CN117736959B - Engineering strains, preparation methods and applications of Zymomonas mobilis - Google Patents

Abstract

The application relates to the technical field of zymomonas mobilis, in particular to an engineering strain of zymomonas mobilis, a preparation method and application thereof. The engineering strain is obtained by knocking out the sdaA gene of zymomonas mobilis; the glyA gene is knocked out; eceamA gene is overexpressed; the serA gene is overexpressed; the serC gene is overexpressed; the serB gene is overexpressed; or a strain in which the pgk gene is overexpressed. The engineering strain can realize accumulation of L-serine while producing ethanol without dissolved oxygen control, can synthesize the two raw materials simultaneously, effectively reduces the production cost, and has wide application prospects in the synthesis of ethanol and L-serine and related fields.

Description

Engineering strains, preparation methods and applications of Zymomonas mobilis

Technical Field

The present application relates to the technical field of Zymomonas mobilis, and in particular to an engineered strain, a preparation method and application of Zymomonas mobilis.

Background technique

Zymomonas mobilis is a natural ethanol-producing facultative anaerobic Gram-negative bacterium with a unique ED metabolic pathway and high sugar fermentation efficiency. It also has the characteristics of an ideal industrial cell factory, such as high ethanol production, low product volume, strong ethanol tolerance, high osmotic pressure resistance, and no need for additional oxygen during the fermentation process. At present, the production and fermentation of products such as PHB, 2,3-butanediol, isobutanol and lactic acid have been achieved in Zymomonas mobilis. In addition, Zymomonas mobilis has a high tolerance to lignocellulosic hydrolysate, and cellulosic ethanol production has been commercialized. At the same time, the research on the mechanism of tolerance to inhibitors in hydrolysates is also relatively mature. In addition, through synthetic biology and metabolic engineering, Zymomonas mobilis can be transformed into chassis cells that use lignocellulosic hydrolysate to produce different platform compounds.

Summary of the invention

The present application discloses an engineered strain, preparation method and application of Zymomonas mobilis. The engineered strain can achieve the accumulation of L-serine while producing ethanol without dissolved oxygen control, can synthesize these two raw materials at the same time, effectively reduce the production cost, and has broad application prospects in the synthesis of ethanol and L-serine and related fields.

Based on this, the embodiments of the present application disclose at least the following technical solutions:

In a first aspect, the embodiment discloses an engineered strain of Zymomonas mobilis. The engineered strain is a strain in which at least one of the sdaA gene of the Zymomonas mobilis is knocked out, the glyA gene is knocked out, the EceamA gene is overexpressed, the SerA gene is overexpressed, the SerC gene is overexpressed, the SerB gene is overexpressed, or the pgk gene is overexpressed.

In a second aspect, the embodiment discloses a method for preparing an engineered strain of Zymomonas mobilis. The preparation method comprises: obtaining Zymomonas mobilis ZM4 as a starting strain; obtaining a first knockout plasmid targeting the sdaA gene, wherein the first knockout plasmid knocks out the sdaA gene of the Zymomonas mobilis; obtaining a second knockout plasmid targeting the glyA gene, wherein the second knockout plasmid knocks out the glyA gene of the Zymomonas mobilis; and transferring at least one of the first knockout plasmid and the second knockout plasmid into the Zymomonas mobilis to obtain the engineered strain.

In a third aspect, an embodiment discloses a method for preparing L-serine, comprising: obtaining the engineered strain described in the first aspect or the engineered strain prepared by the preparation method described in the second aspect; fermenting the engineered strain; and harvesting the L-serine from the fermentation product.

In the fourth aspect, the embodiment discloses the application of the engineered strain described in the first aspect or the engineered strain prepared by the preparation method described in the second aspect. The application is selected from at least one of the following: synthesis of L-serine; synthesis of cycloserine; chiral resolution of serine; preparation of food additives; or preparation of nutritional supplements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing fermentation test results of the ZM4 strain provided in the example and the engineered strains S01 and S02 obtained by genome modification using the ZM4 strain as the starting strain.

Figure 2 is a schematic diagram of the structures of the overexpression plasmids 39-B1, 39-B2 and 39-B3 provided in the examples, a schematic diagram of the construction of the engineering strains S02B1, S02B2 and S02B3, and a diagram of the fermentation test results of the engineering strains S02, S02B1, S02B2 and S02B3.

Figure 3 is a schematic diagram of the construction of overexpression plasmids pEZ-A1, pEZ-A2, pEZ-A3, pEZ-A4, engineered strains S02A1B3, S02A2B3, S02A3B3 and S02A4B3 provided in the examples, and a diagram of the fermentation test results of the engineered strains S02A1B3, S02A2B3, S02A3B3 and S02A4B3.

Figure 4 is an amino acid sequence comparison diagram of the PGDH enzyme of Escherichia coli and the PGDH enzyme from Zymomonas mobilis provided in the example (A), a schematic diagram of the three-dimensional structure of the PGDH enzyme mutant (B), a schematic diagram of the structure of the overexpression plasmids pEZ-A4, pEZ-A5, pEZ-A6, pEZ-A7, pEZ-A8, pEZ-A9 and pEZ-A10 and the corresponding fermentation test results of the engineering strains S02A4B3, S02A5B3, S02A6B3, S02A7B3, S02A8B3, S02A9B3 and S02A10B3 (C), and a structural diagram of the Tc growth rate, glucose consumption and ethanol yield of the engineering strain S02A9B3 (D).

FIG5 is a schematic diagram of the structures of the overexpression plasmids pEZ-A9 and 39-B4 provided in the examples, and the fermentation test results obtained by the engineered strain S02A9B4.

6 is a schematic diagram of the structure of the overexpression plasmid 39-B5 provided in the example and the fermentation test results of the engineering strain S02A9B5 (A), as well as a structural diagram of the Tc growth rate, glucose consumption and ethanol production of the S02A9B5 engineering strain (B).

FIG. 7 is a diagram showing the fermentation test results of the engineered strain S02A9B5 provided in the example under the condition of supplementing nitrogen source.

FIG8 is a schematic diagram of the genome structure comparison of ZM4, engineered strain S02, and engineered strain S02 provided in the examples.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail in conjunction with the examples below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

Serine is a neutral aliphatic polar α-amino acid and a non-essential amino acid. It plays a vital role in biological growth, including one-carbon unit metabolism (one-carbon unit is an important material for synthesizing nucleotides), protein synthesis, purine and pyrimidine synthesis, and the production and processing of cell membranes. As one of the thirty most attractive skeleton compounds in the chemical industry, L-serine is widely used in medical, cosmetics, food, scientific research and other fields. It has a broad market and application prospects, and the annual market demand growth rate is estimated to be 7-10%.

