、yjiP-U-A、yjiP-D-S、yjiP-D-A andyjiP-dapA-S、yjiP-dapAthe-A is used as a primer, the upstream homology arm, the downstream homology arm and the target gene fragment are obtained through PCR amplification, and then the P is obtained through overlap PCR by using the fragment as a template trc -dapA（rph) Gene integration fragments, the gene integrationThe synthetic segment is composed ofyjiPUpstream homology arm, P trc -dapAGenes of interestyjiPDownstream homology arms.
(2) To be used foryjiPpGRB-SyjiPpGRB-A as primer, construction of pGRB by PCR annealing procedureyjiPThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-yjiP。
(3) The P obtained in the steps (1) and (2) is reacted with trc -dapA（yjiP) Gene integration fragment and pGRByjiPThe plasmid is electrotransferred into PIP05 strain, and positive transformant is obtained by screening, pGRB-yjiPThe plasmid was named PIP06.
Example 8
The present embodiment is intended to be described inyciQPseudogene locus use P trc Promoter controllysAThe method comprises the following steps of gene overexpression:
(1) the E.coli W3110 genome was used as a template, respectivelyyciQ-U-S、yciQ-U-A、yciQ-D-S、yciQ-D-A andyciQ-lysA-S、yciQ-lysAthe-A is used as a primer, an upstream homology arm, a downstream homology arm and a target gene fragment are obtained through PCR amplification, and then the P is obtained through overlap PCR by using the primer as a template trc -lysA（yciQ) A gene integration fragment consisting ofyciQUpstream homology arm, P trc -lysAGenes of interestyciQDownstream homology arms.
(2) To be used foryciQpGRB-SyciQpGRB-A as primer, construction of pGRB by PCR annealing procedureyciQThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-yciQ。
(3) The P obtained in the steps (1) and (2) is reacted with trc -lysA（yciQ) Gene integration fragment and pGRByciQThe plasmid is electrotransferred into PIP06 strain, and positive transformant is obtained by screening, pGRB-yciQThe plasmid was designated PIP07.
Example 9
The present embodiment is intended to be described inyeeLPseudogene locus use P trc Promoter controllatThe exogenous gene over-expression step includes the following steps:
(1) the E.coli W3110 genome was used as a template, respectivelyyeeL-U-S、yeeL-U-A、yeeL-D-S、yeeLD-A as primer, and PCR amplification to obtain upstream homology arm and downstream homology armFlavobacterium lutescensGenome is used as template toyeeL-lat-S，yeeL-latThe method comprises the steps of (a) using (a) as a primer, amplifying by PCR to obtain a target gene fragment, performing codon optimization on the target gene fragment, and overlapping PCR by using an upstream homology arm, a downstream homology arm and the target gene fragment subjected to the codon optimization as a template to obtain P trc -lat（yeeL) A gene integration fragment consisting ofyeeLUpstream homology arm, P trc -latGenes of interestyeeLDownstream homology arms.
(2) To be used foryeeLpGRB-SyeeLpGRB-A as primer, construction of pGRB by PCR annealing procedureyeeLThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-yeeL。
(3) The P obtained in the steps (1) and (2) is reacted with trc -lat（yeeL) Gene integration fragment and pGRByeeLThe plasmid is electrotransferred into PIP07 strain, and positive transformant is obtained by screening, pGRB-yeeLThe plasmid was designated PIP08.
Example 10
The present embodiment is intended to be described inmbhAPseudogene locus use P trc Promoter controllatThe exogenous gene over-expression step includes the following steps:
(1) the E.coli W3110 genome was used as a template, respectivelymbhA-U-S、mbhA-U-A、mbhA-D-S、mbhAD-A as primer, and PCR amplification to obtain upstream homology arm and downstream homology armFlavobacterium lutescensGenome is used as template toyeeL-lat-S，yeeL-lat-A as primer, amplified by PCRObtaining target gene fragments by augmentation, carrying out codon optimization on the target gene fragments, and obtaining P by overlapping PCR by taking the target gene fragments with the upstream homology arm, the downstream homology arm and the codon optimized as templates trc -lat（mbhA) A gene integration fragment consisting ofmbhAUpstream homology arm, P trc -latGenes of interestmbhADownstream homology arms.
(2) To be used formbhApGRB-SmbhApGRB-A as primer, construction of pGRB by PCR annealing procedurembhAThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-mbhA。
(3) The P obtained in the steps (1) and (2) is reacted with trc -lat（mbhA) Gene integration fragment and pGRBmbhAThe plasmid is electrotransferred into PIP08 strain, and positive transformant is obtained by screening, pGRB-mbhAThe plasmid was designated PIP09.
