Proline production strain as well as construction method and application thereof

Abstract

The invention provides a proline production strain and a construction method and application thereof, the proline production strain is obtained by further modification on the basis of an original strain E.coli W3110 by a metabolic engineering modification method, specifically, putA, aceA, poxB, ldhA, ackA and ptsG genes are knocked out, the transcriptional levels of proB74, proC, proA, stA, glf, glk and gdhA genes are up-regulated, the transcriptional level of a SucAB gene is down-regulated, and the proline production strain can be used for producing proline. The method comprises the following steps: carrying out heterologous expression on a phosphoketolyase gene BAD0687 and a proline transporter coding gene cgl2622; the strain takes glucose as a carbon source, does not need to add a substrate, is low in production cost, has the advantages of high production rate, short fermentation period and high strain stability, has very high economic benefits, and lays a foundation for large-scale production of proline.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation engineering, and particularly relates to a proline production strain and a construction method and application thereof. BACKGROUND

[0002] L-proline is mainly used as an organic catalyst in the chemical industry, has the characteristics of strong activity and good stereospecificity; is used as a precursor of compounds in the pharmaceutical and cosmetic industries, is a raw material of amino acid drugs; in addition, L-proline is a potential virulence factor of certain pathogenic bacteria and a source of carbon, nitrogen and energy in prokaryotic and eukaryotic cells, and can be accumulated in large amounts to play a role in regulating osmotic pressure and stabilizing the structure of biological macromolecules.

[0003] At present, the main methods for synthesizing proline are chemical synthesis and microbial fermentation. The chemical synthesis method has the problems of serious environmental pollution and low conversion rate; the microbial fermentation method uses glucose as the energy source for microbial growth, and has the potential for industrial production due to the mild fermentation process conditions.

[0004] However, the existing microbial fermentation method needs to add glutamic acid in the production process of proline, and the conversion rate of proline is still low, so it is urgent to develop a proline production strain that can synthesize proline from scratch without adding substrates and has a high conversion rate. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a proline production strain.

[0006] Another technical problem to be solved by the present application is to provide a construction method of the proline production strain.

[0007] Another technical problem to be solved by the present application is to provide an application of the proline production strain.

[0008] To solve the above technical problems, the technical scheme of the present application is as follows: A proline production strain is obtained by further modifying a starting strain W3110 using a metabolic engineering method, specifically: knocking out the genes of proline dehydrogenase, proline permease and proline dehydrogenase, up-regulating the transcription level of the genes of proline permease and proline dehydrogenase, and down-regulating the transcription level of the genes of proline permease and proline dehydrogenase. E. coli W3110, specifically: knocking out the genes of proline dehydrogenase, proline permease and proline dehydrogenase, up-regulating the transcription level of the genes of proline permease and proline dehydrogenase, and down-regulating the transcription level of the genes of proline permease and proline dehydrogenase. putA, aceA, poxB, IdhA, ackA, ptsG genes, up-regulating the transcription level of the genes of proline permease and proline dehydrogenase, and down-regulating the transcription level of the genes of proline permease and proline dehydrogenase. proB74 , proC, proA, sthA, glf, glk, gdhA genes, up-regulating the transcription level of the genes of proline permease and proline dehydrogenase, and down-regulating the transcription level of the genes of proline permease and proline dehydrogenase. SucAB genes, up-regulating the transcription level of the genes of proline permease and proline dehydrogenase, and down-regulating the transcription level of the genes of proline permease and proline dehydrogenase. BAD_0687 phosphoketolase gene and proline transporter coding gene. cgl2622 phosphoketolase gene and proline transporter coding gene.

[0009] Preferably, the proline production strain, the metabolic engineering method is CRISPR-Cas9 gene editing technology.

[0010] Preferably, the above proline-producing strain, the above E. coli W3110 is E. coli W3110 ATCC 27325.

[0011] Preferably, the above proline-producing strain, the above E. coli W3110 genome, respectively, knock out putA gene, so that it does not express; knock out aceA gene, so that it does not express; knock out poxB gene, so that it does not express; knock out ldhA gene, so that it does not express; knock out ackA gene, so that it does not express; knock out ptsG gene, so that it does not express; in yghX P trc promoter control proB74 gene overexpression; in mbhA P trc promoter control proB74 mutant gene overexpression; in yciQ P proA and proC gene and form a mini-operon proAC , and P trc promoter start; in yjiP P trc promoter control sthA gene overexpression; in ylbE P trc promoter control glf gene overexpression; in ydeU P trc promoter control glk gene overexpression; in yncI P trc promoter control gdhA gene overexpression; replace the original promoter of SucAB gene with BBa-j23109 promoter, weaken the expression; in yeep P trc promoter control phosphoketolase gene BAD_ 0687 heterologous expression; in yjgX P trc promoter control proline transporter encoding gene cgl2622 overexpression.