The most widely used methods for producing L-serine are whole-cell catalysis, enzyme catalysis and microbial fermentation. Both whole-cell catalysis and enzyme catalysis require expensive precursors such as glycine as substrates, and methanol as a one-carbon compound supplement. The production cost is relatively expensive and will cause environmental pollution. Therefore, using microorganisms to produce serine from renewable raw materials is more environmentally friendly, more efficient and more attractive. The biosynthesis of L-serine utilizes the metabolism of microorganisms themselves to synthesize intracellularly and is excreted to the extracellular space through transport proteins. However, most microbial fermentation processes for producing L-serine require oxygen and require a large amount of energy to supply oxygen. In addition, oxygenating the fermentation device will increase the continuous production cost and cause a waste of the effective volume of the bioreactor device.

The term "engineered strain" refers to a strain obtained by genetically engineering a wild-type strain, such as modifying its genome, introducing an overexpression plasmid into it, and other technical means. For example, a strain obtained by knocking out or overexpressing a gene in a genome of a wild-type Zymomonas mobilis, introducing an overexpression plasmid into it, and other technical means is called an "engineered strain."

However, in the first aspect, the embodiment discloses an engineered strain of Zymomonas mobilis. The engineered strain is a strain in which at least one of the sdaA gene of the Zymomonas mobilis is knocked out; the glyA gene is knocked out; the EceamA gene is overexpressed; the SerA gene is overexpressed; the SerC gene is overexpressed; the SerB gene is overexpressed; or the pgk gene is overexpressed.

In some embodiments, the engineered strain is a strain in which the sdaA gene and the glyA gene of the Zymomonas mobilis are knocked out. The engineered strain in which the sdaA and glyA genes of Zymomonas mobilis are knocked out blocks the two main degradation pathways of L-serine in the strain, thereby achieving the accumulation of L-serine.

In some embodiments, the engineered strain is an engineered strain in which the sdaA gene of the mobilis Zymomonas is knocked out, the glyA gene is knocked out, and the EceamA gene is overexpressed. In order to enhance the extracellular transport and tolerance of L-serine by mobilis Zymomonas, the transporter gene EceamA in Escherichia coli is introduced into the engineered strain to achieve the transport and tolerance of L-serine.

In some embodiments, the engineered strain is a strain of the Zymomonas mobilis in which the sdaA gene is knocked out, the glyA gene is knocked out, the EceamA gene is overexpressed, the SerA gene is overexpressed, the SerC gene is overexpressed, and the SerB gene is overexpressed. The engineered strain is overexpressed with endogenous genes SerA , SerC , and SerB , which can enhance the synthesis of L-serine.

In some embodiments, the engineered strain is a strain of the Zymomonas mobilis in which the sdaA gene is knocked out, the glyA gene is knocked out, the EceamA gene is overexpressed, the SerA gene is overexpressed, the SerC gene is overexpressed, the SerB gene is overexpressed, and the pgk gene is overexpressed. The engineered strain is overexpressed to enhance the precursor-related gene pgk , which can enhance the synthesis of L-serine.

In a second aspect, the embodiment discloses a method for preparing an engineered strain of Zymomonas mobilis. The preparation method comprises: obtaining Zymomonas mobilis ZM4 as a starting strain; obtaining a first knockout plasmid targeting the sdaA gene, wherein the first knockout plasmid knocks out the sdaA gene of the Zymomonas mobilis; obtaining a second knockout plasmid targeting the glyA gene, wherein the second knockout plasmid knocks out the glyA gene of the Zymomonas mobilis; and transferring at least one of the first knockout plasmid and the second knockout plasmid into the Zymomonas mobilis to obtain the engineered strain.

In some embodiments, the preparation method includes transferring the first knockout plasmid and the second knockout plasmid into the mobilis Zymomonas to obtain the engineered strain. Thus, the engineered strain of mobilis Zymomonas sdaA and glyA genes is knocked out to block the two main degradation pathways of L-serine in the strain, thereby achieving the accumulation of L-serine.

In some embodiments, the preparation method further comprises obtaining a third overexpression plasmid that overexpresses the EceamA gene. The third overexpression plasmid is transferred into an engineered strain in which the sdaA and glyA genes are knocked out, thereby obtaining an engineered strain in which the sdaA gene is knocked out, the glyA gene is knocked out, and the EceamA gene is overexpressed. In this way, the engineered strain can improve its ability to transport and tolerate L-serine.

In some embodiments, the preparation method further comprises obtaining a fourth overexpression plasmid of overexpression elements of SerA gene, SerC gene and SerB gene; and transferring the third overexpression plasmid and the fourth overexpression plasmid into an engineered strain in which the sdaA gene is knocked out, the glyA gene is knocked out and the EceamA gene is overexpressed. In this way, the engineered strain can overexpress SerA , SerC and SerB , and can enhance the synthesis of L-serine.

In some embodiments, the preparation method further comprises obtaining a fifth overexpression plasmid that overexpresses the pgk gene, and transferring the fifth overexpression plasmid into an engineered strain in which the sdaA gene is knocked out, the glyA gene is knocked out, the EceamA gene is overexpressed, and SerA , SerC , and SerB are overexpressed. The engineered strain obtained in this way overexpresses the enhanced precursor-related gene pgk , and can enhance the synthesis of L-serine.

In a third aspect, an embodiment discloses a method for preparing L-serine, which comprises obtaining the engineered strain described in the first aspect or the engineered strain prepared by the preparation method described in the second aspect; fermenting the engineered strain; and harvesting the L-serine from the fermentation product.

In some embodiments, the method for preparing L-serine further comprises fermenting the engineered strain in a culture solution supplemented with a nitrogen source.

In the fourth aspect, the embodiment discloses the application of the engineered strain described in the first aspect or the engineered strain obtained by the preparation method described in the second aspect. Since L-serine can be used as a biochemical reagent and food additive; a nutritional supplement, for example, as a skin nutritional additive in cosmetics; it can be used for biochemical and nutritional research, and can also be used as a raw material for synthesizing cycloserine. Therefore, the application provided in the embodiment is selected from at least one of the following: synthesizing L-serine; synthesizing cycloserine; chiral resolution of serine; preparing food additives; or preparing nutritional supplements.

Preparation of engineered strains with sdaA and/or glyA knockout

In some embodiments, the endogenous I-F CRISPR-Cas gene editing system of Zymomonas mobilis is used to knock out the L-serine dehydratase and hydroxymethylserine transferase (SHMT) encoded by the sdaA and glyA genes in the Zymomonas mobilis genome to block the degradation of L-serine by Zymomonas mobilis to produce pyruvate and glycine. In this way, the engineered strain S01 (ΔsdaA, sdaA is knocked out) with sdaA knocked out and the engineered strain S02 (ΔsdaAΔglyA, sdaA and glyA are knocked out) with glyA knocked out. As shown in Figure 1, the growth delay period of the engineered strains S01 and S02 increased by about 3h, the final biomass remained unchanged, and the accumulation of L-serine increased from 15.3 mg/L to 50.7 mg/L.

