Escherichia coli weakened in uhpT gene expression and method for producing l-serine thereof

Abstract

This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a genetically modified *Escherichia coli* and a method for producing L-serine. The genetically engineered bacterium is a weakened version of *E. coli*. uhpT The gene expression pattern was modified to compare it with unmodified E. coli. uhpT The expression level of the gene is downregulated or not expressed. This invention utilizes modified Escherichia coli to produce L-serine, significantly improving the L-serine production capacity of Escherichia coli without affecting the growth performance of the strain.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a weakening... uhpT Gene-expressing Escherichia coli and a method for producing L-serine using this Escherichia coli. Background Technology

[0002] L-Serine is one of the twenty essential amino acids required by the human body, but it is not an essential amino acid. L-Serine participates in the metabolism of various nutrients during normal cell growth, is a precursor to many nucleosides, amino acids, and adenosine, and directly participates in cell membrane synthesis and fatty acid metabolism. Phosphatidylserine is the only phospholipid in the brain that can regulate the functional state of key cell membrane proteins, thus improving and repairing brain function. L-Serine, as a precursor for the synthesis of phosphatidylserine, is widely used in the pharmaceutical field. Furthermore, because L-Serine has the most significant cell growth-promoting effect among all amino acids, it is often added as a nutrient to compound amino acid injections.

[0003] L-Serine was initially isolated from silkworm silk, and this method was also the original synthesis method for L-serine. First, industrial hydrochloric acid was added to waste silkworm silk to allow for complete hydrolysis. After hydrolysis, the solution was diluted, powdered activated carbon was added, and the mixture was filtered to obtain the hydrolysate. The hydrolysate was then loaded onto a granular activated carbon column and eluted with ammonia to obtain the eluent. Finally, crude L-serine was obtained after rough separation, purification, and refining. However, this method resulted in significant L-serine loss and low purity during production, and was also complex and costly; therefore, this method is rarely used nowadays.

[0004] Currently, strategies for breeding L-serine-producing strains mainly include rational metabolic engineering and non-rational metabolic engineering (such as random mutagenesis). Mutagenesis breeding uses chemical or physical methods to induce mutations in organisms and then selects favorable mutations to cultivate new varieties. However, this breeding method has certain limitations. Most of the mutations produced during the mutagenesis process are unfavorable. In addition, due to the randomness of mutagenesis, the direction of mutation cannot be determined, the results are difficult to control, and the predictability is poor. Therefore, the current strategy for breeding L-serine-producing strains is mainly metabolic engineering. Traditional metabolic engineering strategies for constructing L-serine-producing strains mainly include: blocking L-serine degradation pathways, enhancing the supply of L-serine precursors, strengthening the L-serine transport system, and enhancing cellular tolerance to L-serine. For example, patent CN202211587322.1 systematically modified the L-serine synthesis module, L-serine degradation module, L-serine transport module, and by-product synthesis module of Escherichia coli, and on this basis, improved the strain's tolerance to high concentrations of L-serine.

[0005] In recent years, with the rapid development of omics technologies, a large number of omics technologies have begun to be applied in the field of synthetic biology. Researchers use transcriptomics technology to provide a "panoramic map" of gene expression, revealing the connections between various regulatory networks in cells, laying the foundation for constructing synthetic pathways and building cell factories. Omics technologies are used to identify target sites in metabolic pathways that are closely related to the yield of target products, in addition to genes related to synthetic pathways, and to modify these target sites, thereby achieving the goal of improving product accumulation and efficient product synthesis. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a genetically modified Escherichia coli and a method for producing L-serine.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Firstly, the present invention provides a genetically engineered bacterium for producing L-serine, specifically, belonging to *Escherichia coli*, and more specifically, the genetically engineered bacterium is a weakened version of *Escherichia coli*. uhpT The way genes are expressed has been modified.

[0009] Secondly, the present invention provides the application of genetically engineered bacteria as described in the first aspect in the production of L-serine.