Example 11
The present embodiment is intended to be described inilvGPseudogene locus use P trc Promoter controlproCThe exogenous gene over-expression step includes the following steps:
(1) the E.coli W3110 genome was used as a template, respectivelyilvG-U-S、ilvG-U-A、ilvG-D-S、ilvGThe D-A is used as a primer, an upstream homology arm and a downstream homology arm are obtained by PCR amplification, and the Corynebacterium glutamicum ATCC 13032 genome is used as a template, so thatilvG-proC-S，ilvG-proCThe target gene fragment is obtained by PCR amplification by taking the A as a primer, and the P is obtained by overlapping PCR by taking the upstream homology arm, the downstream homology arm and the target gene fragment as templates trc -proC（ilvG) A gene integration fragment consisting ofilvGUpstream homology arm, P trc -proCGenes of interestilvGDownstream homology arms.
(2) By pGRB-ilvGS and pGRB-ilvGConstructing pGRB by PCR annealing procedure using A as primerilvGThe DNA fragment containing the target sequence is used and converted to Top10 conversion competenceIn the cells, positive transformants are obtained by screening, and plasmid pGRB is extractedilvG。
(3) The P obtained in the steps (1) and (2) is reacted with trc -proC（ilvG) Gene integration fragment and pGRBilvGThe plasmid was electrotransferred into PIP09 strain, and positive transformants were obtained by selection, the PGRB plasmid was deleted and designated PIP10.
Example 12
The present embodiment is intended to be described inygaYPseudogene locus use P trc Promoter controlpntABThe method comprises the following steps of gene overexpression:
(1) the E.coli W3110 genome was used as a template, respectivelyygaY-U-S、ygaY-U-A、ygaY-D-S、ygaY-D-A andygaY-pntAB-S、ygaY-pntABthe-A is used as a primer, the upstream homology arm, the downstream homology arm and the target gene fragment are obtained through PCR amplification, and then the P is obtained through overlap PCR by using the fragment as a template trc -pntAB（ygaY) A gene integration fragment consisting ofygaYUpstream homology arm, P trc -pntABGenes of interestygaYDownstream homology arms.
(2) To be used forygaYpGRB-SygaYpGRB-A as primer, construction of pGRB by PCR annealing procedureygaYThe DNA fragment containing the target sequence is used and transformed into Top10 transformed competent cells, positive transformants are obtained by screening, and plasmid pGRB-ygaY。
(3) The P obtained in the steps (1) and (2) is reacted with trc -pntAB（ygaY) Gene integration fragment and pGRBygaYThe plasmid is electrically transferred into PIP010 strain, positive transformant is obtained through screening, pGRB-ygaYAnd pREDCas9 plasmid and designated PIP11.
Example 13
The strain PIP11 is used as an L-homoproline production strain, and the method for producing L-homoproline by using the production strain is described in the embodiment, wherein a 5L mechanical stirring type fermentation tank is used, and the specific culture mode is as follows:
seed activation: inoculating the preserved strain at-80deg.C on non-activated slant, culturing at 37deg.C for 12 hr, and passaging for 2 times; the activated cells on the inclined plane were eluted with sterilized distilled water and transferred to a 5L mechanical stirring fermenter to start seed culture.
Seed culture: the culture temperature is 35 ℃, the pH of the culture is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 40% by adjusting the stirring rotation speed or ventilation, and the OD is obtained 600nm Meet the inoculation requirement when the number is 20; the seed culture medium adopted is as follows: 30g/L glucose, 5g/L yeast powder, 2g/L peptone, (NH) 4 ) 2 SO 4 1g/L，KH 2 PO 4 3g/L，MgSO 4 ·7H 2 0.8g/L of O, 1g/L of monosodium glutamate and the balance of water.
Fermentation culture: the fermentation inoculation amount is 30%, the culture temperature is 35 ℃, the culture pH is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the culture dissolved oxygen value is maintained at 40% by adjusting the stirring rotation speed or ventilation amount, the glucose concentration in a tank is controlled to be less than or equal to 0.5g/L by feeding 80% (mass volume fraction) glucose solution, meanwhile, 9 mgPLP and 2.25g choline chloride (namely PLP 6mg/L sugar solution and choline chloride 1.5g/L sugar solution) are added into 1.5L 80% (mass volume fraction) glucose solution along with the glucose solution fed, and the fermentation period is 50h; the fermentation medium adopted is: 30g/L glucose, 5g/L yeast powder, 2g/L peptone, (NH) 4 ) 2 SO 4 1g/L，KH 2 PO 4 4g/L，MgSO 4 ·7H 2 O1.5 g/L, methionine 0.3g/L, isoleucine 0.5g/L, feSO 4 ·7H 2 O20 mg/L, and the balance of water.