In some embodiments, knocking out sdaA is achieved by transferring a first knockout plasmid into Zymomonas mobilis to obtain an engineered strain S01.

In some embodiments, the knockout of glyA is achieved by transferring a second knockout plasmid into the engineered strain S01 to obtain the engineered strain S02.

In some embodiments, the first knockout plasmid carries a first targeting element and a first donor sequence. The first targeting element includes two repeating sequences as shown in SEQ ID NO.1 and a first guide sequence as shown in SEQ ID NO.2 located between the two repeating sequences. The first targeting element targets the sdaA gene. The first donor sequence is composed of a 600bp sequence upstream of the sdaA gene and a 600bp sequence downstream of the sdaA gene.

In some embodiments, the second knockout plasmid carries a second targeting element and a second donor sequence. The second targeting element includes two repeating sequences as shown in SEQ ID NO.1 and a second guide sequence as shown in SEQ ID NO.3 located between the two repeating sequences. The second targeting element targets the glyA gene. The second donor sequence is composed of a 600bp sequence upstream of the glyA gene and a 600bp sequence downstream of the sdaA gene.

In some embodiments, the steps of preparing an engineered strain with sdaA and/or glyA knockout include:

1. Preparation of the first and second knockout plasmids

1) First and second guide sequences

A sequence 32 bp downstream of the PAM site CCC site was selected from the sdaA gene in the ZM4 genome as the first guide sequence, as shown in SEQ ID NO. 2. A sequence 32 bp downstream of the PAM site CCC site was selected from the glyA gene in the ZM4 genome as the second guide sequence, as shown in SEQ ID NO. 3.

2) The first and second targeting plasmids

In some embodiments, a first targeting element comprising a first guide sequence is inserted into a base plasmid to generate a first targeting plasmid that targets the sdaA gene.

In some embodiments, a targeting primer sequence (grsdaA-F, shown in SEQ ID NO.4; grsdaA-R (shown in SEQ ID NO.5) is designed according to the first guide sequence to guide the nucleotidase to cut the targeting site; the targeting is annealed and connected to the basic plasmid (pEZ15Asp), and the first targeting plasmid is obtained by screening.

Some methods for constructing the first targeting plasmid specifically include:

The vector pEZ15Asp was digested with restriction endonuclease Bsa Ⅰ to obtain linearized pEZ15Asp; then the primers of the first guide sequence (grsdaA-F, grsdaA-R) were annealed according to the system shown in Table 1 (the annealing system contained 1µL grsdaA-F (10μM), 1µL grsdaA-R (10μM) and 8µL ddH2O in 10µL), denatured at 95°C for 5min, and cooled to room temperature for use; the annealing product (i.e., the first guide sequence) and the linearized pEZ15Asp were ligated at 22°C for 3-6h using T4 ligase according to the system shown in Table 2; the ligation product was transferred into the Escherichia coli cloning strain DH5α for plasmid construction using a common chemical transformation method in the art; the single colony was selected for screening using a spectinomycin plate, and verified by colony PCR using primers pEZ15A-F (shown in SEQ ID NO.6) and pEZ15A-R (shown in SEQ ID NO.7), respectively. The expected band size was verified by sequencing. The T4 reaction system contained 20-40ng linearized pEZ15Asp, 2µL first guide sequence, 0.5µL T4 ligase, 1µL buffer and the balance of ddH ₂ O in 10µL. The reaction program of colony PCR was pre-denaturation at 98℃ for 3min, 1 cycle; denaturation at 98℃ for 10s, annealing at 55℃ for 10s, extension at 72℃, set at 10s/kb according to the fragment length, for a total of 30 cycles; extension at 72℃ for 2min, 1 cycle; storage at 12℃ for 2min, 1 cycle.

In some examples of this embodiment, the spectinomycin knockout plasmid vector (pEZ15Asp) is a spectinomycin encoding gene inserted into pEZ15A as a marker gene. Wherein, pEZ15A is obtained with reference to "Yang S, Mohagheghi A, Franden MA, et al. Metabolic engineering of Zymomonas mobilis for 2,3-butanediolproduction from lignocellulosic biomass sugars [J]. Biotechnol Biofuels, 2016, 9 (1): 189." method. To obtain pEZ15A encoding different genes (such as resistance genes), different marker genes can be inserted therein with reference to "Construction and Application of Plasmid pUC19-CM-D [J]. Anhui Agricultural Science, 2010, Public Methods, Issue 19".

In some embodiments, a second targeting element comprising a second guide sequence is inserted into a base plasmid to obtain a second targeting plasmid, and the first targeting plasmid targets the glyA gene. In some embodiments, a primer (grglyA-F, shown in SEQ ID NO.8; grglyA-R, shown in SEQ ID NO.9) targeting the second guide sequence of the glyA gene is connected to a spectinomycin knockout plasmid vector (pEZ15Asp) containing a CRISPR-IF expression unit to obtain a second targeting plasmid. The preparation process of the second targeting plasmid is the same as that of the first targeting plasmid, and will not be repeated. Among them,

3) Construction of the first knockout plasmid (pL2R-sdaA) and the second knockout plasmid (pL2R-glyA)

In some embodiments, the steps of preparing the first knockout plasmid include:

1) The upstream sequence of the sdaA gene was amplified using primers sdaAUS-F (shown in SEQ ID NO.10) and sdaAUS-R (shown in SEQ ID NO.11). The downstream sequence of the sdaA gene was amplified using primers sdaADS-F (shown in SEQ ID NO.12) and sdaADS-R (shown in SEQ ID NO.13).

2) The sdaA upstream sequence and the sdaA downstream sequence were sequentially connected by Overlap PCR to serve as the first donor fragment (shown in SEQ ID NO. 14).

3) The first targeting plasmid constructed in the previous step was amplified by reverse PCR using primers 15Afk-F (shown in SEQ ID NO.15) and 15Afk-R (shown in SEQ ID NO.16) to obtain the reverse-amplified first targeting plasmid.

4) The first target plasmid and the first donor fragment of the reverse amplification were Gibson assembled and ligated at a ratio of 1:3, and the ligation product was transferred into E. coli competent cells. Screening was performed using spectinomycin plates, and single colonies were picked and verified by colony PCR using primers pEZ15A-F (shown in SEQ ID NO.6) and pEZ15A-R (shown in SEQ ID NO.7), respectively. The band size was consistent with the expected one, and was verified by sequencing. The first knockout plasmid was extracted and isolated from the verified positive clones.

