Escherichia coli engineering strain with high yield of l-serine and application thereof

Abstract

This invention discloses a high-yield L-serine-producing *Escherichia coli* strain and its applications. Through systems metabolic engineering, a comprehensive approach was adopted, employing strategies such as enhancing synthetic pathways, blocking product degradation and internal transport pathways, weakening competitive pathways, and reconstructing the threon-glycine and one-carbon metabolic systems. This resulted in the redistribution of carbon flux and optimization of intracellular homeostasis, yielding a strain capable of efficient and stable industrial-scale production of L-serine. Scale-up fermentation yielded an L-serine production of up to 150 g / L with a sugar conversion rate of 60%, demonstrating stable and high-yield scale-up performance.

Description

(I) Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a high-yielding L-serine-producing *Escherichia coli* obtained through systems metabolic engineering. Escherichia coli Engineered strains and their application in the microbial fermentation preparation of L-serine. (II) Background Technology

[0002] L-Serine is a structurally simple but physiologically vital non-essential amino acid, serving as a key node in the carbon-nitrogen metabolic network of human and most microbial cells. Widely distributed in nature, it not only serves as a basic building block of protein synthesis but also participates in various biosynthetic pathways. It is a precursor to many biomolecules, including purines, pyrimidines, inositol, cysteine, and phospholipids, playing a crucial role as a "metabolic hub" in cellular metabolism and physiological function maintenance. Due to its multiple physiological functions, including protein synthesis, neural regulation, and antioxidant activity, L-Serine is widely used in the pharmaceutical, food, cosmetic, and animal feed industries, with demand increasing annually and a promising market outlook.

[0003] Currently, the main production routes for L-serine include chemical synthesis and microbial fermentation. While chemical synthesis is a mature process, it suffers from drawbacks such as reliance on petrochemical raw materials, demanding reaction conditions, and severe organic solvent pollution, failing to meet the requirements of green development. In contrast, microbial fermentation uses renewable carbon sources as raw materials and utilizes microbial metabolic pathways to synthesize the target product. It offers advantages such as a clean process, high controllability, and high optical purity of the product, gradually replacing traditional methods and becoming a highly promising production technology. However, traditional strains generally face the following technical bottlenecks in the L-serine fermentation process:

[0004] (1) Insufficient main pathway synthesis flux: L-serine is mainly generated via the synthesis pathway with 3-phosphoglycerate as a precursor, but the expression level and catalytic ability of key synthases in this pathway are limited, resulting in low overall synthesis flux. This makes it impossible to meet the demand for target products under high-density culture conditions, becoming the primary bottleneck restricting yield improvement.

[0005] (2) Active product degradation pathway: The product is transported via the L-serine / L-threonine transport system ( sdaC , sstT ) and deaminase ( sdaA, sdaB Further degradation into pyruvate, which flows back to the central carbon for metabolism, resulting in limited product accumulation;

[0006] (3) One-carbon metabolism redox balance is easily disrupted: The synthesis of L-serine and its metabolic conversion with glycine are closely related to intracellular one-carbon metabolism. Traditional strains often have difficulty maintaining the dynamic balance between one-carbon metabolism and redox system, which can easily lead to energy metabolism disorder and thus inhibit its continuous synthesis.

[0007] (4) The strain has weak tolerance and insufficient fermentation stability: Excessive accumulation of intracellular L-serine can easily lead to feedback inhibition and osmotic pressure stress, thereby reducing cell activity and fermentation stability.

[0008] Based on the common problems in microbial fermentation mentioned above, researchers both domestically and internationally have implemented various modification strategies to enhance the biosynthetic capacity of L-serine. For example, by knocking out competing pathway genes (such as...) glyA Reduce carbon flux dispersion, or by overexpressing key enzyme genes (such as...) serA , serB , serC Enhanced synthesis of target products. However, such modifications are mostly focused on a single pathway or local node, failing to take into account the synergistic regulation between carbon metabolism, one-carbon metabolism and amino acid homeostasis, making it difficult to simultaneously improve L-serine synthesis flux, metabolic homeostasis and fermentation stability at the overall metabolic network level.

[0009] In recent years, with the advancements in systems biology and metabolic engineering, constructing efficient synthetic systems based on *E. coli* has gradually become a research hotspot. This strain exhibits significant advantages in carbon source utilization, genetic stability, and industrial scale-up capabilities, providing an ideal host for high-yield amino acid fermentation. However, existing publicly reported L-serine-producing *E. coli* engineered strains mostly employ local metabolic pathway modification strategies, generally exhibiting problems such as a single level of metabolic regulation, uneven distribution of carbon flux and energy metabolism, and insufficient coordination of one-carbon metabolism and redox systems. This makes it difficult to simultaneously achieve high yields while maintaining strain growth status and fermentation process stability, thus limiting its further scale-up applications.

[0010] Therefore, developing a system-engineered L-serine high-yield strain based on Escherichia coli, through multi-dimensional synergistic means such as overall enhancement of the synthetic pathway, blocking product degradation and internal transport pathways, weakening competitive pathways, and reconstructing the threon-glycine and one-carbon metabolic system, can significantly improve the synthetic throughput and maintain intracellular metabolic stability. This has important scientific research significance and industrial application value for realizing the efficient and low-cost industrial production of L-serine. (III) Summary of the Invention

[0011] The purpose of this invention is to provide a high-yield L-serine-producing *Escherichia coli* strain and its applications. Through systems metabolic engineering, a comprehensive approach was adopted, employing strategies such as enhancing synthetic pathways, blocking product degradation and internal transport pathways, weakening competitive pathways, and reconstructing the threon-glycine and one-carbon metabolic systems. This resulted in the redistribution of carbon flux and optimization of intracellular homeostasis, yielding a strain capable of efficient and stable industrial-scale production of L-serine. Scale-up fermentation processes showed that the obtained engineered strain achieved an L-serine yield of up to 150 g / L with a sugar conversion rate of 60%, demonstrating stable and high-yield scale-up performance.

[0012] The technical solution adopted in this invention is:

[0013] This invention provides a high-L-serine-producing engineered strain of *Escherichia coli*, which is constructed by editing the genome of *ZYYZ-1* bacteria with one or more of the following genes: overexpression gene. serA fr , serB , serC , eamA , pgk , tdh ,Will thrABC or gcvTHP The original promoter is replaced with a strong promoter, and the gene is knocked out. sdaA , sdaB , sstT , sdaC or glyA The genotype of the fungus ZYYZ-1 is: E.coli Trc- araE Trc -murC Δ rhtA - yjiM - ompX - opgE - rybA Δ lacI .

[0014] Furthermore, we obtained serA fr The nucleotide sequence is shown in SEQ ID NO.1. serB , serC , eamA , pgk , tdh , thrA, thrB, thrC , gcvT, gcvH, gcvP , sdaA , sdaB , sstT , sdaC and glyA The GeneIDs are 948913, 945527, 946081, 947414, 948139, 945803, 947498, 945198, 947390, 947393, 947394, 946331, 947262, 947605, 947264 and 947022.