[0010] Thirdly, the present invention provides a method for producing L-serine, comprising: culturing a genetically engineered bacterium as described in the first aspect in a culture medium to produce L-serine; and collecting L-serine from the genetically engineered bacterium and / or the culture medium. The *E. coli* described herein has been weakened. uhpT The gene expression pattern was modified to compare it with unmodified E. coli. uhpT The expression level of the gene is downregulated or not expressed.

[0011] The beneficial effects of this invention are as follows: E. coli uhpT The gene encodes a hexose-6-phosphate:phosphate antiporter, the relationship and function of which are unclear regarding the L-serine synthesis pathway. This invention demonstrates that, by weakening... uhpT Modifying gene expression can significantly improve the L-serine production capacity of *E. coli* without affecting the strain's growth performance. This invention provides a method for producing L-serine using modified *E. coli* strains, achieving an L-serine yield that is at least 8%-12% higher than that of control strains. Attached Figure Description

[0012] Figure 1: Plasmid maps of pEcCas9 and pEcgRNA used in the gene editing method of this invention. Detailed Implementation

[0013] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0014] Firstly, the present invention provides a genetically engineered bacterium for producing L-serine, specifically, belonging to *Escherichia coli*, and more specifically, the genetically engineered bacterium is a weakened version of *Escherichia coli*. uhpT The way genes are expressed has been modified.

[0015] Secondly, the present invention provides the application of genetically engineered bacteria as described in the first aspect in the production of L-serine.

[0016] Thirdly, the present invention provides a method for producing L-serine, comprising: culturing a genetically engineered bacterium as described in the first aspect in a culture medium to produce L-serine; and collecting L-serine from the genetically engineered bacterium and / or the culture medium. The *E. coli* described herein has been weakened. uhpT The gene expression pattern was modified to compare it with unmodified E. coli. uhpT The expression level of the gene is downregulated, for example, by at least 50%, 60%, 70%, 80%, 90%, or 100%.

[0017] According to the definition of the present invention, the weakening uhpT Gene expression modes include, but are not limited to: Changes at the DNA level uhpT The genomic DNA sequence of the gene, including the one that makes the gene... uhpT Gene deletion, substitution, insertion, or point mutation (e.g., using gene editing technologies such as CRISPR / Cas9, TALEN, or ZFN to cause frameshifts, introduce premature stop codons, delete key exons or functional domains, or perform gene substitution through homologous recombination); modification of transcriptional regulatory elements of target genes at the transcription initiation level, including modifications to the aforementioned gene. uhpTPhysical disruption, replacement, or epigenetic modification of non-coding regulatory regions such as promoters, enhancers, or insulators of genes (e.g., linking or replacing endogenous strong promoters with weak promoters, inducible promoters, or tissue-specific promoters; inserting transcription termination signals or insulator elements into regulatory regions; or inducing transcriptional silencing through DNA methylation or histone modification); intervention at the post-transcriptional and protein levels of the transcripts or functions of target genes, including promoting the degradation, splicing inhibition, or blocking of target gene mRNA translation through exogenous intervention molecules, and promoting the degradation or inactivation of target proteins (e.g., RNA interference (RNAi) using siRNA, shRNA, or miRNA; mRNA cleavage using antisense oligonucleotides (ASO) or ribozymes; blocking transcriptional elongation using CRISPRi; or targeted protein degradation using PROTAC technology, and inactivation of protein activity using neutralizing antibodies or small molecule inhibitors).

[0018] According to some specific embodiments of the present invention, the weakening uhpT Gene expression occurs through gene knockout. Preferably, gene knockout in *E. coli* is achieved using gene editing techniques (such as the CRISPR / Cas9 gene editing system). uhpT Genes prevent it from being expressed.

[0019] According to the present invention, the *Escherichia coli* can be any *Escherichia coli*, such as those commonly used as hosts in the art. E coli MG1655 E coli W3110 E coli BL21 E.coli BW25113, or Escherichia coli obtained by directional or non-directional modification based on it.