The preparation method of the second knockout plasmid (pL2R-glyA) is the same as the preparation method of the first knockout plasmid. The sequences involved are: glyAUS-F, shown in SEQ ID NO.17. glyAUS-R: shown in SEQ ID NO.18. glyADS-F: shown in SEQID NO.19. glyADS-R: shown in SEQ ID NO.20. The second donor fragment, shown in SEQ ID NO.21, is formed by connecting the glyA upstream sequence and the glyA downstream sequence.

2. Electroporation of the first and second knockout plasmids

Add 1 μg of the first knockout plasmid or the second knockout plasmid to 50 μL competent ZM4 competent S01, mix well and add to a 0.1 cm electroporation cup, and perform electroporation according to the program of 1800V, 25 μF, 200Ω. After electroporation, transfer it to 1 mL RM medium and culture it in a 30°C incubator for 4-6 hours, then take 200 μL and evenly spread it on a 100 μg/ml spectinomycin resistance plate, invert it and culture it in a 30°C incubator for 2-3 days.

3. Screening of engineered strains S01/S02

After colonies grew out, the engineered strains were tested by colony PCR using verification primers sdaAcheck-F (shown in SEQ ID NO.22) and sdaAcheck-R (shown in SEQ ID NO.23). Strains with band sizes consistent with the expected ones were verified by sequencing, and the correct strains were saved for later use and named S01.

The screening of strain S02 was the same as that of strain S01, wherein the screening primers involved were: glyAcheck-F, shown in SEQ ID NO.24; glyAcheck-R, shown in SEQ ID NO.25.

4. Fermentation test of engineered strains S01 and S02

(1) Growth and fermentation performance test of engineered strains:

The L-serine fermentation performance of the engineered strains S01 and S02 described in the above examples at different initial glucose concentrations was tested. The engineered strains S01 and S02 were inoculated into 50 mL conical flasks of RMG5 with a bottling volume of 80%, and 5 g/L of glutamic acid hydrochloride was added, and the mixture was placed in a 30 °C shaker at a shaking speed of 100 rpm for culture. Samples were taken at regular intervals during the fermentation process for HPLC testing, and the samples at different time points were centrifuged and the supernatant was taken (12000 rpm, 2 min) as test samples.

RMG5 enriched medium contained 50 g/L glucose, 10 g/L yeast extract, and 2 g/L KH ₂ PO ₄ .

(2) Detection of fermentation supernatant

1) Detection of glucose, ethanol and acetic acid

Agilent 1100 series high performance liquid chromatograph (LC-20AD) of Shimadzu Trading Co., Ltd. was used; the detector was a differential refractive index detector (RID-10A); the chromatographic column was an organic acid column (Bio-Rad Aminex HPX-87H, 300 mm×7.8 mm); the pool temperature was 40℃, and the column oven temperature was 60℃; the mobile phase was 5 mM sulfuric acid, the flow rate was 0.5 mL/min, and the initial flow rate was set to 0.2 mL/min during instrument operation. After the column pressure stabilized, the flow rate was gradually increased to 0.5 mL/min at 0.1 mL/min; the injection volume was 20 μL. Configuration of mobile phase: 1.41 mL of chromatographic grade concentrated sulfuric acid was taken into a 5 L blue-capped bottle, and the volume was made up to 5 L with ultrapure water, mixed evenly, and filtered using a 0.45 μm pore size aqueous phase filter membrane. The filtered mobile phase was dispensed into a 1 L mobile phase blue-capped bottle for ultrasonic degassing for 20-30 min. It can be used after returning to room temperature.

2) Detection of L-serine

L-serine cannot be detected directly. It needs to react with O-phenylenediaminoketone (OPA) before HPLC injection to form a stable light-absorbing OPA-L-serine derivative. This derivative has strong UV absorption and can be detected and quantified by the UV detector in the HPLC system. L-serine detection was analyzed using SPD-20A through an Agilent Advance bio AAA amino acid analysis column (Agilent, DE, USA), and pre-column derivatization was performed using an Agilent amino acid analysis package. Mobile phase A was 10 mM Na ₂ HPO ₄ and 10 mM Na ₂ B ₄ O ₇ to control the pH to 8.2. Mobile phase B was 45% methanol and 45% acetonitrile. The column temperature was controlled at 50°C and the flow rate was 1.5 ml/min. The mobile phase ratio program was: start A:B (98:2, v/v), 0.35 min A:B (98:2, v/v), 13.4 min A:B (43:57, v/v), 13.5 min A:B (0:100, v/v), 15.7 min A:B (0:100, v/v), 15.8 min A:B (98:2, v/v), and 20 min end program.

D-serine was detected by pre-column derivatization with 260 mM N-isobutyryl-L-cysteine (IBLC reagent) and 170 mM O-phenylenediaminoketone (OPA) and determined by reverse phase detection on an Agilent Advance bio AAA amino acid analysis column. The product was tested to be free of D-serine. The mobile phases used were as follows: Phase A: 50 mM sodium acetate (pH 6.0) Phase B: 45% methanol 45% acetonitrile. The column temperature was controlled at 30°C and the flow rate was 0.7 ml/min. The mobile phase ratio program was: 0 - 2.0 min, 4 %B, 2.0 - 4.0 min, 10%B, 4.0 - 15 min, 20%B, 15 -27 min, 35 %B, 27 - 35 min, 50%B, 35 -37 min, 100 %B, 37 - 42 min, 100 %B, and the program ended.

(2) Results

As shown in Figure 1, the growth delay period of the engineered strains S01 and S02 increased by about 3 h, the final biomass remained unchanged, and the accumulation of L-serine increased from 15.3 mg/L to 50.7 mg/L.

Preparation of engineered strains with introduced transporter proteins

The L-serine transporters that have been found and applied so far include EceamA (NC_000913.3) in Escherichia coli and CgthrE (NC_022040.1) and Cg0580 (NC_021352.1) in Corynebacterium glutamicum. However, there is no gene encoding L-serine transporter in Zymomonas mobilis, and no homologous genes have been found.

Based on this, in the embodiment, CgthrE , Cg0580 and EceamA were amplified from the genomes of Escherichia coli and Corynebacterium glutamicum, respectively, and overexpression plasmids 39p-B1, 39p-B2 and 39p-B3 were constructed using the Ptet promoter, which were respectively electroporated into the S02 strain to obtain the engineered strain S02B1 overexpressing CgthrE, the engineered strain S02B2 overexpressing Cg0580 and the engineered strain S02B3 overexpressing EceamA.

As shown in Figure 2, after tolerance testing, it was found that the engineered strain S02B3 overexpressing EceamA had the highest growth and L-serine accumulation in the presence of 12 g/L RMG5.