[0015] Furthermore, the aforementioned serA fr Inserted in the form of an expression box driven by an artificial strong promoter ygaY and sdaA The nucleotide sequence of the expression cassette is shown in SEQ ID NO.2; serB Inserted in the form of an expression box driven by an artificial strong promoter yeeP The nucleotide sequence of the expression cassette is shown in SEQ ID NO.3; serC Inserted in the form of expression boxes driven by wild-type promoters yghX The site, and the nucleotide sequence, are shown in SEQ ID NO.4. pgk and eamA Inserted in the form of expression boxes driven by artificial strong promoters respectively ycgH and rph The nucleotide sequences of the sites and expression cassettes are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. tdh Inserted in the form of an expression box driven by an artificial strong promoter lafU The nucleotide sequence of the expression cassette is shown in SEQ ID NO.7. thrL The site-directed insertion of the strong promoter Prom1 (nucleotide sequence shown in SEQ ID NO. 8) drives... thrABC Expression. In situ introduction of the strong promoter Prom2 (nucleotide sequence shown in SEQ ID NO.9) for enhancement. gcvTHP Express.

[0016] Furthermore, the engineered Escherichia coli strain was constructed according to the following steps:

[0017] (1) In the genome of the chassis bacteria ygaY, yeeP and yghX Insertion at each site serA fr , serB , serC Expression cassette, constructing engineered bacteria ZYYZ-1 ygaY :: serA fr yeeP :: serB yghX :: serC It was named strain SER-01;

[0018] (2) In the genome of strain SER-01 rph and ycgH Site insertion eamA and pgk Expression cassette, obtained strain SER-01 rph :: eamA ycgH ::pgk The strain was named SER-02;

[0019] (3) In the genome of strain SER-02 sdaA Site insertion serA fr Expression box, knockout sdaB Genes were used to obtain strain SER-02. sdaA :: serA fr Δ sdaB The strain was named SER-03;

[0020] (4) Knockout of genes in the SER-03 genome sstT and sdaC SER-03 Δ strain was obtained sstT Δ sdaC It was named strain SER-04;

[0021] (5) Knockout of the SER-04 genome glyA Genes, in genes lafU Site insertion tdh Expression box, thrL Insert strong promoter Prom1 at the site to enhance thrABC Expression intensity, in-situ insertion of strong promoter Prom2 enhancement gcvTHP Expression, obtain SER-04 Δ glyA lafU :: tdh thrL :: Prom1 Prom2 - gcvTHP It was named SER-05.

[0022] This invention also provides the application of the engineered *Escherichia coli* strain in the fermentation production of L-serine. The method of application is as follows: the engineered *Escherichia coli* strain is inoculated into LB medium and cultured overnight at 37 °C and 200 rpm to obtain a pre-culture; the pre-culture is inoculated into a shake flask containing fermentation medium at a volume concentration of 1-5% (preferably 2%) and cultured at 37 °C. o Fermentation was carried out at 220 rpm for 48 h to obtain a fermentation broth containing L-serine. The fermentation medium consisted of the following: glucose 20 g / L, yeast extract 5 g / L, peptone 3 g / L, FeSO4 10 mg / L, KH2PO4 1.5 g / L, MgSO4 2 g / L, trace element mixture 1 mL / L, and V... BThe mixed solution contains 1 mL / L of phenol red (8 mg / L), 2-3 drops of defoamer, and deionized water as the solvent, with a pH of 7.0-7.2. The trace element mixed solution consists of: FeCl3 27 g / L, CoCl2·6H2O 2 g / L, Na2MoO4 2 g / L, CaCl2 24.6 g / L, CuSO4 1 g / L, H3PO3 0.5 g / L, AlCl3 10 g / L, deionized water as the solvent, and pH adjusted to 7.0 with 1 M HCl. B Mixture composition: V B1 V B3 V B5 V B12 Each 1 g / L, solvent is deionized water.

[0023] Furthermore, the fermentation is carried out using a fed-batch fermenter culture method: the engineered E. coli bacteria are activated and inoculated into a fermenter containing the initial fermentation medium, and the fermentation conditions are set as follows: temperature 37 ℃, pH 7.0 (adjusted by adding ammonia), dissolved oxygen (DO) 20-40%; when the bacterial OD... 600 When the concentration reaches 10-20 g / L, the fermentation broth is transferred to a new fermenter containing fermentation medium at a volume concentration of 10-20%, and fermentation continues under the same conditions. When the residual sugar concentration reaches 1-5 g / L, feed medium is added, and the flow rate is controlled to maintain the glucose concentration in the fermentation broth at 5-10 g / L. When the increase in L-serine production is no longer significant or even decreases, feed is stopped. Fermentation is stopped when the residual sugar concentration is below 2 g / L, yielding a fermentation broth containing L-serine. The initial fermentation medium composition is: glucose 20 g / L, yeast extract 5 g / L, peptone 3 g / L, KH2PO4 1.5 g / L, MgSO4 2 g / L, FeSO4 10 mg / L, V B The mixture was 1 mg / L, with deionized water as the solvent, and the pH was adjusted to 7.0-7.2. The fermentation medium consisted of: 20 g / L glucose, 4 g / L yeast extract, 2 g / L peptone, 5 g / L KH₂PO₄, 3 g / L MgSO₄, 1 mL / L trace element mixture, and V B The mixed solution contained 0.5 mg / L FeSO4, 5 mg / L FeSO4, 10 mg / L MnSO4, and 2 mg / L biotin, in deionized water as the solvent; the trace element mixed solution consisted of: FeCl3 27 g / L, CoCl2·6H2O 2 g / L, Na2MoO4 2 g / L, CaCl2 24.6 g / L, CuSO4 1 g / L, H3PO3 0.5 g / L, and AlCl3 10 g / L, in deionized water as the solvent, with the pH adjusted to 7.0 using 1 M HCl; V B Mixture composition: VB1 V B3 V B5 V B12 Each 1 g / L, with deionized water as the solvent; the feeding medium is a 60% glucose aqueous solution.

[0024] This invention utilizes overexpression serA fr , serB , serC This gene enhances the regulation of the serine biosynthesis pathway, increasing the overall throughput of serine synthesis from sugar metabolism intermediates. This alleviates the yield bottleneck caused by limited serine synthesis steps and low key reaction rates in existing technologies, laying the foundation for efficient serine accumulation. Furthermore, through overexpression... eamA and pgk This gene enhances L-serine secretion and efflux, increases the amount of precursors required for serine synthesis, and mitigates the problems of insufficient precursors or feedback inhibition of key enzymes by products under high-throughput synthesis conditions, enabling the serine synthesis reaction to proceed continuously and efficiently. Simultaneously, by knocking out... sdaA , sdaB Genes can be used to inhibit or block the intracellular degradation pathway of serine, reducing non-productive consumption caused by the conversion of serine into other metabolites, thereby increasing the net accumulation of serine in microbial cells. Furthermore, gene knockout can also be used to... sstT and sdaC, Regulating serine-related transmembrane transport processes reduces ineffective serine transport and metabolic disturbances between intracellular and extracellular spaces, maintaining relative stability of intracellular serine concentration, thereby improving the physiological stability and fermentation performance of the strain under high serine concentration conditions. Furthermore, by knocking out… glyA Genes, and overexpression tdh , thrABC , gcvTHP Genes were used to reconstruct the threo-glycine and one-carbon metabolic network. This reduced the consumption of serine for glycine conversion while maintaining a metabolic balance between cellular one-carbon metabolism and glycine supply, preventing the inhibition of a single pathway from affecting normal cell growth. This achieved a balance between efficient serine synthesis and the physiological needs of the strain.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) This invention enhances the serine biosynthesis pathway as a whole and removes the feedback inhibition of L-serine on 3-phosphoglycerate dehydrogenase, thereby significantly increasing the reaction rate of 3-phosphoglycerate to L-serine synthesis, and increasing the synthesis intensity of L-serine by 10 times compared with the starting strain.

[0027] (2) By synergistically optimizing the carbon metabolism and one-carbon metabolism pathways, the present invention enables a continuous and stable supply of precursors and one-carbon units required for L-serine synthesis, thereby further increasing L-serine production by 37.60%.