[0020] According to the present invention, the preferred host *Escherichia coli* has been modified to enable it to produce L-serine. For example, L-serine degradation pathways are blocked, L-serine synthesis pathways are enhanced, and / or other L-serine metabolic modules are modified by introducing gene mutations or gene knockouts to promote L-serine accumulation.

[0021] According to some specific embodiments of the present invention, the *Escherichia coli* can be selected from... SER7, SER8, SER9 or SER12 (CN202211587322.1) and other engineered strains for L-serine production that have already been modified.

[0022] According to the present invention, the uhpTThe gene is not limited to the nucleotide sequence shown in NCBI GeneID: 948201, but may also include a mutant nucleotide sequence of the sequence shown in NCBI GeneID: 948201 or a mutant gene that is homologous to the sequence shown in NCBI GeneID: 948201 and encodes a phosphohexose transporter (e.g., the amino acid sequence shown in SEQ ID NO: 10). uhpT Genes can be variant nucleotide sequences due to the degeneracy of the genetic code.

[0023] According to a second aspect of the invention, the culture of the genetically engineered bacteria can be carried out using methods conventional in the art. The culture medium used for the production of L-serine can be a synthetic or natural culture medium, such as a typical culture medium containing a carbon source, nitrogen source, sulfur source, inorganic ions, and other required organic and inorganic components.

[0024] According to some specific embodiments of the present invention, the culture medium comprises: glucose 20 g / L, yeast extract 3 g / L, tryptone 3 g / L, K₂HPO₄ 5 g / L, MgSO₄·7H₂O 2 g / L, FeSO₄·7H₂O 20 mg / L, MnSO₄·7H₂O 20 mg / L, VB1, VB3, VB5, and VB6. 12 V H Each 2 mg / L, the remainder being water, pH 7.0-7.2.

[0025] According to a second aspect of the invention, the genetically engineered bacteria can be cultured under aerobic conditions for 12 to 72 hours, or 24 to 60 hours, or 36 to 48 hours; the culture temperature can be controlled at 25 to 45°C, or 30 to 37°C; and the pH can be adjusted between 5.0 and 8.0, or 6.0 and 7.5, or 6.8 and 7.2. The pH can be adjusted by using inorganic or organic acidic or alkaline substances, as well as ammonia.

[0026] After cultivation, solids, such as cells and cell debris, can be removed from the liquid culture medium using conventional techniques (e.g., centrifugation, membrane filtration). L-serine can then be recovered from the fermentation broth using any combination of conventional techniques (e.g., concentration, ion exchange chromatography, crystallization).

[0027] According to the present invention, the L-serine includes not only L-serine in its free form, but also salts or hydrates of L-serine.

[0028] Before inoculation and fermentation, the genetically engineered bacteria may undergo strain activation, seed culture, etc., depending on their storage state. Appropriate conditions and culture media can be selected according to conventional techniques in the field. For example, the seed culture medium can use the same composition as the fermentation culture medium, or it can be appropriately adjusted based on this.

[0029] According to some specific embodiments of the present invention, the method includes: culturing the bacterial strain on an agar slant, seed culture, inoculating it into a glucose fermentation medium, shaking culture, and adding glucose solution as needed to maintain fermentation; wherein the bacterial strain is *Escherichia coli* as described in the first aspect.

[0030] Other specific operational methods involving molecular biology, genetic engineering, etc., can all be implemented using technical manuals, textbooks, or literature reports that are readily available to those in the field, and there is no need to describe the operational process in detail here.

[0031] The present invention will be described in more detail below through specific embodiments. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental methods and equipment described in the following embodiments are conventional methods and equipment.

[0032] (1) Gene editing methods used in the examples The gene editing method used in this invention is based on the literature (Li Y, Lin Z, Huang C, et al Metabolic engineering of Escherichia coli The procedure was performed using CRISPR–Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21. The two plasmid maps used in this method are attached. Figure 1 The pEcCas9 plasmid carries the pEcgRNA elimination system, the λ phage Red recombination system, and the Cas9 protein expression system. It also contains ampicillin resistance (working concentration: 100 ug / mL) and is cultured at 37°C. The pEcgRNA uses pUC18 as its backbone and includes the promoter J23100, the gRNA-Cas9 binding region sequence, and the terminator sequence. It also contains zithromycin resistance (working concentration: 50 ug / mL) and is cultured at 37°C.