In some embodiments, the method for constructing the engineered strain S02B3 overexpressing EceamA comprises:

1. Construction of the third overexpression plasmid (39p-B3)

The EceamA sequence was obtained by PCR amplification of the E. coli genome using primers (EceamA-F, shown in SEQ ID NO.26; EceamA-R, shown in SEQ ID NO.27). The Zymomonas mobilis ZM4 genome was amplified by PCR using primers (Ptet-F, shown in SEQ ID NO.29; Ptet-R, shown in SEQ ID NO.30) to obtain the Ptet strong promoter sequence (shown in SEQ ID NO.28).

The vector pEZ15A was reversely amplified using primers 15Afk-F (shown in SEQ ID NO. 15) and 15Afk-R (shown in SEQ ID NO. 16) to obtain linearized pEZ15A.

These PCR amplification reactions contained 0.5 μL of 10 μM upstream and downstream primers, 10 μL PrimerSTARDNA Polymerase (Takara), 5-10 ng template and the balance of ddH2O in a 20 μL volume. The PCR amplification program was set as follows: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 10 s, 72°C extension (set at 10 s/kb according to the fragment length), for a total of 30 cycles; 72°C was maintained for 5 min after the cycle reaction; the product was purified and stored at -20°C.

The obtained fragment and linearized pEZ15A were mixed at a ratio of 3:1. After the reaction system was prepared according to the table below, it was left to stand on ice for 5 minutes, and then E. coli competent cells were added and transformed using the general method. Screening was performed using spectinomycin resistance plates, and single colonies were picked. The primer pairs 15A-fwd (shown in SEQ ID NO.31) and 15A-rev (shown in SEQ ID NO.32) 1 were used for verification by colony PCR (the PCR amplification program was set as follows: 98℃ pre-denaturation for 3min; 98℃ denaturation for 10 s, 55℃ annealing for 10 s, and 72℃ extension for 80 s, for a total of 30 cycles). The band size was consistent with the expected one, and was verified by sequencing. That is, the third overexpression plasmid 39p-B3 of EceamA was extracted and separated from the verified positive clones. The ligation reaction system of the target fragment of the EceamA gene and the plasmid pEZ15A contained 0.12 pM of the target fragment of the EceamA gene, 0.04 pM of the plasmid pEZ15A, 0.5 μL of 10×Buffer 4 (Thermo), 0.5 UT5 Exonuclease and the balance of ddH ₂ O in 5 μL.

2. Obtaining the engineered strain S02B3 overexpressing EceamA

Prepare competent cells of Zymomonas mobilis S02 obtained in the above example, take out the frozen bacteria from the -80℃ refrigerator, take 100 μL and inoculate in a cryovial containing 1 mL RMG5, and place it in a 30℃ incubator to activate the strain. After culturing until turbidity, transfer it to a 250 mL blue-capped bottle containing 200 mL RMG5 liquid culture medium, so that the initial OD 600nm is in the range of 0.025~0.3, place it in a 30℃ incubator, and when OD 600nm exceeds 0.3, collect the bacteria at room temperature at 100 rpm, then wash once with sterile water, wash twice with 10% glycerol, and finally slowly resuspend the bacteria with 1~2mL 10% glycerol, and dispense 55 μL competent cells into 1.5 mL EP tubes.

Add 1 mg of the third overexpression plasmid 39p-B3 to the electroporation cup containing competent cells and to the 1.5 mL EP tube containing 55 μL competent cells, mix gently and transfer to the 1 mm electroporation cup. The electroporation instrument program settings: 200Ω, capacitance: 25 μF, voltage: 1.6 KV. Place the electroporation cup in the electroporation instrument for electroporation. Immediately after electroporation, add 1 mL of RMG5 liquid culture medium, mix well and transfer to a sterile EP tube, seal it with a sealing film and incubate it in a 30 ℃ constant temperature incubator for 4~6 h. Take 100 μL of bacterial solution and evenly spread it on the RMG5+Spe plate (100 μg/mL spectinomycin). Seal the plate with a sealing film and place it in an inverted culture in a 30 ℃ incubator. Colony PCR verification: After a single colony grows on the plate, PCR verification of the single colony is performed using 15A-fwd/15A-rev primers. The PCR system and PCR procedure are the same as the above colony PCR. The correct positive clones were activated in the RMG5+Spe medium and maintained in glycerol stock.

In some embodiments, the construction methods of the overexpression plasmid 39p-B1 of CgthrE and the overexpression plasmid 39p-B2 of Cg0580 are the same as 39p-B1, respectively. The primers involved are: CgthrE-F, shown in SEQ ID NO.33; CgthrE-R, shown in SEQ ID NO.34; Cg0580-F, shown in SEQ ID NO.35; Cg0580-R, shown in SEQ ID NO.36.

Engineered strains overexpressing SerA1, SerC and SerB genes

L-serine is synthesized from 3-phosphoglycerate (3-PGA) in Zymomonas mobilis by three enzymes: phosphoglycerate dehydrogenase (PGDH, encoded by SerA), phosphoserine aminotransferase (PSAT, encoded by SerC), and phosphoserine phosphatase (PSP, encoded by SerB).

1. Construction of the fourth overexpression plasmid (pEZ-A1~4) and the corresponding engineering strain

In some embodiments, the strong promoter Pgap (shown in SEQ ID NO.37), Peno (shown in SEQ ID NO.38), P_ZMO1980 (shown in SEQ ID NO.39) and the inducible promoter Ptet are used to control the expression of three genes SerA1, SerC, and SerB in the L-serine synthesis pathway. Four plasmids pEZ-A1, pEZ-A2, pEZ-A3, and pEZ-A4 were constructed and electroporated into the competent cells of the S02B3 strain to construct four engineering strains S02A1B3, S02A2B3, S02A3B3, and S02A4B3. The specific preparation process of the four plasmids pEZ-A1, pEZ-A2, pEZ-A3, and pEZ-A4 refers to the preparation process of 39p-B3.

Among them, the pEZ-A1 plasmid is an overexpression plasmid obtained by inserting the expression elements of Pgap, SerA1, RBS, SerC, RBS, and SerB connected in sequence into the vector pEZ15A. The pEZ-A2 plasmid is an overexpression plasmid obtained by inserting the expression elements of Peno, SerA1, RBS, SerC, RBS, and SerB connected in sequence into the vector pEZ15A. The pEZ-A3 plasmid is an overexpression plasmid obtained by inserting the expression elements of P_ZMO1980, SerA1, RBS, SerC, RBS, and SerB connected in sequence into the vector pEZ15A. The pEZ-A4 plasmid is an overexpression plasmid obtained by inserting the expression elements of Ptet, SerA1, RBS, SerC, RBS, and SerB connected in sequence into the vector pEZ15A.

