A proline-producing strain, its construction method and application

Abstract

This invention provides a proline-producing strain, its construction method, and its applications. The strain is derived from a starting strain using metabolic engineering techniques. E. coli It was obtained through further modifications based on W3110, specifically: knockout. putA、aceA、poxB、 ldhA、ackA、ptsG Genes, upregulated proB74 , proC、 proA、sthA、glf、glk、gdhA The transcriptional level of the gene was downregulated. SucAB The transcriptional level of genes, affecting phosphorylketonease genes BAD_0687 and proline transporter encoding genes cgl2622 Heterologous expression was performed; the strain used glucose as a carbon source, requiring no substrate addition, resulting in low production costs, high production rate, short fermentation cycle, and high strain stability, thus offering significant economic benefits and laying the foundation for large-scale proline production.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and in particular to a proline-producing strain, its construction method, and its application. Background Technology

[0002] L-proline is mainly used as an organic catalyst in the chemical industry, characterized by its high activity and good stereospecificity. In the pharmaceutical and cosmetic industries, it is used as a precursor of compounds and a raw material for amino acid drugs. In addition, in prokaryotic and eukaryotic cells, L-proline is a potential virulence factor for certain pathogenic bacteria and a source of carbon, nitrogen and energy. It can also accumulate in large quantities to play a role in regulating osmotic pressure and stabilizing the structure of biological macromolecules.

[0003] Currently, the main methods for proline synthesis are chemical synthesis and microbial fermentation. Chemical synthesis has problems such as significant environmental pollution and low conversion rate; microbial fermentation uses glucose as the energy source for microbial growth, and the fermentation process is mild, making it a promising method for industrial production.

[0004] However, existing microbial fermentation methods require the addition of glutamic acid during proline production, resulting in a low conversion rate of proline. There is an urgent need to develop proline-producing strains that can synthesize proline de novo without the addition of substrates and achieve high conversion rates. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a proline-producing strain.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned proline-producing strain.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned proline-producing strain.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] A proline-producing strain is produced by modifying the starting strain using metabolic engineering methods. E. coli It was obtained through further modifications based on W3110, specifically: knockout. putA、aceA、poxB、ldhA、ackA、ptsG Genes, upregulated proB74 , proC、proA、sthA、glf、glk、gdhA The transcriptional level of the gene was downregulated. SucAB The transcriptional level of genes, affecting phosphorylketonease genes BAD_0687 and proline transporter encoding genes cgl2622 Perform heterologous expression.

[0010] Preferably, the metabolic engineering modification method for the above-mentioned proline-producing strain is CRISPR-Cas9 gene editing technology.

[0011] Preferably, the above-mentioned proline-producing strain, wherein E. coli W3110 is E. coli W3110 ATCC 27325.

[0012] Preferably, the above-mentioned proline-producing strain, in the starting strain E. coli Knockout of W3110 genome putA Genes that are not expressed; knockout aceA Genes that are not expressed; knockout poxB Genes that are not expressed; knockout ldhA Genes that are not expressed; knockout ackA Genes that are not expressed; knockout ptsG Genes, so that they are not expressed; in yghX Pseudogene loci use P trc Starter control proB74 Gene overexpression; in mbhA Pseudogene loci use P trc Starter control proB74 Mutant gene overexpression; in yciQ Pseudogene loci tandem integration proA and proC Genes form a miniature operon proAC and with P trc Startup sub-boot; in yjiP Pseudogene loci use P trc Starter control sthA Gene overexpression; in ylbE Pseudogene loci use P trc Starter control glf Gene overexpression; in ydeU Pseudogene loci use P trc Starter control Glk Gene overexpression; in yncI Pseudogene loci use P trc Starter control gdhA Gene overexpression; replacing the original SucAB gene promoter with the BBa-j23109 promoter to weaken expression; in yeep Pseudogene loci use P trc Promoter controls phosphoryl ketone enzyme genes BAD_ 0687 Heterologous expression; in yjgX Pseudogene loci use P trc Promoters control proline transporter encoding genes cgl2622 Overexpression.