[0033] The specific steps of this method are as follows: 1.1 Construction of pEcgRNA plasmid The purpose of constructing the pEcgRNA plasmid is to transcribe the corresponding gRNA, thereby forming a complex with the Cas9 protein, and achieving a double-strand break in the target DNA through base pairing and PAM recognition of the target gene site. The pEcgRNA plasmid is constructed using a recombination method involving a DNA fragment containing the target sequence and a linearized vector fragment.

[0034] 1.1.1 Target Sequence Design The target sequence (PAM: 5'-NGG-3') was designed using CRISPR RGEN Tools. 1.1.2 Preparation of plasmids containing target sequences The base sequence of the site to be knocked out or integrated is input into CRISPR RGEN Tools. Primers are designed according to the target sequence given by CRISPR RGEN Tools. Using the pECgRNA empty vector plasmid as a template, the optimal N20 sequence is integrated into the pECgRNA plasmid through whole plasmid PCR amplification. 1.1.3 Plasmid Transformation The full plasmid PCR product was slowly added to E. coli DH5α competent cells, gently pipetted and aspirated to mix, then incubated on ice for 20 min, incubated in water at 42℃ for 45 s, then immediately incubated on ice for 3 min, 900 μL of recovery solution was added, and the cells were allowed to recover for 2 h. 200 μL of the recovered bacterial culture was then aspirated and evenly spread onto LB agar plates containing zizomycin, and incubated upside down at 37℃ for 12-15 h. After single colonies grew on the plates, colony PCR was used for identification, and positive recombinants were selected.

[0035] 1.1.6 Cloning Identification PCR-positive colonies were inoculated into LB medium containing 50 ug / mL zizomycin and cultured overnight for preservation. Plasmids were then extracted and identified by enzyme digestion.

[0036] 1.2 Preparation of Recombinant DNA Fragments For uhpT The gene knockout recombination fragment is from [[ID= The gene's upstream and downstream homologous arms are composed (upstream homologous arm - downstream homologous arm). Primer design software Primer 5 was used to... ​ Using the upstream and downstream sequences of the gene as templates, primers for the upstream and downstream homologous arms were designed. The upstream and downstream homologous arms were amplified by PCR, and then the recombinant fragment was prepared by overlap PCR. The PCR system and method are shown in Table 1.

[0037] Table 1

[0038] The system for overlap PCR is shown in Table 2 (Note: The template consists of equimolar amounts of amplified fragments of upstream and downstream homologous arms and the target gene, and the total amount does not exceed 10 ng).

[0039] Table 2

[0040] PCR reaction conditions (Takara Bio PrimeSTAR HS enzyme): Pre-denaturation (95℃) for 5 min; then 30 cycles: denaturation (98℃) for 10 s, annealing ((Tm-3 / 5)℃) for 15 s, extension at 72℃ (this enzyme activity extends by about 1 kb per min); continue extension at 72℃ for 10 min; maintain (4℃).

[0041] 1.3 Transformation of plasmids and recombinant DNA fragments 1.3.1 Conversion of pEcCas9 The pEcCas9 plasmid was electroporated into the electrocompetent cells of the starting strain. After cell resuscitation and culture, the cells were plated on LB agar plates containing ampicillin and incubated overnight at 37°C. Single colonies growing on the antibiotic-resistant plates were subjected to colony PCR using identification primers to screen for positive recombinants.

[0042] 1.3.2 Preparation of electrotransformation competent cells of the target strain containing pEcCas9 Incubate at 37℃ until OD 600 When the concentration reaches 0.1–0.2, add 0.1 M arabinose (to bring the final concentration to 10 mM), and continue culturing until OD reaches 0.2. 600 Competent cells were prepared when the pH was 0.6–0.7. The addition of arabinose was intended to induce the expression of the recombinase on the pEcCas9 plasmid. The culture medium and preparation process for competent cells followed standard operating procedures.