[0012] Preferably, the above proline-producing strain, the above mini-operon proACThe nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing; this microoperon proAC At the genome level, using the trc promoter, tandem integration was achieved. proA and proC The gene is modified by inserting an RBS sequence (the RBS ribosome binding site controls the initiation of transcription by the operon) into the middle of the gene sequence. The transcription is terminated by the same rrnB T1 terminator, forming a miniature artificial operon capable of tandemly expressing glutamate semialdehyde dehydrogenase encoded by the proA gene and pyrroline-5-carboxylic acid reductase encoded by the proC gene. Specifically, the operon gene sequence is P... trc The promoter, RBS ribose binding site, proA, proC, and rrnB T1 terminator are included. The nucleotide sequence of the RBS is shown in SEQ ID NO. 20 of the sequence listing; the nucleotide sequence of the rrnB T1 terminator is shown in SEQ ID NO. 21 of the sequence listing.

[0013] Preferably, the above-mentioned proline-producing strain, wherein P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.1; the nucleotide sequence of the BBa-j23109 promoter is shown in SEQ ID NO.14.

[0014] Preferably, the above-mentioned proline-producing strain, wherein putA The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing; aceA The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing; poxB The nucleotide sequence of the gene is shown in SEQ ID NO.4 of the sequence listing; ldhA The nucleotide sequence of the gene is shown in SEQ ID NO. 5 of the sequence listing; ackA The nucleotide sequence of the gene is shown in SEQ ID NO. 6 of the sequence listing; ptsG The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing; proB74 The nucleotide sequence of the gene is shown in SEQ ID NO. 8 of the sequence listing; proA The nucleotide sequence of the gene is shown in SEQ ID NO. 18 of the sequence listing; proCThe nucleotide sequences of the genes are shown in SEQ ID NO. 19; the nucleotide sequence of the sthA gene is shown in SEQ ID NO. 10; the nucleotide sequence of the glf gene is shown in SEQ ID NO. 11; the nucleotide sequence of the glk gene is shown in SEQ ID NO. 12; the nucleotide sequence of the gdhA gene is shown in SEQ ID NO. 13; the nucleotide sequence of the SucAB gene is shown in SEQ ID NO. 17; the phosphorylketolase gene BAD_0687 The nucleotide sequence of the proline transporter gene cgl12622 is shown in SEQ ID NO.15; the nucleotide sequence of the proline transporter gene cgl12622 is shown in SEQ ID NO.16.

[0015] The above-mentioned method for constructing proline-producing strains involves starting with the proline-producing strain. E. coli Further targeted modifications will be made based on the W3110, with the following specific steps: (1) In the starting strain E. coli Knockout on the W3110 genome putA Genes were obtained from strain Pro01; (2) Starting with strain Pro01, the aceA gene was knocked out to obtain strain Pro02; (3) Starting with strain Pro02, the poxB gene was knocked out to obtain strain Pro03; (4) Starting with strain Pro03, the ldhA gene was knocked out to obtain strain Pro04; (5) Starting with strain Pro04, the ackA gene was knocked out to obtain strain Pro05. (6) Starting with strain Pro05, the ptsG gene was knocked out to obtain strain Pro06; (7) Using strain Pro06 as the starting strain, in yghX Pseudogene loci use P trc Starter control proB74 Gene overexpression yielded strain Pro07; (8) Using strain Pro07 as the starting strain, in mbhA Pseudogene loci use P trc Starter control proB74 Gene overexpression yielded strain Pro08; (9) Using strain Pro08 as the starting strain, in yciQ Pseudogene loci tandem integration proA, proC Genes form a miniature operon proAC and with P trc The promoter was used to start the strain, resulting in strain Pro09. (10) Take strain Pro09 as the starting strain, use P yjiP P trc Take strain Pro10 as the starting strain, use P (11) Take strain Pro10 as the starting strain, use P trc Take strain Pro11 as the starting strain, use P (12) Take strain Pro11 as the starting strain, use P ydeU P trc Take strain Pro12 as the starting strain, use P (13) Take strain Pro12 as the starting strain, use P ncI P trc Take strain Pro13 as the starting strain, use P (14) Take strain Pro13 as the starting strain, replace the original promoter of SucAB gene with BBa-j23109 promoter to obtain strain Pro14; (15) Take strain Pro14 as the starting strain, use P yeep P trc Take strain Pro15 as the starting strain, use P BAD_0687 Take strain Pro16 as the starting strain, use P (16) Take strain Pro15 as the starting strain, use P yjgX P trc Take strain Pro16 as the starting strain, use P cgl2622 The obtained strain Pro16 is the target strain after successful modification.