[0013] Preferably, the above-mentioned proline-producing strain, the microoperon 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.

[0014] 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.

[0015] 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.

[0016] 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:

[0017] (1) In the starting strain E. coli Knockout on the W3110 genome putA Genes were obtained from strain Pro01;

[0018] (2) Starting with strain Pro01, the aceA gene was knocked out to obtain strain Pro02;

[0019] (3) Starting with strain Pro02, the poxB gene was knocked out to obtain strain Pro03;

[0020] (4) Starting with strain Pro03, the ldhA gene was knocked out to obtain strain Pro04;

[0021] (5) Starting with strain Pro04, the ackA gene was knocked out to obtain strain Pro05.

[0022] (6) Starting with strain Pro05, the ptsG gene was knocked out to obtain strain Pro06;

[0023] (7) Using strain Pro06 as the starting strain, in yghX Pseudogene loci use P trc Starter control proB74 Gene overexpression yielded strain Pro07;

[0024] (8) Using strain Pro07 as the starting strain, in mbhA Pseudogene loci use P trc Starter control proB74 Gene overexpression yielded strain Pro08;

[0025] (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.

[0026] (10) Using strain Pro09 as the starting strain, in yjip Pseudogene loci use P trc Strain Pro10 was obtained by controlling the overexpression of the sthA gene via the promoter.

[0027] (11) Using strain Pro10 as the starting strain, P was used at the ylbE pseudogene site. trc The promoter controlled the overexpression of the glf gene to obtain strain Pro11;

[0028] (12) Using strain Pro11 as the starting strain, in ydeU Pseudogene loci use P trc Strain Pro12 was obtained by controlling glk gene overexpression via promoter control.

[0029] (13) Using strain Pro12 as the starting strain, in y ncI Pseudogene loci use P trc Strain Pro13 was obtained by controlling gdhA gene overexpression via promoter control.

[0030] (14) Starting with strain Pro13, replace the original promoter of the SucAB gene with the BBa-j23109 promoter to obtain strain Pro14;

[0031] (15) Using strain Pro14 as the starting strain, in yeep Pseudogene loci use P trc Starter control BAD_0687 Gene overexpression yielded strain Pro15;

[0032] (16) Using strain Pro15 as the starting strain, in yjgX Pseudogene loci use P trc Starter control cgl2622 Gene overexpression yielded strain Pro16, which is the successfully modified target strain.

[0033] Preferably, in the method for constructing the above-mentioned proline-producing strain, the proline oxidase gene... putA Derived from E. coli, knocking out this gene can effectively reduce the catabolism of proline, thereby achieving the goal of proline accumulation; isocitrate lyase gene. aceADerived from *E. coli*, knocking out this gene can effectively reduce the catabolism of isocitrate, providing more α-ketoglutarate for proline production; pyruvate dehydrogenase gene. poxB Derived from E. coli, knocking out this gene can effectively reduce the catabolism of pyruvate, providing more pyruvate for proline production; D-lactate dehydrogenase gene ldhA Derived from E. coli, knocking out this gene can effectively reduce the catabolism of pyruvate, providing more pyruvate for the production of proline; acetylkinase gene. ackA Derived from E. coli, knocking out this gene can effectively reduce the catabolism of acetyl-CoA, providing more citrate for the production of proline; a core component gene of the PTS system. ptsG Derived from E. coli, knocking out this gene allows the bacteria to simultaneously utilize multiple carbon sources, improving substrate utilization; glutamate kinase gene. proB74 It originates from E. coli, and is produced by proB The gene was obtained through mutation, encoding the first enzyme in proline synthesis. The mutation removes negative feedback inhibition, allowing this enzyme to effectively increase proline production; glutamate semialdehyde dehydrogenase gene. proA Derived from *E. coli*, it encodes the second enzyme in proline synthesis, which further enhances the proline biosynthetic pathway; pyrrololine-5-carboxylic acid reductase gene. proC The gene *sthA*, derived from *E. coli*, encodes the third enzyme in proline synthesis, which enhances the proline biosynthetic pathway; the gene *sthA*, encoding pyridine nucleotide transhydrogenase, also from *E. coli*, enhances intracellular NADPH supply; and the gene *UDP-galactose mutase*... glf Derived from Escherichia coli, this enzyme enables the simultaneous utilization of mixed sugars, improving substrate conversion efficiency; glucokinase gene. Glk Derived from Escherichia coli, this enzyme can improve glucose conversion efficiency and reduce metabolic burden; glutamate dehydrogenase gene gdhA Derived from *E. coli*, this enzyme can provide more glutamate for proline production; the α-ketoglutarate dehydrogenase gene *SucAB*, to weaken the *E. coli* gene, replaced the original promoter of the *SucAB* gene with the BBa-j23109 promoter, which weakens the expression of α-ketoglutarate in the TCA cycle and enhances proline metabolic flux; phosphorylketase... BAD_0687 The gene originates from the codon optimization of Bifidobacterium adolescentis. BAD_0687 The gene encodes a key enzyme that catalyzes the production of acetyl-CoA; a proline transporter. cgl2622 The gene, derived from Corynebacterium glutamicum, promotes the transport of the intracellular product proline to the extracellular space.