Example 14
The effect of PLP and choline chloride in L-homoproline fermentation applications is illustrated in this example using PIP11 as a producer strain. The fermentation culture was conducted in accordance with example 13, except that four control groups were provided with the addition or non-addition of PLP and choline chloride to the 80% glucose solution, and the results of the fermentation in the four fermenter groups for 50 hours were shown in Table 2.
TABLE 2 influence of PLP and choline chloride on biomass of cells and L-homoproline production
Group 1 Group 2 Group 3 Group 4
PLP addition (mg/L sugar solution) 0 0 6 6
Choline chloride addition (g/L sugar solution) 0 1.5 0 1.5
Bacterial biomass OD 600nm 151.7 159.2 154.9 157.5
L-high proline yield g/L 29.8 33.4 36.1 40.5
In conclusion, the L-homoproline producing strain is obtained by directionally modifying the metabolic synthesis path of L-homoproline, and in the constructed L-homoproline producing strain, lysine decarboxylase genecadAAndldcCis derived from Escherichia coli, and can catalyze L-lysine to be decomposed and metabolized in cells to generate pentanediamine and carbon dioxide, so as to reducecadAAndldcCthe expression level of the gene can effectively improve the accumulation of L-lysine which is a precursor of L-homoproline. Phosphoenolpyruvate carboxylaseppcFrom Escherichia coli, the enzyme coded by the enzyme can catalyze the conversion of phosphoenolpyruvic acid into oxaloacetic acid, and promote the accumulation of L-lysine which is a precursor of L-homoproline. Aspartokinase genelysCAspartate semialdehyde dehydrogenase geneasd，Dihydropyridine dicarboxylic acid synthase genedapA，Diaminopimelate decarboxylase geneslysAAll are derived from escherichia coli, and the coded enzyme can effectively improve the accumulation of L-lysine which is a precursor of L-homoproline. L-lysine-6-aminotransferase genelatDerived fromFlavobacterium lutescensCodon optimizedlatThe gene codes for a key enzyme catalyzing the production of L-homoproline, and the cofactor alpha-ketoglutarate can be used for ammonifying L-lysine into alpha-aminoadipic semialdehyde. And (V) 1 Pyrroline-5-carboxylate reductase geneproCFrom E.coli, the enzyme encoded thereby catalyzes the production of L-homoproline. Membrane boundary inducerpntABThe gene can improve the intracellular NADPH conversion rate and the L-high proline yield.
The L-homoproline production strain takes glucose as a carbon source, L-homoproline is directly synthesized from the head in a fermentation tank, pyridoxal phosphate (PLP) and choline chloride are added in the fermentation process of the strain, so that on one hand, the catalytic requirement of L-lysine 6-aminotransferase can be ensured, on the other hand, the activity of the strain can be ensured, the production benefit is improved, the production cost in the fermentation process is low, the heredity is stable, and the high economic benefit is realized, thereby laying a foundation for realizing the large-scale production of L-homoproline.
The foregoing is merely illustrative of the preferred embodiments of this invention, and it will be appreciated by those skilled in the art that variations and modifications of the invention and strain changes, which are carried out by or based on the methods of this invention, may be made without departing from the spirit of this invention.

Claims (10)

1. An L-homoproline producing strain, characterized in that: is prepared from the original strain by metabolic engineeringE.coliThe W3110 is obtained by further modification, and specifically comprises the following steps:
knockout on genomecadAThe gene is not expressed by the gene,
knockout on genomeldcCThe gene is not expressed by the gene,
at the position ofycdNPseudogene locus use P trc Promoter controlppcThe gene is over-expressed and the expression of the gene is improved,
at the position ofrphPseudogene locus use P trc Promoter controllysCThe gene is over-expressed and the expression of the gene is improved,
at the position offhiaPseudogene locus use P trc Promoter controlasdThe gene is over-expressed and the expression of the gene is improved,
at the position ofyjiPPseudogene locus use P trc Promoter controldapAThe gene is over-expressed and the expression of the gene is improved,
at the position ofyciQPseudogene locus use P trc Promoter controllysAThe gene is over-expressed and the expression of the gene is improved,
at the position ofyeeLPseudogene locus use P trc Promoters control exogenous sourceslatThe gene is over-expressed and the expression of the gene is improved,
at the position ofmbhAPseudogene locus use P trc Promoters control exogenous sourceslatThe gene is over-expressed and the expression of the gene is improved,
at the position ofilvGPseudogene locus use P trc Promoters control exogenous sourcesproCThe gene is over-expressed and the expression of the gene is improved,
at the position ofygaYPseudogene locus use P trc Promoter controlpntABThe gene is over-expressed.