[0043] 1.3.3 Transformation of pEcgRNA and Recombinant DNA Fragments pEcgRNA and donor DNA fragments were simultaneously electroporated into electrocompetent cells containing pEcCas9. The revived cells after electroporation were plated on LB agar plates containing ampicillin and zithromycin and incubated overnight at 37°C. Colony PCR was performed using specially designed identification primers to verify the colonies, screen for positive recombinants, and maintain the cells.

[0044] 1.4 Plasmid Elimination 1.4.1 Elimination of pEcgRNA Positive recombinants were placed in LB medium containing 1 M rhamnose, ampicillin resistance, and kanamycin resistance and cultured overnight. After appropriate dilution, they were spread onto LB plates containing ampicillin resistance and cultured overnight at 37°C. For LB plates containing ampicillin and zicromycin resistance, single colonies that did not grow on zicromycin-resistant plates but grew on ampicillin-resistant plates were selected and preserved.

[0045] 1.4.2 Elimination of pEcCas9 plasmid The positive recombinant was transferred to LB liquid medium containing kanamycin resistance and incubated overnight at 37°C. After appropriate dilution, it was spread onto LB plates containing kanamycin resistance and incubated overnight at 37°C. Single colonies that did not grow on ampicillin-resistant plates but grew on kanamycin-resistant plates were selected and preserved.

[0046] (2) Primers used in the examples The primers and their sequences involved in the strain construction process are shown in Table 3.

[0047] Table 3

[0048] Example 1: This embodiment is used to illustrate the construction of the genetically engineered bacteria for producing L-serine according to the present invention. Specifically, it can be the genetically engineered bacteria SER0405, SER0406, SER0407 or SER0408.

[0049] L-serine production engineered bacteria such as *Escherichia coli* SER7, SER8, SER9, or SER12 (all from CN202211587322.1) that have already been modified were selected, and their production was weakened. ​ The mode of gene expression has been modified, preferably by gene editing to knock out gene expression. ​ Genetic modification.

[0050] Among them, SER7 is in ​ MG1655 does not express genes ​ (NCBI-GeneID:946331) ​ (NCBI-GeneID:947262) ​ (NCBI-GeneID: 2847724), and overexpressed gene. ​ H344A / N364A (SEQ ID NO:7) ​ (NCBI-GeneID:948913) ​ (NCBI-GeneID:945527) 、​ (NCBI-GeneID:946081). The SER8 described is an variant of SER7 that does not express the gene. ​ (NCBI-GeneID:947605). The SER9 variant does not express genes, unlike the SER8 variant. ​ (NCBI-GeneID:947264). The SER12 mentioned is... ​ Overexpression based on ​ The gene encoding 2-hydroxyglutarate dehydrogenase from this source. ​ (SEQ ID NO:8), and the gene encoding 7-cyano-7-dehydroguanine reductase in Escherichia coli itself. ​ The nucleotide sequence of (NCBI-GeneID:947270) is mutated as shown in SEQ ID NO:9.

[0051] Using the Escherichia coli genome as a template, based on its ​ Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI GeneID: 948201). ​ -S、UP- ​ -A) and downstream homologous arm primer (DN- ​ -S、DN- ​ -A); the above fragments were fused using overlap PCR to obtain the target gene (upstream homologous arm - downstream homologous arm). pEcgRNA was constructed. ​ The DNA fragment containing the target sequence used is transmitted via primer gRNA- ​ -S and gRNA- ​ -A is obtained by annealing.

[0052] To prepare competent cells of the engineered strain SER7, follow the steps in 1.3 and 1.4 above to construct cells with knockout antibodies. ​ The engineered strain SER0405.

[0053] To prepare competent cells of the engineered strain SER8, follow the steps in 1.3 and 1.4 above to construct cells with knockout antibodies. ​ The engineered strain SER0406.

[0054] To prepare competent cells of the engineered strain SER9, follow the steps in 1.3 and 1.4 above to construct cells that knock out the strain. ​ The engineered strain SER0407.