[0028] (3) By inhibiting the L-serine degradation pathway, this invention effectively reduces the proportion of L-serine that is degraded or converted into other metabolites in the cell, reduces non-productive consumption, and enables the synthesized L-serine to accumulate stably in the cell, thereby further increasing the L-serine production by 32.44%.

[0029] (4) By regulating the transport pathways related to L-serine, this invention reduces the ineffective circulation of L-serine between the intracellular and extracellular spaces, alleviates metabolic disturbances, maintains intracellular metabolic homeostasis, and enables the strain to maintain good growth and fermentation performance under high serine concentration conditions, further increasing L-serine production by 21.23%.

[0030] (5) While reducing the consumption of L-serine to glycine transport, this invention promotes the one-carbon cycle and replenishes glycine supply by reconstructing the threon-glycine and one-carbon metabolic network, avoiding the impact of single metabolic pathway inhibition on normal cell growth, thereby achieving coordination between efficient L-serine synthesis and the physiological needs of the strain, and further increasing L-serine production by 20.17%.

[0031] (6) The multi-module synergistic metabolic process strategy adopted in this invention does not rely on a single gene or a specific host system, is applicable to a variety of industrial microorganisms, has a stable genetic structure, is suitable for scale-up fermentation and continuous production, and has good industrial application prospects.

[0032] (7) The high-yield L-serine engineered strain of this invention has a scalable fermentation process for industrial production. Through batch feeding culture, the synthesis rate is maximized while ensuring cell growth, achieving high yield, low by-products and high tolerance to fermentation. The final L-serine yield can reach 150 g / L, and the sugar-acid conversion rate is 60%. (iv) Description of the attached drawings

[0033] Figure 1 This is a schematic diagram of the metabolic pathway modification of the L-serine strain of the present invention.

[0034] Figure 2 OD of engineered bacteria in Example 1 600 And the L-serine content in the fermentation broth supernatant.

[0035] Figure 3 OD of engineered bacteria in Example 2 600 And the L-serine content in the fermentation broth supernatant.

[0036] Figure 4 OD of engineered bacteria in Example 3 600 And the L-serine content in the fermentation broth supernatant.

[0037] Figure 5 OD of engineered bacteria in Example 4 600 And the L-serine content in the fermentation broth supernatant.

[0038] Figure 6 OD of engineered bacteria in Example 5 600 And the L-serine content in the fermentation broth supernatant.

[0039] Figure 7 The growth curves and L-serine accumulation of the engineered strain in Example 6 are shown in a 5 L fermenter. (V) Detailed Implementation Methods

[0040] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0041] Example 1: Construction of Chasmophyton ZYYZ-1

[0042] 1. Recombinant Escherichia coli E.coli Trc- araE T rc -murC Construction:

[0043] Using the CRISPR-Cas9 system to introduce the promoter Trc expression into E. coli genes araE Genes, the specific steps are as follows:

[0044] (1) Using pTarget plasmid (Addgene number 62226) as a template, PCR amplification was performed (primers) araE -PTTB-F and araE -PTTB-R), for sgRNA N 20 Site-directed mutagenesis of sequences for targeted purposes araE Promoter portion. PCR products were digested with DpnI. The digested products were then transferred to... E.coli In DH5α, the cells were plated on LB agar plates containing 50 mg / L spectinomycin and incubated overnight at 37°C. Single colonies were picked for sequencing verification (Table 4 primers pT-YZ-F and pT-YZ-R). Successfully mutated pTarget- araE Plasmid.

[0045] (2) In the pattern E. coli Using the DSM 5911 genome as a template, primers were used... araE -Up-F and araE -Up-R, araE -Down-F andaraE -Down-R, perform PCR amplification to obtain araE The upstream and downstream fragments are each 500bp. Additionally, primers were used... araE - trc -F and araE - trc -R, obtained by PCR amplification araE Expression Box (Trc Promoter and) araE The linker consists of the Trc promoter nucleotide sequence shown in SEQ ID NO.10. araE The nucleotide sequences are shown in Table 1. The three DNA fragments were fused using fusion PCR to obtain the fragment Donor-. araE .

[0046] (3) The pattern E. coli DSM 5911 was prepared into chemically competent cells. The pCas plasmid (Addgene number 62225) was transformed into the chemically competent cells via chemical transformation. The cells were then plated on LB agar plates containing 50 mg / L kanamycin resistance and incubated overnight at 30°C to obtain the strain. E. coli / pCas.

[0047] (4) The strain E. coli / pCas was prepared as electrocompetent states. The plasmid pTarget- araE Donor- )]araE Electricity transfer E. coli After / pCas electrocompetency, the cells were plated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin dual resistance, and cultured for 30 minutes. o Incubate inverted culture overnight, then pick single colonies for PCR verification (primers). araE -JYZ-YZ-F and araE -JYZ-YZ-R), successfully screened for overexpression araE strains.

[0048] (5) Pick a single positive colony from step (4) and inoculate it into an LB tube containing 10 mM IPTG and 50 mg / L kanamycin. Incubate overnight at 30°C. Streak the colony on an LB plate containing 50 mg / L kanamycin and incubate at 30°C for 24 h. Pick a single colony and streak it on an LB plate containing 50 mg / L spectinomycin and incubate at 30°C for 24 h. Single colonies that cannot grow on an LB plate containing 50 mg / L spectinomycin have pTarget- araE Plasmid successfully eliminated. Pick pTarget- araESingle colonies with successfully eliminated plasmids were cultured overnight at 42 °C in antibiotic-free LB agar plates. The next day, the bacterial culture was streaked onto antibiotic-free LB agar plates and cultured overnight at 37 °C. Single colonies were then picked and streaked onto LB agar plates containing 50 mg / L kanamycin and cultured overnight at 37 °C. Single colonies that could not grow on LB agar plates containing 50 mg / L kanamycin had their pCas plasmids successfully eliminated, resulting in plasmid-free bacterial strains. E. coli Trc -araE .

[0049] Using the same method as described above, murC Expression Box (Trc Promoter and) murC Connection composition, murC (Genes are shown in Table 1) Inserted into pseudogenes yjiT In the location, the strain was obtained E. coli Trc -araE yjiT:: Trc -murC .

[0050] 2. Recombinant Escherichia coli E.coli Trc- araE Trc -murC Δ rhtA - yjiM - ompX - opgE - rybA Δ lacI Construction of (i.e., the chassis bacteria ZYYZ-1):

[0051] strain in step 1 E. coli Trc -araE yjiT:: Trc -murC Based on this, knock out rhtA - yjiM - ompX - opgE - rybA Genes, the specific steps are as follows:

[0052] (1) Using pTarget plasmid as a template, PCR amplification was performed (primers) ryoor -PTTB-F and ryoor -PTTB-R), for sgRNA N 20 Site-directed mutagenesis of sequences for targeted purposes ​ Genes. PCR products were digested with DpnI. The digested products were then transferred to... ​ In DH5α, the cells were plated on LB agar plates containing 50 mg / L spectinomycin and incubated overnight at 37 °C. Single colonies were picked for sequencing verification (primers pT-YZ-F and pT-YZ-R). Successfully mutated pTarget- ​ Plasmid.

[0053] (2) In the pattern ​ Using the DSM 5911 genome as a template, primers were used... ​ -Up-F and ​ -Up-R, ​ - Down-F and ​-Down-R, perform PCR amplification to obtain ​ The upstream and downstream segments of the gene fragment are each 500 bp. These two DNA fragments are fused using fusion PCR to obtain the Donor- fragment. ​ .