2. The L-homoproline producing strain of claim 1, which is characterized in that: the starting strainE.coliW3110 was deposited under ATCC 273250.
3. The L-homoproline producing strain of claim 1, which is characterized in that: the P is trc The nucleotide sequence of the promoter is shown in a sequence table SEQ ID NO. 1; the saidcadAThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 2; the saidldcCThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 3; the saidppcThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 4; the saidlysCThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 5; the saidasdThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 6; the saiddapAThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 7; the saidlysAThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 8; the saidlatThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 9; the saidproCThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 10; the saidpntABThe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 11.
4. A method of constructing an L-homoproline producing strain according to any one of claims 1 to 3, characterized in that: in the original strainE.coliThe method for directionally modifying the W3110 comprises the following specific steps:
(1) In the original strainE.coliW3110 genomic knockoutcadAThe gene is obtained into a strain PIP01;
(2) Based on strain PIP01, knockout on genomeldcCThe gene is obtained into a strain PIP02;
(3) Based on strain PIP02, inycdNPseudogene locus use P trc Promoter controlppcThe gene is over-expressed to obtain a strain PIP03;
(4) Based on strain IP03, inrphPseudogene locus use P trc Promoter controllysCThe gene is over-expressed to obtain a strain PIP04;
(5) Based on strain PIP04, infhiaPseudogene locus use P trc Promoter controlasdThe gene is over-expressed to obtain a strain PIP05;
(6) Based on strain PIP05, inyjiPPseudogene locus use P trc Promoter controldapAThe gene is over-expressed to obtain a strain PIP06;
(7) Based on strain PIP06, inyciQPseudogene locus use P trc Promoter controllysAThe gene is over-expressed to obtain a strain PIP07;
(8) Based on strain PIP07, inyeeLPseudogene locus use P trc Promoters control exogenous sourceslatThe gene is over-expressed to obtain a strain PIP08;
(9) Starting from strain PIP08, inmbhAPseudogene locus use P trc Promoters control exogenous sourceslatThe gene is over-expressed to obtain a strain PIP09;
(10) Starting from strain PIP09, inilvGPseudogene locus use P trc Promoters control exogenous sourcesproCThe gene is over-expressed to obtain a strain PIP10;
(11) Starting from strain PIP10, inygaYPseudogene locus use P trc Promoter controlpntABGene overexpression gives strain PIP11.
5. Use of an L-homoproline producing strain according to any one of claims 1 to 3 for the fermentative production of L-homoproline.
6. The use of the strain for producing L-homoproline according to claim 5, wherein: the L-homoproline is produced by fermentation in a mechanically stirring type fermentation tank, and is synthesized by seed culture and fermentation culture and glucose as a substrate.
7. The use of the strain for producing L-homoproline according to claim 6, characterized in that: the method comprises the following specific steps:
(1) Seed culture: the culture temperature was 35℃by automatic flowAdding 25% ammonia water solution to maintain culture pH at 7.0+ -0.2, regulating stirring speed or ventilation rate to maintain dissolved oxygen value at 40%, and obtaining OD 600nm Meet the inoculation requirement when the number is 20;
(2) Fermentation culture: the inoculation amount is 30%, the culture temperature is 35 ℃, the pH is controlled to be 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained to be 40% by adjusting the stirring speed or ventilation, and the glucose concentration in the tank is controlled to be less than or equal to 0.5g/L by feeding 80% glucose solution.
8. The use of the strain for producing L-homoproline according to claim 7, characterized in that: inoculating the preserved strain at-80deg.C on non-activated slant, culturing at 37deg.C for 12 hr, and passaging for 2 times; eluting the activated thalli on the inclined plane by sterilized distilled water, and transferring the thalli into a mechanical stirring type fermentation tank to start seed culture.
9. The use of the strain for producing L-homoproline according to claim 7, characterized in that: the seed culture medium adopted in the seed culture comprises: 30g/L glucose, 5g/L yeast powder, 2g/L peptone, (NH) 4 ) 2 SO 4 1g/L，KH 2 PO 4 3g/L，MgSO 4 ·7H 2 0.8g/L of O, 1g/L of monosodium glutamate and the balance of water; the fermentation culture medium adopted in the fermentation culture comprises the following components: 30g/L glucose, 5g/L yeast powder, 2g/L peptone, (NH) 4 ) 2 SO 4 1g/L，KH 2 PO 4 4g/L，MgSO 4 ·7H 2 O1.5 g/L, methionine 0.3g/L, isoleucine 0.5g/L, feSO 4 ·7H 2 O20 mg/L, and the balance of water.
10. The use of the strain for producing L-homoproline according to claim 7, characterized in that: during fermentation culture, pyridoxal phosphate and choline chloride are fed along with the glucose solution.
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