The primers involved are: serA1-F, shown in SEQ ID NO.40; serA1-R, shown in SEQ ID NO.41; serC-F, shown in SEQ ID NO.42; serC-R, shown in SEQ ID NO.43; serB-F, shown in SEQ ID NO.44; serB-R, shown in SEQ ID NO.45.

Taking the construction of S02A4B3 as an example, the construction method includes: using primers to amplify the endogenous serA1, serC and serB genes from the ZM4 genome, and using the pEZ15A plasmid containing the Ptet promoter as a template to reversely amplify the vector. Finally, the three gene fragments are overlapped and connected into a large fragment, and finally connected to the vector, and transformed into Escherichia coli competent DH5α competent cells, screened using spectinomycin resistance (100 μg/mL) plates, picking single colonies, and verifying them by PCR using appropriate primers. The correct transformant pEZ-A4 is obtained. Then it is electroporated into the competent state of the S02B3 strain to obtain the S02A4B3 engineered strain.

As shown in FIG3 , fermentation tests on the four engineered strains S02A1B3, S02A2B3, S02A3B3, and S02A4B3 showed that the engineered strain S02A4B3 had the highest L-serine production of 260.3 mg/L.

2. Construction of the fourth overexpression plasmid (pEZ-A5~10) and the corresponding engineering strain

The pEZ-A5 plasmid is an overexpression plasmid obtained by inserting the expression elements of Ptet, BlSerA1, RBS, SerC, RBS, and SerB connected in sequence into the vector pEZ15A. BlSerA1 is the endogenous SerA of Bacillus licheniformis DW2 (Bacillus licheniformis), and the sequence is shown in SEQ ID NO.58. The primers involved are: BlserA1-F, shown in SEQ ID NO.46; BlserA1-R, shown in SEQ ID NO.47.

The pEZ-A6 plasmid is an overexpression plasmid obtained by inserting the expression elements of Ptet, BsSerA1, RBS, SerC, RBS, and SerB connected in sequence into the vector pEZ15A. BsSerA1 is the endogenous SerA of Bacillus subtilis 168, and the sequence is shown in SEQ ID NO.59. The primers involved are: BsserA1-F, shown in SEQ ID NO.48; BsserA1-R, shown in SEQID NO.49.

The pEZ-A7 plasmid is an overexpression plasmid obtained by inserting the expression elements of Ptet, EcSerAmut, RBS, SerC, RBS, and SerB sequentially connected into the vector pEZ15A. EcSerAmut is derived from Escherichia coli. Refer to "Mundhada, H., Schneider, K., Christensen, H.B., Nielsen, A.T., 2016. Engineering of highyield production of L-serine in Escherichia coli. Biotechnol. Bioeng. 113(4), 807-816." The primers involved are EcserA1mut1-F, shown in SEQ ID NO.50; EcserA1mut1-R, shown in SEQ ID NO.51.

The pEZ-A8 plasmid is an overexpression plasmid obtained by inserting the expression elements of Ptet, CgSerAmut, RBS, SerC, RBS, and SerB sequentially connected into the vector pEZ15A. CgSerAmut is derived from Corynebacterium glutamicum. Refer to "Zhang, X., Gao, Y., Chen, Z., Xu, G., Zhang, X., Li, H., Shi, J., Koffas, M.A.G., Xu, Z., 2020. High-yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacteriumglutamicum. Microb. Cell Fact. 19(1), 115." The primers involved are CgSerAmut-F, shown in SEQ ID NO.52; CgSerAmut-R, shown in SEQ ID NO.53.

In some embodiments, the phosphoglycerate dehydrogenase PGDH encoding gene derived from Zymomonas mobilis ZM4 is subjected to directed mutation as shown in FIG. 4A , that is, the amino acids at positions 475, 477, and 495 are mutated to alanine, and the mutant sequences ZmserAmut1 (shown in SEQ ID NO.54) and ZmserAmut2 (shown in SEQ ID NO.55) are obtained, and the phosphoglycerate dehydrogenase PGDH with the molecular structure shown in FIG. 4B is obtained. In this way, the two mutants ZmSerAmut1 and ZmSerAmut2 can relieve the feedback inhibition of L-serine of the wild-type gene SerA. The sequences of ZmserAmut1 and ZmserAmut2 were synthesized by Qingke Company, thereby constructing the overexpression plasmids pEZ9 and pEZ10 of ZmSerAmut1 and ZmSerAmut2.

The pEZ-A9 plasmid is an overexpression plasmid obtained by inserting the expression elements of Ptet, ZmSerAmut1, RBS, SerC, RBS, and SerB connected in sequence into the vector pEZ15A. The primers involved are: ZmSerAmut1-F, shown in SEQ ID NO.60, and ZmSerAmut1-R, shown in SEQ ID NO.61.

The pEZ-A10 plasmid is an overexpression plasmid obtained by inserting the expression elements of Ptet, ZmSerAmut2, RBS, SerC, RBS, and SerB connected in sequence into the vector pEZ15A. The primers involved are: ZmSerAmut2-F, shown in SEQ ID NO.62, and ZmSerAmut2-R, shown in SEQ ID NO.63.

pEZ-A4, pEZ-A5, pEZ-A6, pEZ-A7, pEZ-A8, pEZ-A9 and pEZ-A10 were respectively transferred into the engineering strain S02B3, and the engineering strains S02A4B3, S02A5B3, S02A6B3, S02A7B3, S02A8B3, S02A9B3 and S02A10B3 were obtained in sequence. These engineering strains were subjected to fermentation tests, and as shown in Figures 4C and 4D, the engineering strain S02A9B3 had the highest L-serine production (536.7 mg/L) and the highest ethanol production (20.4 g/L).

Among them, the construction methods of overexpression plasmids pEZ-A1, pEZ-A2, pEZ-A3, pEZ-A4, pEZ-A5, pEZ-A6, pEZ-A7, pEZ-A8, pEZ-A9 and pEZ-A10 can refer to the preparation process of 39p-B3.

Enhanced expression of SerB using a strong promoter and increased copy number

As shown in FIG5 , based on the above examples, an EcSerA sequence with a strong promoter Ptet and a SerB sequence with a strong promoter Pgap were synthesized and inserted into the vector pEZ15A to obtain an overexpression plasmid 39p-B4. The specific preparation process refers to the preparation process of 39p-B3.

In some embodiments, the overexpression plasmid 39p-B4 and the overexpression plasmid pEZ-A9 were simultaneously transferred into the engineered strain S02B4 to obtain the engineered strain S02A4B4-1. As shown in FIG5, after fermentation test, the engineered strain S02A4B3-1 had an L-serine yield of 625.6 mg/L.