[0016] Preferably, in the construction method of the proline-producing strain, the proline oxidase gene putA is derived from Escherichia coli, and knocking out the gene can effectively reduce the catabolism of proline to achieve the purpose of proline accumulation; the isocitrate lyase gene aceA is derived from Escherichia coli, and knocking out the gene can effectively reduce the catabolism of isocitric acid to provide more alpha-ketoglutaric acid for the production of proline; the pyruvate dehydrogenase gene poxB is derived from Escherichia coli, and knocking out the gene can effectively reduce the catabolism of pyruvic acid to provide more pyruvic acid for the production of proline; the D-lactate dehydrogenase gene ldhA is derived from Escherichia coli, and knocking out the gene can effectively reduce the catabolism of pyruvic acid to provide more pyruvic acid for the production of proline; the acetate kinase gene ackA, which is derived from Escherichia coli, and knocking out the gene can effectively reduce the catabolism of acetyl-CoA, providing more citric acid for the production of proline; the PTS system core component gene ptsG , which is derived from Escherichia coli, and knocking out the gene can make the bacterial cells simultaneously utilize multiple carbon sources, improving substrate utilization rate; glutamate kinase gene proB74 , which is derived from Escherichia coli, and is obtained by mutating the proB gene, which encodes the first enzyme for proline synthesis, and after mutation, the negative feedback inhibition effect is removed, and the enzyme can effectively improve the yield of proline; glutamate semialdehyde dehydrogenase gene proA , which is derived from Escherichia coli, and encodes the second enzyme for proline synthesis, which can further strengthen the proline synthesis metabolic pathway; pyrroline-5-carboxylate reductase gene proC , which is derived from Escherichia coli, and encodes the third enzyme for proline synthesis, which can strengthen the proline synthesis metabolic pathway; pyridine nucleotide transhydrogenase encoding gene sthA, which is derived from Escherichia coli, and the enzyme can strengthen the supply of NADPH in cells; UDP galactose mutarotase gene glf , which is derived from Escherichia coli, and the enzyme can realize the simultaneous utilization of mixed sugars, improving substrate conversion efficiency; glucose kinase gene glk , which is derived from Escherichia coli, and the enzyme can improve glucose conversion efficiency and reduce metabolic burden; glutamate dehydrogenase gene gdhA , which is derived from Escherichia coli, and the enzyme can provide more glutamic acid for the production of proline; alpha-ketoglutarate dehydrogenase gene SucAB, which is a weakened Escherichia coli gene, and the original promoter of SucAB gene is replaced by BBa-j23109 promoter, which weakens the expression of alpha-ketoglutarate in TCA cycle and enhances the proline metabolic flow; phosphoketolase BAD_0687 gene, which is derived from Bifidobacterium adolescentis and is codon-optimized BAD_0687 gene, which encodes a key enzyme for catalyzing acetyl-CoA generation; proline transporter cgl2622 gene, which is derived from Corynebacterium glutamicum, and which can promote the transport of intracellular product proline to the extracellular.

[0017] The above-mentioned proline production strain is applied in the fermentation production of proline.

[0018] Preferably, the above-mentioned application has the following specific steps: (1) Strain activation: inoculate the bacterial liquid from the bacterial preservation tube into the slant medium to activate the culture for 12h; inoculate the colonies from the slant medium into the LB medium to continue the activation culture for 12h, and the culture temperature is 37℃; (2) Inoculate into the flask containing the fermentation medium at 10%-15% inoculation amount, seal with nine layers of gauze, cultivate at 37℃ with 220 r / min shaking, maintain pH at 7.0-7.2 by adding ammonia water during the fermentation process, add 60% glucose solution to maintain the fermentation, and the fermentation cycle is 22-26h.

[0019] Preferably, in the above application, the LB medium used in step (1) is: yeast extract powder 5g / L, peptone 10g / L, NaCl 10g / L, agar powder 20g / L, and the rest is water.

[0020] Preferably, in the above application, the slant medium used in step (1) is: glucose 2g / L, yeast extract powder 5g / L, peptone 10g / L, NaCl 2.5g / L, KH2PO4 1g / L, MgSO4·7H2O 0.2g / L, agar powder 2.5g / L, and the rest is water.