[0054] (3) The pattern ​ Trc ​ Trc ​ Chemically competent cells were prepared, and the pCas plasmid was transformed into them via chemical transformation. The cells were then plated on LB agar plates containing 50 mg / L kanamycin resistance and incubated overnight at 30°C to obtain the strain. ​ Trc ​ Trc ​ / pCas.

[0055] (4) The strain ​ Trc ​ Trc ​ / pCas was prepared as an electrocompetent state. The plasmid pTarget-ryoor and the fragment Donor- ​ Electricity transfer ​ Trc -araE yjiT:: Trc -murC After / pCas electrocompetency, the cells were plated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin dual resistance, and cultured for 30 minutes. o Incubate inverted culture overnight, then pick single colonies for PCR verification (primers). ryoor -JYZ-YZ-F and ryoor -JYZ-YZ-R), successful screening and knockout rhtA-yjiM-ompX-opgE-rybA Strains with the gene (the corresponding gene sequence is shown in Table 1).

[0056] (5) Pick positive single colonies from step (4) and inoculate them into LB tubes containing 10 mM IPTG and 50 mg / L kanamycin. Incubate overnight at 30 ℃. Streak the colonies onto LB plates containing 50 mg / L kanamycin and incubate at 30 ℃ for 24 h. Pick single colonies and streak them onto LB plates containing 50 mg / L spectinomycin and incubate at 30 ℃ for 24 h. Single colonies that cannot grow on LB plates containing 50 mg / L spectinomycin have pTarget- ryoor Plasmid successfully eliminated. Pick pTarget- ryoorSingle colonies with successfully eliminated plasmids were cultured overnight at 42 °C in antibiotic-free LB agar plates. The next day, the bacterial culture was streaked onto antibiotic-free LB agar plates and cultured overnight at 37 °C. Single colonies were then picked and streaked onto LB agar plates containing 50 mg / L kanamycin and cultured overnight at 37 °C. Single colonies that could not grow on LB agar plates containing 50 mg / L kanamycin had their pCas plasmids successfully eliminated, resulting in plasmid-free bacterial strains. E. coli Trc- araE yjiT ::Trc - murC Δ rhtA-yjiM-ompX-opgE-rybA .

[0057] Knockout using the same method described above. lacI Genes (gene sequences are shown in Table 1), strains obtained E.coli Trc- araE Trc -murC Δ rhtA-yjiM-ompX-opgE-rybA Δ lacI (i.e., the basal bacteria ZYYZ-1), all gene descriptions and required primers are shown in Table 1 and Table 2, respectively.

[0058] Table 1. Genes involved in gene editing and their corresponding descriptions

[0059]

[0060] Table 2. Primers

[0061]

[0062] Example 2: Construction of L-serine synthesis pathway enhanced strain SER-01

[0063] In *E. coli*, the synthesis of L-serine begins with 3-phosphoglycerate and proceeds through three steps. These three steps are determined by genes. serA, serC and serB Encoded protein control. Among them, E. coli... serA The gene-encoded 3-phosphoglycerate dehydrogenase is subject to feedback inhibition by L-serine. To promote L-serine production, it is necessary to address the key enzyme in serine synthesis. Therefore, mutants that remove feedback inhibition are needed. serA fr Introduced. By driving an artificial strong promoter serA fr and serB Expression boxes and those driven by wild-type promoters serC Expression cassettes are inserted into the genome. ygaY, yeeP and yghX The location of the gene is determined, increasing the expression level of the aforementioned key genes and promoting L-serine synthesis. This is driven by an artificial strong promoter. serAfr , serB and driven by wild-type promoters serC The nucleotide sequences of the expression cassette are as shown in SEQ ID NO.2 (1-267 bp represent the promoter, 268-1500 bp represent...). serA fr ), SEQ ID NO.3 (1-76 bp represents the promoter, 77-1045 bp represents serB 1046-1132 bp represents the rrnB T1 terminator) and SEQ ID NO.4 (1-260 bp represents the promoter, 261-1349 bp represents the...) serC (1350-1399 bp represents a non-specific sequence introduced by primers, as shown in the figure).

[0064] 1. Construction of strain SER-01

[0065] (1) Using pTarget plasmid as a template, PCR amplification was performed (primers) ygaY -PTTB-F and ygaY -PTTB-R), for sgRNA N 20 Site-directed mutagenesis of sequences for targeted purposes [[ID=E=34]]ygaY The PCR products were digested with DpnI. The digested products were then transferred to... E.coli In DH5α, the cells were plated on LB agar plates containing 50 mg / L spectinomycin and incubated overnight at 37 °C. Single colonies were picked for sequencing verification (primers pT-YZ-F and pT-YZ-R). Successfully mutated pTarget- ygaY Plasmid.

[0066] (2) Using the ZYYZ-1 genome as a template, primers were used to... ygaY -Up-F and ygaY -Up-R, ygaY - Down-F and ygaY -Down-R, perform PCR amplification to obtain pseudogenes. ygaY The upstream and downstream fragments are each 500bp. Additionally, primers were used... ygaY - serA fr -F and ygaY - serA fr -R, PCR amplification to obtain artificial strong promoter-driven... serA fr Expression cassette (nucleotide sequence as shown in SEQ ID NO.2). The three DNA fragments were fused using fusion PCR to obtain the Donor- fragment. ygaY- serAfr .

[0067] (3) Prepare ZYYZ-1 as a chemically competent cell, and transform the pCas plasmid into the ZYYZ-1 chemically competent cell by chemical transformation. Spread the plasmid on LB plates containing 50 mg / L kanamycin resistance and incubate overnight at 30 °C to obtain the ZYYZ-1 / pCas strain.

[0068] (4) Prepare electrocompetent cells from strain ZYYZ-1 / pCas. Use plasmid pTarget- ygaY Donor- ygaY-serA fr After electrotransferring to ZYYZ-1 / pCas electrocompetent cells, they were plated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin dual resistance. o Incubate inverted culture overnight, then pick single colonies for PCR verification (primers). ygaY - serA fr -JYZ-YZ-F and ygaY - serA fr -JYZ-YZ-R), selected and successfully edited strains.

[0069] (5) Pick positive single colonies from step (4) and inoculate them into LB tubes containing 10 mM IPTG and 50 mg / L kanamycin. Incubate overnight at 30 ℃. Streak the colonies onto LB plates containing 50 mg / L kanamycin and incubate at 30 ℃ for 24 h. Pick single colonies and streak them onto LB plates containing 50 mg / L spectinomycin and incubate at 30 ℃ for 24 h. Single colonies that cannot grow on LB plates containing 50 mg / L spectinomycin have pTarget- ygaY Plasmid successfully eliminated. Pick pTarget- ygaY Single colonies with successfully eliminated plasmids were cultured overnight at 42 °C in antibiotic-free LB agar plates. The next day, the bacterial culture was streaked onto antibiotic-free LB agar plates and cultured overnight at 37 °C. Single colonies were then picked and streaked onto LB agar plates containing 50 mg / L kanamycin and cultured overnight at 37 °C. Single colonies that could not grow on LB agar plates containing 50 mg / L kanamycin had their pCas plasmids successfully eliminated, resulting in the plasmid-free strain ZYYZ-1. ygaY :: serA fr .

[0070] Based on the above method, the artificially driven strong starter will be sequentially... [[ID=E=59]]serB Expression cassette (nucleotide sequence as shown in SEQ ID NO. 3) and artificial strong promoter driven serCExpression cassettes (nucleotide sequences shown in SEQ ID NO.4) are inserted into pseudogenes. yeeP and yghX In the gene location, strain ZYYZ-1 was obtained. ygaY :: serA fr yeeP :: serB yghX :: serC It was named SER-01, and all the gene descriptions and required primers are shown in Tables 3 and 4, respectively.