[0034] The application of the above-mentioned proline-producing strains in the fermentation production of proline.

[0035] Preferably, the above application follows these steps:

[0036] (1) Activation of bacterial strain: Inoculate the bacterial solution from the preservation tube into the slant medium and activate it for 12h; transfer the colonies from the slant medium to LB medium and continue to activate and culture for 12h at a temperature of 37℃;

[0037] (2) Inoculate 10%-15% into the Erlenmeyer flask containing the fermentation medium, seal with nine layers of gauze, and culture at 37℃ and 220 r / min with shaking. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; 60% glucose solution is added to maintain the fermentation. The fermentation cycle is 22-26h.

[0038] Preferably, in the above application, the LB culture medium used in step (1) is: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, 20 g / L agar powder, and the remainder is water.

[0039] Preferably, in the above application, the slant culture medium used in step (1) is: glucose 2g / L, yeast extract 5g / L, peptone 10g / L, NaCl 2.5g / L, KH2PO4 1g / L, MgSO4·7H2O 0.2g / L, agar powder 2.5g / L, and the remainder is water.

[0040] Preferably, in the above application, the fermentation medium used in step (2) 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.

[0041] All of the above-mentioned culture media can be prepared using standard methods.

[0042] Beneficial effects:

[0043] The aforementioned proline-producing strain was obtained through a de novo directed synthesis method using the proline metabolic pathway. This strain uses glucose as a carbon source, requires no substrate addition, has low production costs, and boasts advantages such as high production rate, short fermentation cycle, and high strain stability, resulting in significant economic benefits and laying the foundation for large-scale proline production. In application, fermentation was conducted in Erlenmeyer flasks. The strain efficiently and stably synthesized proline de novo, achieving a proline yield of 26.35 g / L after 24 hours of fermentation, thus laying the foundation for large-scale proline production. Attached Figure Description

[0044] Figure 1 A diagram illustrating the de novo synthesis pathway gene modification process for proline-producing strains. Detailed Implementation

[0045] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0046] Unless otherwise specified, the percentage sign "%" in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0047] The starting strain used in the examples is E. coli The primers used in the construction of strain W3110 ATCC 27325 are shown in Table 1.

[0048] Table 1 Primers used in strain construction

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] The gene editing method used is based on 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 the engineered plasmids pREDCas9 and pGRB. pREDCas9 carries the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of λ phage, the Cas9 protein expression system, and zebumycin resistance (working concentration: 100 mg / L). pGRB uses pUC18 as its backbone and includes the promoter J23100, the gRNA-Cas9 binding region sequence, the terminator sequence, and ampicillin resistance (working concentration: 100 mg / L). All technical terms used in the following examples are explained in this article.