[0021] Preferably, in the above application, the fermentation medium used in step (2) is: glucose 15g / L, yeast extract powder 3.5g / L, peptone 1g / L, (NH4)2SO4 2g / L, K2HPO4·3H2O 2g / L, MgSO4·7H2O 2g / L, glutamic acid 2g / L, methionine 0.5g / L, MnSO4·H2O 10mg / L, FeSO4·7H2O 30mg / L.

[0022] The above-mentioned culture media can be prepared by standard methods.

[0023] Beneficial effects: The above-mentioned proline-producing strain is obtained by the de novo synthesis of the proline metabolic synthesis pathway using the directed modification method, the strain uses glucose as the carbon source, does not need to add substrate, has low production cost, high production rate, short fermentation cycle, and high strain stability, has high economic benefits, and lays a foundation for large-scale production of proline. In the application process, the strain efficiently and stably synthesizes proline de novo, and the yield of proline is 26.35g / L after 24h of fermentation, which lays a foundation for large-scale production of proline. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Process diagram for genetic modification of the de novo synthesis pathway of the proline-producing strain. DETAILED DESCRIPTION

[0025] In order for those skilled in the art to better understand the technical solutions of the present application, the technical solutions of the present application will be further described in detail below in combination with specific embodiments.

[0026] The percentage sign "%" involved in the examples, if not specifically stated, refers to the mass percentage, the percentage of the solution refers to the grams of solute contained in 100 mL, and the percentage between liquids refers to the volume ratio of the solution at 25°C.

[0027] The starting strain used in the examples is E. coli W3110 ATCC 27325, the primers used in the strain construction process are shown in Table 1.

[0028] Table 1 Primers involved in the strain construction process

[0029]

[0030]

[0031]

[0033]

[0034]

[0035]

[0036] The gene editing method used refers to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). This method involves engineering plasmids pREDCas9, pGRB, wherein pREDCas9 carries a gRNA expression plasmid pGRB elimination system, a Red recombination system of lambda phage, a Cas9 protein expression system and a spectinomycin resistance (working concentration: 100 mg / L); pGRB takes pUC18 as the backbone, including promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence and ampicillin resistance (working concentration: 100 mg / L). The professional terms involved in the following examples can be explained in this article.

[0037] Example 1 This example aims to illustrate the steps of knocking out genes in the specific construction of the strain. In particular, if there are the same type of gene operation methods in the examples, only one is provided and annotated, and no more details are added.

[0038] (1) With E. coli W3110 genome as template, and with putA-Q-1, putA-Q-2 and putA-Q-3, putA-Q-4 as primers, the upstream homologous arm and the downstream homologous arm are obtained by HS enzyme PCR amplification, and then with the upstream homologous arm and the downstream homologous arm as templates, the ΔputA gene knockout fragment is obtained by HS enzyme overlap PCR. A The gene integration fragment is composed of putA the upstream homologous arm and putA the downstream homologous arm.

[0039] (2) With pGRB-putA-S and pGRB-putA-A as primers, a DNA fragment containing the target sequence used by pGRB-putA is constructed by PCR annealing program, and then the DNA fragment is transformed into DH5α competent cells, and positive transformants are screened to extract plasmid pGRB-putA. (3) The ΔputA gene knockout fragment obtained in the above step is electroporated into putA W3110 strain with pGRB-putA plasmid, and positive transformants are screened and named as Pro01. E. coli

[0040] (4) Knockout of the genomic aceA gene: the same operation method as in steps (1)-(3) is used, except that the primers used are aceA-Q-1, aceA-Q-2, aceA-Q-3, aceA-Q-4, pGRB-aceA-S, pGRB-aceA-A. The competent cells are Pro01, and the strain Pro02 is obtained.

[0041] (5) Knockout of the genomic poxB gene: the same operation method as in steps (1)-(3) is used, except that the primers used are poxB-up-S, poxB-up-A, poxB-down-S, poxB-down-A, PGRB-poxB-UP, PGRB-poxB-DN. The competent cells are Pro02, and the strain Pro03 is obtained.

[0042] (6) Knockout of the genomic ldhA gene: the same operation method as in steps (1)-(3) is used, except that the primers used are ldhA-Q-1, ldhA-Q-2, ldhA-Q-3, ldhA-Q-4, pGRB-ldhA-S, pGRB-ldhA-A. The competent cells are Pro03, and the strain Pro04 is obtained.