[0071] 2. Cultivation of strain SER-01

[0072] The SER-01 strain was inoculated into 10 mL of LB medium and cultured overnight at 37 °C and 200 rpm to obtain a preculture. 1 mL of the preculture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium and fermented at 37 °C and 200 rpm for 48 h. The fermentation medium consisted of the following: glucose 20 g / L, yeast extract 5 g / L, peptone 3 g / L, FeSO4 10 mg / L, KH2PO4 1.5 g / L, MgSO4 2 g / L, trace element mixture 1 mL / L, and V... B The mixed solution contains 1 mL / L of phenol red (8 mg / L), 2-3 drops of defoamer, and deionized water as the solvent, with a pH of 7.0-7.2. The trace element mixed solution consists of: FeCl3 27 g / L, CoCl2·6H2O 2 g / L, Na2MoO4 2 g / L, CaCl2 24.6 g / L, CuSO4 1 g / L, H3PO3 0.5 g / L, AlCl3 10 g / L, deionized water as the solvent, and pH adjusted to 7.0 with HCl. B Mixture composition: V B1 V B3 V B5 V B12 Each was 1 g / L, and the solvent was deionized water. After fermentation, samples were taken to detect OD. 600 Another sample was centrifuged at 12,000 rpm for 2 min, and the supernatant of the fermentation broth was used to determine the L-serine content by high performance liquid chromatography. Strain ZYYZ-1 was used as a control.

[0073] The concentration of L-serine was determined using a 2,4-dinitrofluorobenzene pre-column derivatization method within a Thermo Fisher Scientific high-performance liquid chromatography (HPLC) system. The chromatographic column was a C18 column (4.6 × 250 mm, 5 μm); the UV detector wavelength was 260 nm; the injection volume was 10 μL; the column temperature was 30 ℃; the flow rate was 0.8 mL / min; and the mobile phase consisted of two phases, A and B. Phase A was pure acetonitrile, and phase B was 50 mM HAc-NaAc buffer:acetonitrile:triethylamine = 82.8:17:0.2 (v / v), pH = 4.9. A calibration curve was established using L-serine standards, and the linear correlation coefficient R² > 0.999.

[0074] The results are as follows Figure 2 The accumulation of L-serine in strain SER-01 was 10 times higher than that in the control group ZYYZ-1, indicating that the enhancement of key genes for L-serine synthesis significantly improved serine production.

[0075] Table 3. Genes involved in gene editing and their corresponding descriptions

[0076]

[0077] Table 4. Primers

[0078]

[0079] Example 3: Construction of product efflux and precursor supply enhanced strain SER-02

[0080] overexpression eamA and pgk By driving an artificial strong promoter pgk and eamA Expression cassettes are inserted into genes on the genome. rph and ycgH The location of the artificial strong promoter enhances product efflux and precursor supply, promoting increased L-serine fermentation yield. pgk and eamA The nucleotide sequences of the expression cassette are as shown in SEQ ID NO.5 (1-74 bp represent the promoter, 75-1238 bp represent...). pgk (1239-1325 bp represents the rrnB T1 terminator) and as shown in SEQ ID NO.6 (1-74 bp represents the promoter, 75-974 bp represents the terminator). eamA 975-1061bp represents the rrnB T1 terminator.

[0081] 1. Construction of strain SER-02

[0082] (1) Using pTarget plasmid as a template, PCR amplification was performed (primers)rph -PTTB-F and rph -PTTB-R), for sgRNA N 20 Site-directed mutagenesis of sequences for targeted purposes rph The PCR products were digested with DpnI. The digested products were then transferred to... E.coli In DH5α, the cells were plated on LB agar plates containing 50 mg / L spectinomycin and incubated overnight at 37 °C. Single colonies were picked for sequencing verification (primers pT-YZ-F and pT-YZ-R). Successfully mutated pTarget- rph Plasmid.

[0083] (2) Using the ZYYZ-1 genome as a template, primers were used to... rph -Up-F and rph -Up-R, rph -Down-F and rph -Down-R, perform PCR amplification to obtain pseudogenes. rph The upstream and downstream fragments are each 500bp. Additionally, primers were used... eamA -F and eamA -R, used for PCR amplification to obtain eamA Expression cassette fragment. The three DNA fragments were fused using fusion PCR to obtain the fragment Donor- rph-eamA .

[0084] (3) Prepare the SER-01 strain into a chemically competent state, and transform the pCas plasmid into the SER-01 chemically competent state by chemical transformation. Spread the pCas plasmid on an LB plate containing 50 mg / L kanamycin resistance to obtain the SER-01 / pCas strain.

[0085] (4) Prepare electrocompetent cells from strain SER-01 / pCas. Use plasmid pTarget- rph Donor- rph-eamA After electrotransferring to SER-01 / pCas competent cells, they were plated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin dual resistance. o Incubate inverted culture overnight, then pick single colonies for PCR verification (primers). rph-eamA -JYZ-YZ-F and rph-eamA -JYZ-YZ-R), selected and successfully edited strains.

[0086] (5) Pick positive single colonies from step (4) and inoculate them into LB tubes containing 10 mM IPTG and 50 mg / L kanamycin. Incubate overnight at 30 ℃. Streak the colonies onto LB plates containing 50 mg / L kanamycin and incubate at 30 ℃ for 24 h. Pick single colonies and streak them onto LB plates containing 50 mg / L spectinomycin and incubate at 30 ℃ for 24 h. Single colonies that cannot grow on LB plates containing 50 mg / L spectinomycin have pTarget- rph Plasmid successfully eliminated. Pick pTarget- rph Single colonies with successfully eliminated plasmids were cultured overnight at 42 °C in antibiotic-free LB agar plates. The next day, the bacterial culture was streaked onto antibiotic-free LB agar plates and incubated at 37 °C for 12 h. Single colonies were then picked and streaked onto LB agar plates containing 50 mg / L kanamycin and incubated at 37 °C for 12 h. Single colonies that could not grow on LB agar plates containing 50 mg / L kanamycin had their pCas plasmids successfully eliminated, resulting in the plasmid-free strain SER-01. rph :: eamA .

[0087] Based on the above method, the gene pgk Expression cassette insertion into pseudogene ycgH In the location, strain SER-01 was obtained. rph :: eamA ycgH :: pgk It was named SER-02, and all the gene descriptions and required primers are shown in Tables 5 and 6, respectively.

[0088] 2. Cultivation of strain SER-02

[0089] The SER02 strain was inoculated into 10 mL of LB medium and cultured overnight at 37 °C and 200 rpm to obtain a preculture. 1 mL of the preculture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium (same as in Example 1) and fermented at 37 °C and 200 rpm for 48 h. After fermentation, samples were taken, and the OD values ​​of the samples were analyzed using the method in Example 1. 600 The L-serine content in the fermentation broth supernatant was detected. Results are as follows: Figure 3 Compared with SER-01, strain SER-02 showed a 37.60% increase in L-serine accumulation, indicating that enhanced carbon metabolism pathways provide sufficient carbon skeletons for serine synthesis.