[0055] To prepare competent cells of the engineered strain SER12, follow the steps in 1.3 and 1.4 above to construct cells with knockout antibodies. ​ The engineered strain SER0408.

[0056] Example 2: This embodiment illustrates the method for producing L-serine according to the present invention, specifically using the weakened method constructed in Example 1. ​ Genetically engineered bacteria SER0405, SER0406, SER0407 or SER0408 expressing genes are cultured under suitable conditions and in a suitable culture medium to produce L-serine; and L-serine is collected from the genetically engineered bacteria and / or the culture medium.

[0057] The specific steps of the method in this embodiment are as follows: Slant culture: Streaking the -80℃ preserved strain onto the activated slant, incubating at 37℃ for 12 h, and subculturing once.

[0058] Shake-flask seed culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into a 500mL Erlenmeyer flask containing 30mL of seed culture medium. Seal the flask with nine layers of gauze and incubate at 37℃ and 200rpm for 8 hours.

[0059] Shake-flask fermentation: Inoculate 10% of the seed culture volume into a 500 mL Erlenmeyer flask containing fermentation medium (final volume 30 mL), seal with nine layers of gauze, and incubate at 37℃ with shaking at 200 r / min. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia; add 60% ( ​ Fermentation was maintained using glucose solution; the fermentation cycle was 20 hours.

[0060] The preferred composition of the slant culture medium is: 1 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 1 g / L NaCl, 25 g / L agar, and the remainder is water, with a pH of 7.0-7.2.

[0061] The preferred seed culture medium composition is: glucose 20 g / L, yeast extract 3 g / L, tryptone 3 g / L, KH2PO4·3H2O 3 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 20 mg / L, MnSO4·7H2O 20 mg / L, VB1, VB3, VB5, VB6 12 V H Each 2 mg / L, the remainder being water, pH 7.0-7.2.

[0062] The preferred fermentation medium composition is: glucose 20 g / L, yeast extract 3 g / L, tryptone 3 g / L, K₂HPO₄ 5 g / L, MgSO₄·7H₂O 2 g / L, FeSO₄·7H₂O 20 mg / L, MnSO₄·7H₂O 20 mg / L, VB1, VB3, VB5, VB 12 V H Each 2 mg / L, the remainder being water, pH 7.0-7.2.

[0063] Methods for determining L-serine: Take 1 mL of fermentation broth and transfer it to an EP tube. Centrifuge at 13000 rpm for 3 min. Transfer the supernatant to a new EP tube and dilute the sample to a suitable concentration (within the standard curve range). Detect the L-serine concentration in the sample using the 2,4-dinitrofluorobenzene pre-column derivatization method. The specific steps are as follows: Take 10 μL of the sample to be tested into a new EP tube, add 200 μL of derivatization buffer and 300 μL of derivatizing agent to the EP tube, and incubate in a dark place at 65℃ for 1 h. Add 690 μL of diluent buffer, shake well, and pass through a 0.2 μm organic syringe filter. Finally, detect the L-serine in the sample using high performance liquid chromatography. The detection conditions were as follows: the mobile phase column was an Agilent ZORBAX Eclipse AAA (4.6 mm × 250 mm, 5-Micron). The derivatized sample was eluted by a binary gradient high-pressure gradient with 50% acetonitrile solution and 50 mM sodium acetate aqueous solution (containing 10 mL N,N-dimethylformamide per liter), at a flow rate of 1 mL / min, a detection wavelength of 360 nm, and a column temperature of 33 ℃.

[0064] Comparative Example 1: Comparison of knockout ​ Differences in L-serine production performance between genetically modified SER0405 and the original strain SER7.

[0065] SER0405 and SER7 cells were cultured in shake flasks using the method described in Example 2. The concentration of L-serine in the fermentation supernatant and cell growth (OD) were measured. 600 The results are shown in Table 4.

[0066] Table 4

[0067] Table 1 shows the results of weakening. ​Gene expression did not significantly affect the growth of Escherichia coli; however, it increased the L-serine concentration in SER0409 from 14.2 g / L to 15.3 g / L, and increased the L-serine yield by 7.8%.