[0043] ​(7) Knockout of ackA gene in the genome: the same operation method as in steps (1)-(3) was used, except that the primers used were ackA-Q-1, ackA-Q-2, ackA-Q-3, ackA-Q-4, pGRB-ackA-S, pGRB-ackA-A. The competent cells were Pro04, and the strain Pro05 was obtained.

[0044] (8) Knockout of ptsG gene in the genome: the same operation method as in steps (1)-(3) was used, except that the primers used were ptsG-Q-1, ptsG-Q-2, ptsG-Q-3, ptsG-Q-4, pGRB-ptsG-S, pGRB-ptsG-A. The competent cells were Pro05, and the strain Pro06 was obtained.

[0045] Example 2 This example is intended to illustrate the steps of overexpressing genes in the specific construction of the strain. In particular, if there are the same type of gene operation methods in the example, only one is provided, and it is annotated, and no more is described.

[0046] (1) The genomic DNA of E. coli W3110 was used as the template, and yghX-UP-S, yghX-UP-A, yghX-DN-S, yghX-DN-A, and proB-trc-S, proB-trc-A were used as primers to obtain the upstream homologous arm, the downstream homologous arm, and the target gene fragment by HS enzyme PCR amplification. Then, the genomic DNA of E. coli W3110 was used as the template, and the above-mentioned fragments were used as primers to obtain the P trc -proB( yghX ) gene integration fragment by HS enzyme overlap PCR. The gene integration fragment consisted of yghX the upstream homologous arm, the trc - proB target gene, and yghX the downstream homologous arm.

[0047] (2) The primers pGRB-yghX-S and pGRB-yghX-A were used to construct the DNA fragment containing the target sequence for pGRB-yghX by a PCR annealing program, and the DNA fragment was transformed into DH5a competent cells. Positive transformants were screened, and the plasmid pGRB-yghX was extracted.

[0048] (3) The P trc -proB( yghX ) gene integration fragment obtained in the above step was electroporated into the Pro06 strain together with the pGRB-yghX plasmid, and positive transformants were screened and named Pro07.

[0049] (4) Introducing proB74 gene at the site of mbhA pseudogene and overexpressing: the same operation method as in steps (1)-(3) is used, except that the primers used are mbhA-UP-S, mbhA-UP-A, mbhA-DN-S, mbhA-DN-A, PGRB-mbhA-S, PGRB-mbhA-A. The competent cell is Pro07, and the strain obtained is Pro08.

[0050] (5) Introducing sthA gene at the site of yjiP pseudogene and overexpressing: the same operation method as in steps (1)-(3) is used, except that the primers used are yjiP-UP-S, yjiP-UP-A, yjiP-DN-S, yjiP-DN-A, PGRB-yjiP-S, PGRB-yjiP-A, sthA-trc-UP, sthA-trc-DN. The competent cell is Pro09, and the strain obtained is Pro10. yciQ trc Introducing proB74 gene at the site of mbhA pseudogene and overexpressing: the same operation method as in steps (1)-(3) is used, except that the primers used are mbhA-UP-S, mbhA-UP-A, mbhA-DN-S, mbhA-DN-A, PGRB-mbhA-S, PGRB-mbhA-A. The competent cell is Pro07, and the strain obtained is Pro08. proAC Expression: ① Using the genome of E. coli W3110 as a template, the upper and lower homologous arms are amplified by PCR using primers yciQ-UP-S, yciQ-UP-A and yciQ-DN-S, yciQ-DN-A, respectively. The proA and proC fragments are amplified by PCR using primers proA-1, proA-2 and proC-1, proC-2, and proA-2 and proC-1 have homologous sequences. The trc-proAC fragment is obtained by overlap PCR using the proA and proC fragments as templates and primers proA-1 and proC-2. The overlap fragment P-trc-proAC(yciQ) is obtained by overlap PCR using the upper and lower homologous arms and the target gene fragment as templates and primers yciQ-UP-S and yciQ-DN-A. trc -proAC(yciQ); ② The DNA fragment containing the target sequence used for constructing pGRB-yciQ is obtained by PCR annealing program using primers PGRB-yciQ-S and PGRB-yciQ-A, and is transformed into DH5α competent cells. Positive transformants are screened, and the plasmid pGRB-yciQ is extracted. ③ The P-trc-proAC(yciQ) gene integration fragment obtained in the above step is electroporated into the Pro08 strain together with the pGRB-yciQ plasmid, and positive transformants are screened and named Pro09. trc -proAC(yciQ) gene integration fragment obtained in the above step is electroporated into the Pro08 strain together with the pGRB-yciQ plasmid, and positive transformants are screened and named Pro09.