[0090] Table 5. Genes involved in gene editing and their corresponding descriptions

[0091]

[0092] Table 6. Primers

[0093]

[0094] Example 4: Construction of the serine degradation pathway blocking strain SER-03

[0095] Serine in Escherichia coli can be derived from... sdaA, sdaB The gene-encoded serine deaminase degrades serine into pyruvate. To increase serine accumulation, the gene is knocked out... sdaA (Insert simultaneously) serA fr Expression box) and sdaB The gene can block the degradation of L-serine, thus enabling the accumulation of serine.

[0096] 1. Construction of strain SER-03

[0097] (1) Using pTarget plasmid as a template, PCR amplification was performed (primers) sdaA -PTTB-F and sdaA -PTTB-R), for sgRNA N 20 Site-directed mutagenesis of sequences for targeted purposes sdaA The PCR products were digested with DpnI, and the digested products were then transferred to... E.coli In DH5α, the cells were plated on LB agar plates containing 50 mg / L spectinomycin and incubated overnight at 37 °C. Single colonies were picked for sequencing verification (primers pT-YZ-F and pT-YZ-R). Successfully mutated pTarget- sdaA Plasmid.

[0098] (2) Using the ZYYZ-1 genome as a template, primers were used to... sdaA -Up-F and sdaA -Up-R, sdaA - Down-F and sdaA -Down-R, perform PCR amplification to obtain pseudogenes. sdaA The upstream and downstream fragments, each 500 bp, were fused using fusion PCR to obtain the Donor- fragment. sdaA Additionally, through primers sdaA-serA fr -F and sdaA-serA fr -R, perform PCR amplification to obtain the gene. serA fr Expression cassette (SEQ ID NO.2). The three DNA fragments were fused using fusion PCR to obtain the Donor- fragment. sdaA-serA fr .

[0099] (3) Prepare the SER-02 strain into a chemically competent state, and transform the pCas plasmid into the SER-02 chemically competent state by chemical transformation. Spread the pCas plasmid on an LB plate containing 50 mg / L kanamycin resistance to obtain the SER-02 / pCas strain.

[0100] (4) Prepare electrocompetent cells from strain SER-02 / pCas. Use plasmid pTarget- sdaA Donor- sdaA-serA fr After electroporation to SER-02 / pCas competent cells, the cells were plated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin, and incubated overnight at 30°C. Single colonies were then picked for PCR verification (primers...). sdaA - serA fr -JYZ-YZ-F and sdaA - serA fr -JYZ-YZ-R), selected and successfully edited strains.

[0101] (5) Pick positive single colonies from step (4) and inoculate them into LB tubes containing 10 mM IPTG and 50 mg / L kanamycin. Incubate overnight at 30 ℃. Streak the colonies onto LB plates containing 50 mg / L kanamycin and incubate at 30 ℃ for 24 h. Pick single colonies and streak them onto LB plates containing 50 mg / L spectinomycin and incubate at 30 ℃ for 24 h. Single colonies that cannot grow on LB plates containing 50 mg / L spectinomycin have pTarget- sdaA Plasmid successfully eliminated. Pick pTarget- sdaA Single colonies with successfully eliminated plasmids were cultured overnight at 42°C in antibiotic-free LB agar plates. The next day, the bacterial culture was streaked onto antibiotic-free LB agar plates and cultured overnight at 37°C. Single colonies were then picked and streaked onto LB agar plates containing 50 mg / L kanamycin and cultured overnight at 37°C. Single colonies that could not grow on LB agar plates containing 50 mg / L kanamycin had their pCas plasmids successfully eliminated, resulting in plasmid-free SER-02. sdaA :: serA fr .

[0102] Based on the methods described above, continue to knock out [the virus] in the genome. sdaB The difference lies in the fact that, using the ZYYZ-1 genome as a template, and through primers Donor- sdaB -UP-F and Donor- sdaB -UP-R, Donor- sdaB - Down-F and Donor- sdaB-Down-R, perform PCR amplification to obtain the knockout. sdaB Each of the upstream and downstream segments of the gene is 500 bp. These two DNA fragments are fused using fusion PCR to obtain the Donor- fragment. sstT You can then edit the file to obtain strain SER-02. sdaA :: serA fr Δ sdaB It is named SER-03. All gene descriptions and required primers are shown in Tables 7 and 8, respectively.

[0103] 2. Cultivation of strain SER-03

[0104] Strain SER-03 was inoculated into 10 mL of LB medium and cultured overnight at 37 °C and 200 rpm to obtain a preculture. 1 mL of the preculture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium (same as in Example 1) and fermented at 37 °C and 200 rpm for 48 h. Strain SER-02 was used as a control. After fermentation, samples were taken, and the OD of the samples was analyzed using the method in Example 1. 600 The L-serine content in the fermentation broth supernatant was detected. Results are as follows: Figure 4 The results showed that the L-serine yield of strain SER-03 reached 1.59 g / L, which was 32.44% higher than that of SER-02. This result indicates that knocking out the product degradation gene can effectively increase the accumulation of the product.

[0105] Table 7. Genes involved in gene editing and their corresponding descriptions

[0106]

[0107] Table 8. Primers

[0108]

[0109] Example 5: Construction of the serine transport pathway blocking strain SER-04

[0110] Transport systems play a crucial role in amino acid production. Promoting product efflux and weakening or blocking product uptake pathways are important measures to improve product synthesis efficiency. High concentrations of serine can be physiologically toxic to cells. Blocking the serine ingestion pathway prevents the strain from taking up serine from the extracellular environment, maintaining a normal intracellular serine concentration level, and thus promoting normal cell growth. Therefore, this example focuses on genes involved in the serine ingestion pathway. sstT and sdaC It was knocked out.

[0111] 1. Construction of strain SER-04

[0112] (1) Using pTarget plasmid as a template, PCR amplification was performed (primers) sstT -PTTB-F and sstT -PTTB-R), for sgRNA N 20 Sequence for site-directed mutagenesis targeting sstT The PCR products were digested with DpnI. The digested products were then transferred to... E.coli In DH5α, the cells were plated on LB agar plates containing 50 mg / L spectinomycin and incubated overnight at 37 °C. Single colonies were picked for sequencing verification (primers pT-YZ-F and pT-YZ-R). Successfully mutated pTarget- sstT Plasmid.

[0113] (2) Using the ZYYZ-1 genome as a template, primers were used to... sstT -Up-F and sstT -Up-R, sstT - Down-F and sstT -Down-R, perform PCR amplification to obtain pseudogenes. sstT Each of the upstream and downstream fragments is 500bp. The two DNA fragments are fused using fusion PCR to obtain the Donor- fragment. sstT .

[0114] (3) Prepare the SER-03 strain into a chemically competent state, and transform the pCas plasmid into the SER-03 chemically competent state by chemical transformation. Spread the pCas plasmid on an LB plate containing 50 mg / L kanamycin resistance to obtain the SER-03 / pCas strain.

[0115] (4) Prepare electrocompetent cells from strain SER-03 / pCas. Use plasmid pTarget- sstT Donor- sstT After electroporation to SER-03 / pCas competent cells, the cells were plated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin, and single colonies were picked for PCR verification (primers). sstT -JYZ-YZ-F and sstT -JYZ-YZ-F), the successfully edited strain was screened and cultured overnight at 30 ℃ to obtain strain SER-03Δ sstT .