[0068] Comparative Example 2: Comparison of knockout ​ Differences in L-serine production performance between genetically modified SER0406 and the original strain SER8.

[0069] SER0406 and SER8 were cultured in shake flasks using the culture method described in Example 2. The concentration of L-serine in the fermentation supernatant and cell growth (OD) were measured. 600 The results are shown in Table 5.

[0070] Table 5

[0071] Table 5 shows the results of weakening. ​ Gene expression did not significantly affect the growth of Escherichia coli; however, it increased the L-serine concentration in the experimental strain SER0406 from 15.4 g / L to 17.1 g / L, and the L-serine yield was 11.0% higher than that of the control strain.

[0072] Comparative Example 3: Comparison of knockout ​ Differences in L-serine production performance between genetically modified SER0407 and the original strain SER9.

[0073] SER0407 and SER9 cells were cultured in shake flasks using the method described in Example 2. The concentration of L-serine in the fermentation supernatant and cell growth (OD) were measured. 600 The results are shown in Table 6.

[0074] Table 6

[0075] Table 6 shows the results of weakening. ​ Gene expression did not significantly affect the growth of Escherichia coli; however, it increased the L-serine concentration in the experimental strain SER0407 from 16.3 g / L to 18.3 g / L, and the L-serine yield was 12.3% higher than that of the control strain.

[0076] Comparative Example 4: Comparison of knockout ​ Differences in L-serine production performance between genetically modified SER0408 and the original strain SER12.

[0077] SER0408 and SER12 cells were cultured in shake flasks using the method described in Example 2. The concentration of L-serine in the fermentation supernatant and cell growth (OD) were measured. 600 The results are shown in Table 7.

[0078] Table 7

[0079] Table 7 shows the results of weakening. ​ Gene expression did not significantly affect the growth of Escherichia coli; however, it increased the L-serine concentration in the experimental strain SER0408 from 18.7 g / L to 20.8 g / L, and the L-serine yield was 11.2% higher than that of the control strain.

[0080] The results in summary indicate that a weakened approach is effective against Escherichia coli. ​ Modifying gene expression can significantly improve L-serine production in Escherichia coli without affecting the strain's growth performance.

Claims

1. A genetically engineered bacterium for producing L-serine, characterized in that: The genetically engineered bacteria are a weakened version of Escherichia coli. uhpT The way genes are expressed has been modified.

2. The genetically engineered bacteria as described in claim 1, characterized in that: Compared with unmodified Escherichia coli, the genetically engineered bacteria are... uhpT The expression level of the gene is downregulated or not expressed.

3. The genetically engineered bacteria as described in claim 2, characterized in that: The genetically engineered bacteria do not express the aforementioned... uhpT Gene.

4. The genetically engineered bacteria as described in claim 3, characterized in that: The genetically engineered bacteria do not express the Escherichia coli gene. sdaA , sdaB , tdcG And overexpression of E. coli genes serA H344A / N364A , serB , serC、eamA .

5. The genetically engineered bacteria as described in claim 4, characterized in that: The genetically engineered bacteria do not express the Escherichia coli gene. sstT .

6. The genetically engineered bacteria as described in claim 5, characterized in that: The genetically engineered bacteria do not express the Escherichia coli gene. sdaC .

7. The genetically engineered bacterium as described in claim 6, characterized in that: The genetically engineered bacteria also overexpressed Brevibacterium flavum The gene encoding 2-hydroxyglutarate dehydrogenase from this source. D2HGDH Furthermore, the nucleotide sequence of the gene queF encoding 7-cyano-7-denitroguanine reductase in Escherichia coli is mutated as shown in SEQ ID NO:

9.

8. The use of the genetically engineered bacteria according to any one of claims 1-7 in the production of L-serine.

9. A method for producing L-serine, comprising: The genetically engineered bacteria as described in any one of claims 1-7 are cultured in a culture medium to produce L-serine; In addition, L-serine is collected from the genetically engineered bacteria and / or the culture medium.

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