Overexpression of pgk enhances the production of precursor 3-phosphoglycerate

Phosphoglycerate kinase (encoded by pgk, AVZ41530.1) catalyzes 3-phosphoglyceraldehyde to 3-phosphoglycerate. Overexpression of pgk can enhance carbon flow to 3-phosphoglycerate.

Based on this, as shown in Figure 6, the embodiment uses Pgk-F (shown in SEQ ID NO.56) and Pgk-R (shown in SEQ ID NO.57) to amplify the pgk sequence from the genome of Zymomonas mobilis ZM4 and insert it into the overexpression plasmid 39p-B4 to obtain the fifth overexpression plasmid (39p-B5). The specific preparation process refers to the preparation process of 39p-B3.

In some embodiments, the overexpression plasmid 39p-B5 is electrotransformed into the engineered strain S02A9B5 in the competent state of the S02A9 strain. The test L-serine yield is shown in Figure 6A, which is 687.6 mg/L. The consumption of glucose, the production of ethanol and the production of acetic acid are shown in Figure 6B. The production of acetic acid is reduced from 1.08 g/L to 0.528 g/L, and the production of ethanol is 20.4 g/L.

Supplementation of nitrogen sources to enhance L-serine production

It is very necessary to supplement nitrogen source in the production process of amino acids. Nitrogen source is one of the nutrients necessary for microbial growth. Supplementing sufficient nitrogen source can promote the growth and reproduction of strains, thereby increasing the production of amino acids. Different nitrogen sources may cause different metabolic pathways to be activated or inhibited, thereby affecting the synthesis and accumulation of amino acids.

In some embodiments, RMG5 medium containing 5 g/L nitrogen source glutamate hydrochloride and RMG5 medium containing 5 g/L ammonium sulfate are prepared. The engineered strain S02A9B5 is inoculated into these two culture media for fermentation tests, and it is found (Figure 7) that the growth of the strain is improved, and the L-serine production is further increased to 855.6 mg/L.

The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims (9)