[0116] (5) Pick a positive single colony from step (4) and inoculate it into an LB tube containing 10 mM IPTG and 50 mg / L kanamycin. Incubate overnight at 30 ℃. Streak the colony onto an LB agar plate containing 50 mg / L kanamycin and incubate at 30 ℃ for 24 h. Pick a single colony and streak it onto an LB agar plate containing 50 mg / L spectinomycin and incubate at 30 ℃ for 24 h. Single colonies that cannot grow on LB agar plates containing 50 mg / L spectinomycin have pTarget- sstT Plasmid successfully eliminated. Pick pTarget- sstT Single colonies with successfully eliminated plasmids were cultured overnight at 42 °C in antibiotic-free LB agar plates. The next day, the bacterial culture was streaked onto antibiotic-free LB agar plates and incubated at 37 °C for 12 h. Single colonies were then picked and streaked onto LB agar plates containing 50 mg / L kanamycin and incubated at 37 °C for 12 h. Single colonies that could not grow on LB agar plates containing 50 mg / L kanamycin had their pCas plasmids successfully eliminated, resulting in plasmid-free SER-03 Δ. sstT .

[0117] Based on the methods described above, continue to knock out [the virus] in the genome. sdaC Genes were used to obtain strain SER-03 Δ sstT Δ sdaC It is named SER-04. All gene descriptions and required primers are shown in Tables 9 and 10, respectively.

[0118] 2. Cultivation of strain SER-04

[0119] Strain SER-04 was inoculated into 10 mL of LB medium and cultured overnight at 37 °C and 200 rpm to obtain a preculture. 1 mL of the preculture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium (same as in Example 1) and fermented at 37 °C and 200 rpm for 48 h. SER-03 was used as a control. After fermentation, samples were taken, and the OD of the samples was analyzed using the method in Example 1. 600 The L-serine content in the fermentation broth supernatant was detected. Results are as follows: Figure 5 Compared with SER-03, the accumulation of L-serine in the experimental strain was increased by 21.23% compared with the control group. This result indicates that modifying the transport system directly affects product accumulation, and weakening the internal transport system is a conventional strategy to increase product yield.

[0120] Table 9. Genes involved in gene editing and their corresponding descriptions

[0121]

[0122] Table 10. Primers

[0123]

[0124] Example 6: Threon-glycine and one-carbon metabolic network reconstructed strain SER-05

[0125] This embodiment aims to reconstruct the threo-glycine and one-carbon metabolic network of the host strain, thereby reducing the consumption of serine to glycine conversion and establishing a glycine replenishment system. This ensures the metabolic balance of cellular one-carbon metabolism and glycine supply, and avoids affecting normal cell growth due to inhibition of a single pathway, thus achieving coordination between efficient serine synthesis and the physiological needs of the strain.

[0126] 1. Construction of strain SER-05

[0127] (1) Blocking the serine degradation pathway: Serine can be degraded by serine hydroxymethyltransferase gene glyA Degradation to glycine limits the accumulation of serine. The method described in Example 4 was used to knock out serine using the CRISPR-Cas9 system. glyA Genes that block the degradation of serine into glycine promote the accumulation of serine.

[0128] (2) Glycine supplementation strategy: To compensate for insufficient glycine synthesis, the supplementation pathway for the conversion of threonine to glycine was modified under the above background. The method in Example 1 was adopted, and the glycine was supplemented in the following ways: lafU Site insertion driven by artificial strong promoter tdh Expression cassette (nucleotide sequence as shown in SEQ ID NO.7, 1-74 bp represents the promoter, 75-1100 bp represents the expression cassette). tdh 1101-1187 bp represents the rrnB T1 terminator. thrL The site-directed artificial promoter Prom1 (nucleotide sequence shown in SEQ ID NO. 8) drives the process. thrABC Gene expression ultimately leads to the formation of a threo-glycine supplementation network.

[0129] (3) Reconstructing one-carbon metabolic pathways: Studies have found that knocking out glyA The gene not only increases serine accumulation but also leads to an imbalance in the intracellular one-carbon metabolic network, causing poor cell growth and production limitations. Therefore, to coordinate the balance between glycine and one-carbon units, the artificial strong promoter Prom2 (nucleotide sequence shown in SEQ ID NO.9) was used to drive the process according to the method in Example 1. gcvTHP Gene expression was enhanced to improve the glycine cleavage system, maintaining cell morphology and viability and ensuring the continuous accumulation of intracellular serine. SER-04 was obtained through these modifications. Δ glyA lafU :: tdh thrL :: Prom1 Prom2::gcvTHP It is named SER-05. All gene descriptions and required primers are shown in Tables 11 and 12, respectively.

[0130] 2. Cultivation of strain SER-05

[0131] The method described in Example 1 was used to verify the fermentation of strain SER-05, and the results are as follows: Figure 6 Compared with SER-04, the engineered strain showed a significantly increased specific growth rate, and the accumulation of L-serine was 20.17% higher than the control group and 2.22 g / L higher than the chassis strain. This indicates that the reconstruction of the threonine-glycine and one-carbon metabolic network not only promoted the accumulation of serine but also maintained the intracellular one-carbon metabolic balance.

[0132] Table 11. Genes involved in gene editing and their corresponding descriptions

[0133]

[0134] Table 12. Primers

[0135]

[0136] Example 7: Validation in a 5 L fermenter

[0137] The preferred strain SER-05 was subjected to fed-batch fermentation with 5 L of feed. The specific fermentation steps are as follows:

[0138] (1) Activation of strain: The strain SER-05 was taken out under the conditions of a clean bench and streaked on the small slant of LB test tube and the large slant of eggplant bottle at 37 ℃. The strain was cultured at 37 ℃ for a total of about 27 h. After the bacteria have grown sufficiently to cover the surface of the culture medium, it was ready for use.

[0139] (2) Preparation before fermentation: After cleaning the 5 L fermenter and feed bottle, sterilize them at 121 °C for 20 min. At the same time, prepare the initial fermentation medium, fermentation medium, and feed medium, and add them to the sterilized fermenter and feed bottle respectively. Install dissolved oxygen electrode, pH electrode, and air pipeline with filter membrane, connect the feed tube, and sterilize again at 115 °C for 30 min. After sterilization, cool and introduce sterile air to prepare for fermentation.

[0140] (3) Fermentation culture: The activated strain was washed with sterile ddH2O and then inoculated into the initial fermentation medium. The fermentation conditions were set as follows: temperature 37 ℃, pH 7.0 (adjusted by adding ammonia), dissolved oxygen (DO) 40%. When the bacterial OD 600When the temperature reaches approximately 10-20°C, 20% of the fermentation broth is transferred to a fermenter containing 2 L of fermentation medium, and fermentation continues under the same conditions. When the residual sugar concentration is below 5 g / L, feed medium is added, and the flow rate is controlled to keep the glucose concentration in the fermentation broth below 10 g / L. When the increase in L-serine production is no longer significant or even decreases, feed is stopped. Fermentation is stopped when the residual sugar concentration is below 2 g / L, yielding a fermentation broth containing L-serine.

[0141] The initial fermentation medium consisted of: 20 g / L glucose, 5 g / L yeast extract, 3 g / L peptone, 1.5 g / L KH₂PO₄, 2 g / L MgSO₄, 10 mg / L FeSO₄, and 1 mg / L LV. B Adjust the pH of the mixture to 7.0-7.2;

[0142] The fermentation medium consisted of: 20 g / L glucose, 4 g / L yeast extract, 2 g / L peptone, 5 g / L KH₂PO₄, 3 g / L MgSO₄, 1 mL / L trace element mixture, and 0.5 mg / L LV. B The mixture contained 5 mg / L FeSO4, 10 mg / L MnSO4, and 2 mg / L biotin.

[0143] The trace element mixture consists of: FeCl3 27 g / L, CoCl2·6H2O 2 g / L, Na2MoO4 2 g / L, CaCl2 24.6 g / L, CuSO4 1 g / L, H3PO3 0.5 g / L, AlCl3 10 g / L, deionized water as solvent, and pH adjusted to 7.0 with HCl.