[0051] (5) Introducing sthA gene at the site of yjiP pseudogene and overexpressing: the same operation method as in steps (1)-(3) is used, except that the primers used are yjiP-UP-S, yjiP-UP-A, yjiP-DN-S, yjiP-DN-A, PGRB-yjiP-S, PGRB-yjiP-A, sthA-trc-UP, sthA-trc-DN. The competent cell is Pro09, and the strain obtained is Pro10.

[0052] ​(7) Introduce and overexpress the glf gene at the ylbE pseudogene site: The operation method is the same as in steps (1) to (3), except that the primers used are ylbE-UP-S, ylbE-UP-A, ylbE-DN-S, ylbE-DN-A, PGRB-ylbE-S, PGRB-ylbE-A, glf-trc-UP, and glf-trc-DN. The competent cells are Pro10, and strain Pro11 is obtained.

[0053] (8) Introduce and overexpress the glk gene at the ydeU pseudogene site: The operation method is the same as in steps (1) to (3), except that the primers used are ydeU-US, ydeU-UA, ydeU-DS, ydeU-DA, PGRB-ydeU-UP, PGRB-ydeU-DN, glk-trc-UP, and glk-trc-DN. The competent cells are Pro11, and strain Pro12 is obtained.

[0054] (9) Introduce and overexpress the gdhA gene at the yncI pseudogene site: The operation method is the same as in steps (1) to (3), except that the primers used are yncI-US, yncI-UA, yncI-DS, yncI-DA, PGRB-yncI-UP, PGRB-yncI-DN, gdhA-trc-UP, and gdhA-trc-DN. The competent cells are Pro12, and strain Pro13 is obtained.

[0055] (10) Replace the original promoter of the SucAB gene with the BBa-j23109 promoter: ① Using the Escherichia coli W3110 genome as a template, respectively with SucAB -UPS, SucAB -UP-A、 SucAB -DN-S、 SucAB Using DN-A as primers, upstream and downstream homologous arms were obtained by HS enzyme PCR amplification. Then, using these as templates, P was obtained by HS enzyme overlap PCR. BBa_j23109 - SucAB Gene integration fragment, said gene integration fragment is composed of SucAB Upstream homologous arm and SucAB Downstream homologous arm composition; ② Using pGRB- SucAB -S and pGRB- SucAB -A is the primer, and pGRB- is constructed using a PCR annealing procedure. SucAB The DNA fragment containing the target sequence was used and transformed into DH5α competent cells. Positive transformants were obtained by screening, and plasmid pGRB- was extracted. SucAB ; (3) The PGRB-yeeP-UP and PGRB-yeeP-DN were amplified by PCR from the pGRB-yeeP plasmid, and then the PGRB-yeeP-UP and PGRB-yeeP-DN were digested by restriction enzyme Ndel and Xhol, and then the PGRB-yeeP-UP and PGRB-yeeP-DN were inserted into the pGRB-yeeP plasmid which had been digested by restriction enzyme Ndel and Xhol, and then the pGRB-yeeP-UP-DN was obtained. BBa_j23109 - SucAB The pGRB-yeeP-UP-DN was transformed into the Pro13 strain by electroporation, and then the positive transformant was obtained by screening, and was named as Pro14. SucAB The pGRB-yeeP-UP-DN was transformed into the Pro13 strain by electroporation, and then the positive transformant was obtained by screening, and was named as Pro14.

[0056] (11) The BAD_0687 gene was introduced into the yeeP pseudogene site and overexpressed: the same operation method was used in steps (1)-(3), except that the primers used were yeeP-UP-S, yeeP-UP-A, yeeP-DN-S, yeeP-DN-A, PGRB-yeeP-UP, PGRB-yeeP-DN, BAD_0687-trc-UP, BAD_0687-trc-DN. The competent cells were Pro14, and the strain Pro15 was obtained.

[0057] (12) The Cgl2622 gene was introduced into the yjgX pseudogene site and overexpressed: the same operation method was used in steps (1)-(3), except that the primers used were yjgX-UP-S, yjgX-UP-A, yjgX-DN-S, yjgX-DN-A, PGRB-yjgX-UP, PGRB-yjgX-DN, Cgl2622-trc-UP, Cgl2622-trc-DN. The competent cells were Pro15, and the strain Pro16 was obtained.