[0057] Example 1

[0058] This embodiment aims to illustrate the steps involved in gene knockout during the specific construction of the strain. In particular, if a similar gene manipulation method is used in the embodiment, it will only be provided once and annotated, without further elaboration.

[0059] (1) Using the genome of Escherichia coli W3110 as a template, upstream and downstream homologous arms were obtained by HS enzyme PCR using putA-Q-1, putA-Q-2 and putA-Q-3, putA-Q-4 as primers, respectively. Then, using these as templates, Δput was obtained by HS enzyme overlap PCR. A Gene knockout fragment, the gene integration fragment is composed of putA Upstream homologous arm and putA Downstream homologous arm composition.

[0060] (2) Using pGRB-putA-S and pGRB-putA-A as primers, construct the DNA fragment containing the target sequence used by pGRB-putA through PCR annealing program, and transform it into DH5α competent cells, screen to obtain positive transformants, and extract plasmid pGRB-putA;

[0061] (3) The Δ obtained in the above steps putA Gene knockout fragment and pGRB-putA plasmid electroporation E. coli Positive transformants were obtained from strain W3110 after screening and named Pro01.

[0062] (4) Knockout of the aceA gene in the genome: The operation method is the same as in steps (1) to (3), except that the primers used are aceA-Q-1, aceA-Q-2, aceA-Q-3, aceA-Q-4, pGRB-aceA-S, and pGRB-aceA-A. The competent cells are Pro01, and strain Pro02 is obtained.

[0063] (5) Knockout of the poxB gene in the genome: The operation method is the same as in steps (1) to (3), except that the primers used are poxB-up-S, poxB-up-A, poxB-down-S, poxB-down-A, PGRB-poxB-UP, and PGRB-poxB-DN. The competent cells are Pro02, and strain Pro03 is obtained.

[0064] (6) Knockout of the ldhA gene in the genome: The operation method is the same as in steps (1) to (3), except that the primers used are ldhA-Q-1, ldhA-Q-2, ldhA-Q-3, ldhA-Q-4, pGRB-ldhA-S, and pGRB-ldhA-A. The competent cells are Pro03, and strain Pro04 is obtained.

[0065] (7) Knockout of the ackA gene in the genome: The operation method is the same as in steps (1) to (3), except that the primers used are ackA-Q-1, ackA-Q-2, ackA-Q-3, ackA-Q-4, pGRB-ackA-S, and pGRB-ackA-A. The competent cells are Pro04, and strain Pro05 is obtained.

[0066] (8) Knockout of the ptsG gene in the genome: The operation method is the same as in steps (1) to (3), except that the primers used are ptsG-Q-1, ptsG-Q-2, ptsG-Q-3, ptsG-Q-4, pGRB-ptsG-S, and pGRB-ptsG-A. The competent cells are Pro05, and strain Pro06 is obtained.

[0067] Example 2

[0068] This embodiment aims to illustrate the steps involved in the specific construction of the strain for overexpressing genes. In particular, if there are similar gene manipulation methods in the embodiment, they will only be provided once and annotated, without further elaboration.

[0069] (1) Using the Escherichia coli W3110 genome as a template, and using yghX-UP-S, yghX-UP-A, yghX-DN-S, yghX-DN-A and proB-trc-S, proB-trc-A as primers, upstream homologous arms, downstream homologous arms, and the target gene fragment were amplified by HS enzyme PCR. Then, using this as a template, P was obtained by HS enzyme overlap PCR. trc -proB ( yghX ) gene integration fragment, said gene integration fragment is composed of yghX Upstream homologous arm, P trc - proB Target gene and yghX Downstream homologous arm composition.

[0070] (2) Using pGRB-yghX-S and pGRB-yghX-A as primers, the DNA fragment containing the target sequence used by pGRB-yghX was constructed by PCR annealing program, and it was transformed into DH5α competent cells. Positive transformants were screened and plasmid pGRB-yghX was extracted.

[0071] (3) Take the P obtained in the above steps trc -proB ( yghX The gene integration fragment and pGRB-yghX plasmid were electroporated into the Pro06 strain. Positive transformants were obtained after screening and named Pro07.