[0144] V B Mixture composition: V B1 V B3 V B5 V B12 Each 1 g / L, solvent is deionized water.

[0145] The feeding medium was a 60% glucose aqueous solution.

[0146] Fermentation results as follows Figure 7 At 44 hours, the strain reached its maximum growth and acid production, accumulating 150 g / L of L-serine and 9.9 g / L of glutamic acid, with a sugar-acid conversion rate of 60%. The fermentation process was stable, and the L-serine yield was consistent with the biomass, indicating that the strain has good scale-up stability and industrialization potential.

Claims

1. A high-L-serine-producing engineered strain of *Escherichia coli*, characterized in that, The engineered strain was obtained by editing the genome of *Bacteroides zebrina* ZYYZ-1 using one or more of the following gene edits: overexpression gene serA fr , serB , serC , eamA , pgk , tdh ,Will thrABC or gcvTHP The original promoter is replaced with a strong promoter, and the gene is knocked out. sdaA , sdaB , sstT , sdaC or glyA The genotype of the fungus ZYYZ-1 is: E. coli Trc- araE Trc -murC Δ rhtA-yjiM- ompX-opgE-rybA Δ lacI .

2. The engineered Escherichia coli strain as described in claim 1, characterized in that, The serA fr The nucleotide sequence is shown in SEQ ID NO.

1.

3. The engineered Escherichia coli strain as described in claim 1, characterized in that, The serA fr Inserted in the form of an expression box driven by an artificial strong promoter ygaY and sdaA The nucleotide sequence of the expression cassette at the site is shown in SEQ ID NO.2; The serB Inserted in the form of an expression box driven by an artificial strong promoter yeeP The nucleotide sequence of the expression cassette at the site is shown in SEQ ID NO. 3; The serC Inserted in the form of expression boxes driven by wild-type promoters yghX The nucleotide sequence of the expression cassette at the site is shown in SEQ ID NO.4; The eamA and pgk Inserted in the form of expression boxes driven by artificial strong promoters respectively rph and ycgH The nucleotide sequences of the expression cassettes at the sites are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The tdh Inserted in the form of an expression box driven by an artificial strong promoter lafU The nucleotide sequence of the expression cassette at the site is shown in SEQ ID NO.

7.

4. The engineered Escherichia coli strain as described in claim 1, characterized in that, exist thrL The site-inserted nucleotide sequence is driven by the strong promoter Prom1, as shown in SEQ ID NO.

8. thrABC The expression; and in gcvTHP In situ introduction of the strong promoter Prom2, as shown in SEQ ID NO.9, at the site to enhance... gcvTHP The expression.

5. The engineered Escherichia coli strain as described in claim 1, characterized in that, The engineered Escherichia coli strain was constructed according to the following steps: (1) In the genome of the chassis bacteria ygaY, yeeP and yghX Insertion at each site serA fr , serB , serC Expression cassette, constructing engineered bacteria ZYYZ-1 ygaY :: serA fr yeeP :: serB yghX :: serC It was named strain SER-01; (2) In the genome of strain SER-01 rph and ycgH Site insertion eamA and pgk Expression cassette, obtained strain SER-01 rph :: eamA ycgH :: pgk The strain was named SER-02; (3) In the genome of strain SER-02 sdaA Site insertion serA fr Expression box, and knock it out at the same time sdaB Genes were used to obtain strain SER-02. sdaA :: serA fr Δ sdaB The strain was named SER-03; (4) Knockout of genes in the SER-03 genome sstT and sdaC SER-03 Δ strain was obtained sstT Δ sdaC It was named strain SER-04; (5) Knockout in the genome of strain SER-04 glyA Genes, and in genes lafU Site insertion tdh Expression box, at the same time thrL Insert strong promoter Prom1 at the site to enhance thrABC Expression intensity, and in situ introduction of the strong promoter Prom2 for enhancement. gcvTHP The expression of SER-04Δ was obtained. glyA lafU :: tdh thrL :: Prom1 Prom2 - gcvTHP It was named strain SER-05.

6. The application of the engineered Escherichia coli strain of claim 1 in the fermentation production of L-serine.

7. The application as described in claim 6, characterized in that, The application method is as follows: the engineered Escherichia coli is inoculated into LB medium and cultured overnight at 37 °C and 200 rpm to obtain a pre-culture; the pre-culture is inoculated into a shake flask containing fermentation medium at a volume concentration of 1-5% and incubated at 37 °C. o Fermentation was carried out at 220 rpm for 48 h to obtain a fermentation broth containing L-serine. The fermentation medium consisted of the following: glucose 20 g / L, yeast extract 5 g / L, peptone 3 g / L, FeSO4 10 mg / L, KH2PO4 1.5 g / L, MgSO4 2 g / L, trace element mixture 1 mL / L, and V... B The mixed solution contains 1 mL / L of phenol red (8 mg / L), 2-3 drops of defoamer, and deionized water as the solvent, with a pH of 7.0-7.

2. The trace element mixed solution consists of: FeCl3 27 g / L, CoCl2·6H2O 2 g / L, Na2MoO4 2 g / L, CaCl2 24.6 g / L, CuSO4 1 g / L, H3PO3 0.5 g / L, AlCl3 10 g / L, deionized water as the solvent, and pH adjusted to 7.0 with 1 M HCl. B Mixture composition: V B1 V B3 V B5 V B12 Each 1 g / L, solvent is deionized water.

8. The application as described in claim 6, characterized in that, The fermentation was carried out using fed-batch culture in a fermenter: the engineered E. coli strain was activated and inoculated into a fermenter containing the initial fermentation medium. The fermentation conditions were set as follows: temperature 37℃, pH 7.0, dissolved oxygen 20-40%; when the bacterial OD... 600 When the concentration is 10-20 g / L, the fermentation broth is transferred to a new fermenter containing fermentation medium at a volume concentration of 10-20%, and fermentation continues under the same conditions. When the residual sugar concentration is below 1-5 g / L, feed medium is added, and the flow rate is controlled to keep the glucose concentration in the fermentation broth below 5-10 g / L. When the increase in L-serine production is no longer significant or even decreases, feed is stopped. Fermentation is stopped when the residual sugar concentration is below 2 g / L, yielding a fermentation broth containing L-serine. The initial fermentation medium consisted of: glucose 20 g / L, yeast extract 5 g / L, peptone 3 g / L, KH₂PO₄ 1.5 g / L, MgSO₄ 2 g / L, FeSO₄ 10 mg / L, and V. B The mixture was prepared at a concentration of 1 mg / L, using deionized water as the solvent, and the pH was adjusted to 7.0-7.

2. The fermentation medium consisted of: 20 g / L glucose, 4 g / L yeast extract, 2 g / L peptone, 5 g / L KH₂PO₄, 3 g / L MgSO₄, 1 mL / L trace element mixture, and V. B The mixed solution contained 0.5 mg / L FeSO4, 5 mg / L FeSO4, 10 mg / L MnSO4, and 2 mg / L biotin, in deionized water as the solvent; the trace element mixed solution consisted of: FeCl3 27 g / L, CoCl2·6H2O 2 g / L, Na2MoO4 2 g / L, CaCl2 24.6 g / L, CuSO4 1 g / L, H3PO3 0.5 g / L, and AlCl3 10 g / L, in deionized water as the solvent, with the pH adjusted to 7.0 using 1 M HCl; V B Mixture composition: V B1 V B3 V B5 V B12 Each 1 g / L, with deionized water as the solvent; the feeding medium is a 60% glucose aqueous solution.

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