[0058] Example 3 The strain Pro16 was used as a proline production strain, and the present embodiment aimed to illustrate the method for producing proline by using the production strain Pro16, and the specific culture method was as follows: (1) Strain activation: the bacteria liquid was inoculated from the bacteria preservation tube to the slant medium for activation culture for 12 h; the colony was inoculated from the slant medium to the LB medium for continuous activation culture for 12 h, the culture temperature was 37℃, and the LB medium used was: yeast extract powder 5 g / L, protein peptone 10 g / L, NaCl 10 g / L, agar powder 20 g / L, and the rest was water; the slant medium was: glucose 2 g / L, yeast extract powder 5 g / L, protein peptone 10 g / L, NaCl 2.5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, agar powder 2.5 g / L, and the rest was water.

[0059] (2) Inoculate into a flask with fermentation medium at an inoculation amount of 10%-15%, seal with nine layers of gauze, and the fermentation medium is: glucose 15 g / L, yeast powder 3.5 g / L, peptone 1 g / L, (NH4)2SO4 2 g / L, K2HPO4·3H2O 2 g / L, MgSO4·7H2O 2 g / L, glutamic acid 2 g / L, methionine 0.5 g / L, MnSO4·H2O 10 mg / L, FeSO4·7H2O 30 mg / L.

[0060] With wild-type E. coli W3110 as a control group, after 24 h fermentation verification, the wild-type E. coli W3110 failed to produce proline, and the engineered bacteria Pro16 accumulated 26.35 g / L of proline, proving the effectiveness of the strain described in the application.

[0061] Example 4 This example aims to illustrate the influence of the mini artificial operon ProAC on the proline production capacity, and the strain Pro09 is taken as a chassis strain, and the strain Pro10 yciQ is obtained by using P trc promoter to control the overexpression of the ProA gene. is taken as a chassis strain, and the strain Pro16 is obtained by using P ybeQ promoter to control the overexpression of the ProC gene. trc The method described in Example 3 is used to produce proline, and the proline yield is shown in Table 2.

[0062] Table 2

[0063] As can be seen from Table 2, the proline yield of the strain Pro09 with a mini artificial operon is significantly improved compared with Pro08. By further overexpressing sthA, glf, glk, gdhA, BAD_0687 and cgl2622 genes and replacing the original promoter of the SucAB gene with the BBa-j23109 promoter, the proline yield of the obtained strain Pro16 is significantly improved again.

[0064] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and the technical personnel in the art make improvements and refinements such as strain modification based on the method of the present application, which are all considered within the protection scope of the present application.

Claims

1. A proline-producing strain, characterized by: is obtained by further modification of W3110 based on the starting strain E. coli W3110, specifically, the transcription level of putA, aceA, poxB, ldhA, ackA, ptsG gene is knocked out, the transcription level of proB74 , proC, proA, sthA, glf, glk, gdhA gene is up-regulated, the transcription level of SucAB gene is down-regulated, and the phosphoketolase gene BAD_0687 and the proline transporter coding gene cgl2622 are expressed heterologously.

2. The proline-producing strain according to claim 1, characterized by: The E. coli W3110 is E. coli W3110 ATCC 27325.

3. The proline-producing strain according to claim 1, characterized by: Use P trc Promoter control proB74 Gene overexpression; use P trc Promoter control proB74 Mutant gene overexpression; use P proA and proC Genes are integrated in tandem proAC and are under the control of P trc Promoter control; use P trc Promoter control sthA Gene overexpression; use P trc Promoter control glf Gene overexpression; use P trc Promoter control glk Gene overexpression; use P trc Promoter control gdhA Gene overexpression; replace original promoter of SucAB genes with BBa-j23109 promoter; use P trc Promoter control phosphoketolase genes BAD_0687 Heterologous expression; use P trc Promoter control proline transporter encoding genes cgl2622 Overexpression.

4. The proline-producing strain according to claim 3, characterized by: The micro- operon proAC The nucleotide sequence of the P trc The nucleotide sequence of the BBa-j23109 promoter is shown in the sequence listing SEQ ID NO.

14.

5. The proline-producing strain according to claim 1, characterized by: The putA The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing; aceA The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing; poxB The nucleotide sequence of the gene is shown in SEQ ID NO.4 of the sequence listing; ldhA The nucleotide sequence of the gene is shown in SEQ ID NO. 5 of the sequence listing; ackA The nucleotide sequence of the gene is shown in SEQ ID NO. 6 of the sequence listing; ptsG The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing.