[0072] (4) Introduce and overexpress the proB74 gene at the mbhA pseudogene site: The operation method is the same as in steps (1) to (3), except that the primers used are mbhA-UP-S, mbhA-UP-A, mbhA-DN-S, mbhA-DN-A, PGRB-mbhA-S, and PGRB-mbhA-A. The competent cells are Pro07, and strain Pro08 is obtained.

[0073] (5) In yciQ Pseudogene loci use P trc Promoter controls artificial micromanipulators proAC Express:

[0074] ① Using the E. coli W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using primers yciQ-UP-S, yciQ-UP-A and yciQ-DN-S, yciQ-DN-A, respectively. Then, proA-1, proA-2 and proC-1, proC-2 were used for PCR amplification to obtain proA and proC fragments, with proA-2 sharing homologous sequences with proC-1. Using proA and proC fragments as templates, overlap PCR was performed using primers proA-1 and proC-2 to obtain the trc-proAC fragment. Using the upstream and downstream homologous arms and the target gene fragment as templates, overlap PCR was performed using primers yciQ-UP-S and yciQ-DN-A to obtain the overlap fragment P. trc -proAC(yciQ);

[0075] ② Using PGRB-yciQ-S and PGRB-yciQ-A as primers, a DNA fragment containing the target sequence for pGRB-yciQ was constructed using a PCR annealing program, and then transformed into DH5α competent cells. Positive transformants were screened and the plasmid pGRB-yciQ was extracted.

[0076] ③ Take the P obtained in the above steps trc The -proAC(yciQ) gene integration fragment and the pGRB-yciQ plasmid were electroporated into the Pro08 strain. After screening, positive transformants were obtained and named Pro09.

[0077] (6) Introduce and overexpress the sthA gene at the yjiP pseudogene site: The operation method is the same as in steps (1) to (3), 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, and sthA-trc-DN. The competent cells are Pro09, and strain Pro10 is obtained.

[0078] (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.

[0079] (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.

[0080] (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.

[0081] (10) Replace the original SucAB gene promoter with the BBa-j23109 promoter:

[0082] 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;

[0083] 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 ;

[0084] ③ Take the P obtained in the above steps BBa_j23109 - SucAB Gene integration fragments and pGRB- SucAB The plasmid was electroporated into the Pro13 strain, and positive transformants were obtained after screening and named Pro14.

[0085] (11) Introduce and overexpress the BAD_0687 gene at the yeeP pseudogene site: The operation method is the same as in steps (1) to (3), except that the primers used are yeeP-UP-S, yeeP-UP-A, yeeP-DN-S, yeeP-DN-A, PGRB-yeeP-UP, PGRB-yeeP-DN, BAD_0687-trc-UP, and BAD_0687-trc-DN. The competent cells are Pro14, and strain Pro15 is obtained.

[0086] (12) Introduce and overexpress the Cgl2622 gene at the yjgX pseudogene site: The operation method is the same as in steps (1) to (3), except that the primers used are yjgX-UP-S, yjgX-UP-A, yjgX-DN-S, yjgX-DN-A, PGRB-yjgX-UP, PGRB-yjgX-DN, Cgl2622-trc-UP, and Cgl2622-trc-DN. The competent cells are Pro15, and strain Pro16 is obtained.

[0087] Example 3

[0088] Using strain Pro16 as the proline production strain, this example aims to illustrate the method of producing proline using this strain Pro16. The specific culture method is as follows:

[0089] (1) Activation of bacterial strain: The bacterial solution was inoculated from the preservation tube and then transferred to the slant medium for activation culture for 12 h; the colonies were transferred from the slant medium to LB medium for further activation culture for 12 h at a culture temperature of 37℃. The LB medium used was: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, agar powder 20 g / L, and the remainder was water; the slant medium was: glucose 2 g / L, yeast extract 5 g / L, 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 remainder was water.

[0090] (2) Inoculate 10%-15% of the culture medium into an Erlenmeyer flask containing fermentation medium, seal the flask 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.