6. The proline-producing strain according to claim 1 or 3, characterized by: The proB74 The nucleotide sequence of the gene is shown in SEQ ID NO. 8 of the sequence listing; proA The nucleotide sequence of the gene is shown in SEQ ID NO. 18 of the sequence listing; proC The nucleotide sequences of the genes are shown in SEQ ID NO. 19; the nucleotide sequence of the sthA gene is shown in SEQ ID NO. 10; the nucleotide sequence of the glf gene is shown in SEQ ID NO. 11; the nucleotide sequence of the glk gene is shown in SEQ ID NO. 12; the nucleotide sequence of the gdhA gene is shown in SEQ ID NO. 13; the nucleotide sequence of the SucAB gene is shown in SEQ ID NO. 17; the phosphorylketolase gene BAD_0687 The nucleotide sequence of the proline transporter gene cgl12622 is shown in SEQ ID NO.15; the nucleotide sequence of the proline transporter gene cgl12622 is shown in SEQ ID NO.

16.

7. A method for constructing a proline-producing strain according to any one of claims 1 to 6, characterized in that: In the starting strain E. coli Further directed modification was performed on the basis of W3110, and the specific steps were as follows: (1) In the starting strain E. coli Knocking out of the W3110 genome putA The gene obtained strain Pro01; (2) Taking strain Pro01 as the starting strain, the aceA gene was knocked out to obtain strain Pro02; (3) Taking strain Pro02 as the starting strain, the poxB gene was knocked out to obtain strain Pro03; (4) Taking strain Pro03 as the starting strain, the ldhA gene was knocked out to obtain strain Pro04; (5) Taking strain Pro04 as the starting strain, the ackA gene was knocked out to obtain strain Pro05, (6) Taking strain Pro05 as the starting strain, the ptsG gene was knocked out to obtain strain Pro06; (7) Using strain Pro06 as the starting strain, P trc Promoter control proB74 Gene overexpression to obtain strain Pro07; (8) Using strain Pro07 as the starting strain, P trc Promoter control proB74 Gene overexpression to obtain strain Pro08; (9) Using strain Pro08 as the starting strain, tandem integration was performed. proA, proC Genes form a miniature operon proAC and with P trc The promoter was used to start the strain, resulting in strain Pro09. (10) Using strain Pro09 as the starting strain, P trc The strain Pro10 is obtained by controlling the overexpression of the s thA gene by a promoter; (11) Using strain Pro10 as the starting strain, P trc Strain Pro11 is obtained by using a promoter to control glf gene overexpression; (12) Using strain Pro11 as the starting strain, P trc Strain Pro12 is obtained by using the promoter to control glk gene overexpression; (13) Using strain Pro12 as the starting strain, P trc Strain Pro13 is obtained by controlling the gdhA gene overexpression with a promoter; (14) Taking strain Pro13 as the starting strain, the original promoter of SucAB gene was replaced by BBa-j23109 promoter to obtain strain Pro14; (15) Using strain Pro14 as the starting strain, P trc Promoter control BAD_0687 Gene overexpression to obtain strain Pro15; (16) Using strain Pro15 as the starting strain, P trc Promoter control cgl2622 Gene overexpression, i.e.

8. The application of the proline-producing strain of any one of claims 1-6 in the fermentation production of proline.

9. Use according to claim 8, characterized in that: The specific steps are as follows: (1) Strain activation: inoculate the bacteria from the bacteria preservation tube into the slant medium for activation culture; inoculate the colonies from the slant medium into the LB medium for further activation culture, and the culture temperature is 37°C; (2) Inoculate into the triangular flask containing the fermentation medium at an inoculation amount of 10%-15%, and culture at 37°C with 220 r / min shaking; maintain the pH at 7.0-7.2 by adding ammonia water during the fermentation process; and maintain the fermentation by adding glucose solution.

10. Use according to claim 9, characterized in that: The LB culture medium used is: yeast extract powder 5 g / L, protein peptone 10 g / L, NaCl 10 g / L, and agar powder 20 g / L, and the rest is water; the slant culture medium used is: glucose 2 g / L, yeast extract powder 5 g / L, protein peptone 10 g / L, NaCl 2.5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, and agar powder 2.5 g / L, and the rest is water; and the fermentation culture medium used is: glucose 15 g / L, yeast powder 3.5 g / L, protein peptone 1 g / L, (NH4)2SO4 2 g / L, K2HPO4·3H2O 2 g / L, MgSO4·7H2O 2 g / L, glutamic acid 2 g / L, methionine 0.5 g / L, MnSO4·H2O 10 mg / L, and FeSO4·7H2O 30 mg / L.

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