[0091] Using wild-type E. coli W3110 as the control group, after 24 hours of fermentation verification, wild-type E. coli W3110 failed to produce proline, while engineered strain Pro16 accumulated 26.35 g / L of proline, proving the effectiveness of the strain described in this invention.

[0092] Example 4

[0093] This embodiment aims to illustrate the effect of the micro-artificial operon ProAC on proline production capacity, using strain Pro09. Using strain Pro08 as the chassis bacteria, in yciQ Pseudogene loci use P trc Pro10 strain obtained by overexpression of the ProA gene controlled by the promoter. For strain Pro09 For the chassis bacteria, in ybeQ Pseudogene loci use P trc Proline was obtained by overexpressing the ProC gene under promoter control. Proline was produced using the method described in Example 3, and the proline yield is shown in Table 2.

[0094] Table 2

[0095]

[0096] As shown in Table 2, the proline production of strain Pro09, which incorporated a miniature artificial operon, was significantly increased compared to Pro08. Further analysis revealed that... sthA、glf、glk、gdhA、BAD_0687 and cgl2622 Overexpression of the gene and replacement of the original SucAB gene promoter with the BBa-j23109 promoter resulted in a significant increase in proline production in the obtained strain Pro16.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A proline-producing strain, characterized in that: It utilizes metabolic engineering techniques to modify the starting strain E. coli It was obtained through further modifications based on W3110, specifically: knockout. putA, aceA, poxB, ldhA, ackA, ptsG Genes, using P trc Starter control proB74 Gene overexpression, using P trc Starter control proB74 Gene overexpression, tandem integration proA and proC Genes form a miniature operon proAC and with P trc Startup sub-boot, using P trc Starter control sthA Gene overexpression, using P trc Starter control glf Gene overexpression, using P trc Starter control glk Gene overexpression, using P trc Starter control gdhA Gene overexpression, thereby upregulating proB74 , proC, proA, sthA, glf, glk, gdhA The transcriptional level of the gene; replacing the original promoter of the SucAB gene with the BBa-j23109 promoter, thereby downregulating... SucAB The transcriptional level of genes, using P trc Promoter controls phosphoryl ketone enzyme genes BAD_0687 Heterologous expression; using P trc Promoters control proline transporter encoding genes cgl2622 Heterogeneous overexpression.

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

3. The proline-producing strain according to claim 1, characterized in that: The micromanipulator proAC The nucleotide sequence is shown in SEQ ID NO. 9 of the sequence listing; the 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.

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4. The proline-producing strain according to claim 1, characterized in that: 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.

5. The proline-producing strain according to claim 1, characterized in that: 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.

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6. The method for constructing the proline-producing strain according to any one of claims 1-5, characterized in that: In the originating strain E. coli Further targeted modifications will be made based on the W3110, with the following specific steps: In the originating 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, P trc Starter control proB74 Gene overexpression yielded strain Pro07; (8) Using strain Pro07 as the starting strain, P trc Starter control proB74 Gene overexpression yielded 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 Strain Pro10 was obtained by controlling the overexpression of the sthA gene via the promoter. (11) Using strain Pro10 as the starting strain, P trc The promoter controlled the overexpression of the glf gene to obtain strain Pro11; (12) Using strain Pro11 as the starting strain, P trc Strain Pro12 was obtained by controlling glk gene overexpression via promoter control. (13) Using strain Pro12 as the starting strain, P trc Strain Pro13 was obtained by controlling gdhA gene overexpression via promoter control. (14) Starting with strain Pro13, replace the original promoter of the SucAB gene with the BBa-j23109 promoter to obtain strain Pro14; (15) Using strain Pro14 as the starting strain, P trc Starter control BAD_0687 Gene overexpression yielded strain Pro15; (16) Using strain Pro15 as the starting strain, P trc Starter control cgl2622 Gene overexpression, i.e.

7. The use of the proline-producing strain according to any one of claims 1-5 in the fermentation production of proline.

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