1. An engineered strain, wherein the engineered strain is a strain in which the sdaA gene of Zymomonas mobilis ZM4 is knocked out. 2. An engineered strain, wherein the engineered strain is a strain in which the sdaA gene and the glyA gene of Zymomonas mobilis ZM4 are knocked out. 3. An engineered strain, wherein the engineered strain is a strain of Zymomonas mobilis ZM4 in which the sdaA gene is knocked out, the glyA gene is knocked out, and the EceamA gene is overexpressed. 4. An engineered strain, wherein the engineered strain is a strain of Zymomonas mobilis ZM4 in which the sdaA gene is knocked out, the glyA gene is knocked out, the EceamA gene is overexpressed, the SerA gene is overexpressed, the SerC gene is overexpressed, and the SerB gene is overexpressed. 5. A method for preparing an engineered strain, comprising: Zymomonas mobilis ZM4 was obtained as the starting strain; Obtaining a first knockout plasmid pL2R-sdaA targeting the sdaA gene, wherein the first knockout plasmid pL2R-sdaA knocks out the sdaA gene of the starting strain; obtaining a second knockout plasmid pL2R-glyA targeting the glyA gene, wherein the second knockout plasmid pL2R-glyA knocks out the glyA gene of the starting strain; and Transforming the first knockout plasmid pL2R-sdaA into the starting strain, or simultaneously transforming the first knockout plasmid pL2R-sdaA and the second knockout plasmid pL2R-glyA into the starting strain to obtain the engineered strain; The steps of constructing the first knockout plasmid pL2R-sdaA include: constructing a first targeting plasmid; The upstream sequence of sdaA was amplified using primers sdaAUS-F and sdaAUS-R, and the downstream sequence of sdaA was amplified using primers sdaADS-F and sdaADS-R, wherein the sdaAUS-F is shown in SEQ ID NO.10, the sdaAUS-R is shown in SEQ ID NO.11, the sdaADS-F is shown in SEQ ID NO.12, and the sdaADS-R is shown in SEQ ID NO.13; Connecting the sdaA upstream sequence and the sdaA downstream sequence by Overlap PCR to obtain a first donor fragment as shown in SEQ ID NO.14; Reverse PCR amplification of the first targeting plasmid using primers 15Afk-F and 15Afk-R to obtain a reverse amplification fragment of the first targeting plasmid, wherein the 15Afk-F is shown in SEQ ID NO.15, and the 15Afk-R is shown in SEQ ID NO.16; and The reverse-amplified fragment of the first targeting plasmid and the first donor fragment were Gibson assembled and ligated at a ratio of 1:3, and the ligation product was transferred into Escherichia coli competent cells; screening was performed using spectinomycin plates, and single colonies were picked and verified by colony PCR using pEZ15A-F and pEZ15A-R primers, respectively, and the band size was verified by sequencing if it was consistent with the expected one; the first knockout plasmid pL2R-sdaA was extracted and separated from the verified positive clones, wherein the pEZ15A-F is shown in SEQ ID NO.6, and the pEZ15A-R is shown in SEQ ID NO.7; The construction steps of the first targeting plasmid include: synthesizing primers grsdaA-F and grsdaA-R to guide nucleotidase to cut the targeting site; annealing the grsdaA-F and the grsdaA-R and connecting them to the basic plasmid pEZ15Asp, and obtaining the first targeting plasmid by screening; the grsdaA-F is shown in SEQ ID NO.4, and the grsdaA-R is shown in SEQ ID NO.5; The steps of constructing the second knockout plasmid pL2R-glyA include: constructing a second targeting plasmid; The glyA upstream sequence was amplified using primers glyAUS-F and glyAUS-R, and the glyA downstream sequence was amplified using primers glyADS-F and glyADS-R, wherein the glyAUS-F is shown in SEQ ID NO.17, the glyAUS-R is shown in SEQ ID NO.18, the glyADS-F is shown in SEQ ID NO.19, and the glyADS-R is shown in SEQ ID NO.20; Connecting the glyA upstream sequence and the glyA downstream sequence by Overlap PCR to obtain a second donor fragment as shown in SEQ ID NO.21; Reverse PCR amplification of the second targeting plasmid using the primers 15Afk-F and 15Afk-R to obtain a reverse amplification fragment of the second targeting plasmid; The reverse-amplified fragment of the second targeting plasmid and the second donor fragment were Gibson assembled and connected at a ratio of 1:3, and the connection product was transferred into Escherichia coli competent cells; the single colony was selected for screening using a spectinomycin plate, and the pEZ15A-F and the pEZ15A-R were used as primer pairs for colony PCR verification, and the band size was consistent with the expected one, which was verified by sequencing; the second knockout plasmid pL2R-glyA was extracted and separated from the verified positive clones; Among them, the construction steps of the second targeting plasmid include: synthesizing primers grglyA-F and grglyA-R to guide the cutting of the targeting site by nucleotidase; annealing the grglyA-F and the grglyA-R and connecting them to the basic plasmid pEZ15Asp, and obtaining the second targeting plasmid by screening; the grglyA-F is shown in SEQ ID NO.8, and the grglyA-R is shown in SEQ ID NO.9. 6. A method for preparing an engineered strain, comprising: Zymomonas mobilis ZM4 was obtained as the starting strain; Obtaining a first knockout plasmid pL2R-sdaA targeting the sdaA gene, wherein the first knockout plasmid pL2R-sdaA knocks out the sdaA gene of the starting strain; Obtaining a second knockout plasmid pL2R-glyA targeting the glyA gene, wherein the second knockout plasmid pL2R-glyA knocks out the glyA gene of the starting strain; Obtaining the third overexpression plasmid 39p-B3 overexpressing the EceamA gene; The first knockout plasmid pL2R-sdaA is transferred into the starting strain, or the first knockout plasmid pL2R-sdaA and the second knockout plasmid pL2R-glyA are transferred into the starting strain respectively, or the first knockout plasmid pL2R-sdaA, the second knockout plasmid pL2R-glyA and the third overexpression plasmid 39p-B3 are transferred into the starting strain respectively; and To obtain the engineered strain; The steps of constructing the first knockout plasmid pL2R-sdaA include: constructing a first targeting plasmid; The upstream sequence of sdaA was amplified using primers sdaAUS-F and sdaAUS-R, and the downstream sequence of sdaA was amplified using primers sdaADS-F and sdaADS-R, wherein the sdaAUS-F is shown in SEQ ID NO.10, the sdaAUS-R is shown in SEQ ID NO.11, the sdaADS-F is shown in SEQ ID NO.12, and the sdaADS-R is shown in SEQ ID NO.13; Connecting the sdaA upstream sequence and the sdaA downstream sequence by Overlap PCR to obtain a first donor fragment as shown in SEQ ID NO.14; Reverse PCR amplification of the first targeting plasmid using primers 15Afk-F and 15Afk-R to obtain a reverse amplification fragment of the first targeting plasmid, wherein the 15Afk-F is shown in SEQ ID NO.15, and the 15Afk-R is shown in SEQ ID NO.16; and The reverse-amplified fragment of the first targeting plasmid and the first donor fragment were Gibson assembled and ligated at a ratio of 1:3, and the ligation product was transferred into Escherichia coli competent cells; screening was performed using spectinomycin plates, and single colonies were picked and verified by colony PCR using pEZ15A-F and pEZ15A-R primers, respectively, and the band size was verified by sequencing if it was consistent with the expected one; the first knockout plasmid pL2R-sdaA was extracted and separated from the verified positive clones, wherein the pEZ15A-F is shown in SEQ ID NO.6, and the pEZ15A-R is shown in SEQ ID NO.7; The construction steps of the first targeting plasmid include: synthesizing primers grsdaA-F and grsdaA-R to guide nucleotidase to cut the targeting site; annealing the grsdaA-F and the grsdaA-R and connecting them to the basic plasmid pEZ15Asp, and obtaining the first targeting plasmid by screening; the grsdaA-F is shown in SEQ ID NO.4, and the grsdaA-R is shown in SEQ ID NO.5; The steps of constructing the second knockout plasmid pL2R-glyA include: constructing a second targeting plasmid; The upstream sequence of glyA was amplified using primers glyAUS-F and glyAUS-R; the downstream sequence of glyA was amplified using primers glyADS-F and glyADS-R; the glyAUS-F was shown in SEQ ID NO.17, the glyAUS-R was shown in SEQ ID NO.18, the glyADS-F was shown in SEQ ID NO.19, and the glyADS-R was shown in SEQ ID NO.20; Connecting the glyA upstream sequence and the glyA downstream sequence by Overlap PCR to obtain a second donor fragment as shown in SEQ ID NO.21; Reverse PCR amplification of the second targeting plasmid using the primers 15Afk-F and 15Afk-R to obtain a reverse amplification fragment of the second targeting plasmid; The reverse-amplified fragment of the second targeting plasmid and the second donor fragment are Gibson assembled and connected at a ratio of 1:3, and the connection product is transferred into Escherichia coli competent cells; the single colony is selected for screening using a spectinomycin plate, and the pEZ15A-F and the pEZ15A-R are used as primer pairs for colony PCR verification, and the band size is consistent with the expected one, which is verified by sequencing; the second knockout plasmid pL2R-glyA is extracted and separated from the verified positive clones; The construction steps of the second targeting plasmid include: synthesizing primers grglyA-F and grglyA-R to guide nucleotidase to cut the targeting site; annealing the grglyA-F and the grglyA-R and connecting them to the basic plasmid pEZ15Asp, and obtaining the second targeting plasmid by screening; the grglyA-F is shown in SEQ ID NO.8, and the grglyA-R is shown in SEQ ID NO.9; Wherein, the construction steps of the third overexpression plasmid 39p-B3 include: The E. coli genome was amplified by PCR using primers EceamA-F and EceamA-R to obtain the EceamA sequence; the Zymomonas mobilis ZM4 genome was amplified by PCR using primers Ptet-F and Ptet-R to obtain the Ptet strong promoter sequence shown in SEQ ID NO.28, wherein the EceamA-F is shown in SEQ ID NO.26, the EceamA-R is shown in SEQ ID NO.27, the Ptet-F is shown in SEQ ID NO.29, and the Ptet-R is shown in SEQ ID NO.30; Connecting the EceamA sequence to the Ptet sequence by Overlap PCR to obtain a connection sequence of Ptet and EceamA; The vector pEZ15A was reversely amplified using primers 15Afk-F and 15Afk-R to obtain linearized pEZ15A; The Ptet and EceamA connection sequence was mixed with the linearized pEZ15A at a ratio of 3:1. After the reaction system was prepared according to the table below, it was allowed to stand on ice for 5 minutes, and then E. coli competent cells were added and transformed using the general method; Screening was performed using spectinomycin-resistant plates, and single colonies were picked. The PCR amplification programs for primer pairs 15A-fwd and 15A-rev were set as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 80 s, for a total of 30 cycles. Verification was performed by colony PCR, and the band size consistent with the expected value was verified by sequencing; that is, the third overexpression plasmid 39p-B3 was extracted and separated from the correctly verified positive clones; the 15A-fwd is shown in SEQ ID NO.31, and the 15A-rev is shown in SEQ ID NO.32. 7. A method for preparing L-serine, comprising: Obtaining the engineered strain according to any one of claims 1 to 4 or the engineered strain prepared by the preparation method according to any one of claims 5 or 6; fermenting the engineered strain; and The L-serine is harvested from the fermentation product. 8. The method according to claim 7, further comprising fermenting the engineered strain in a culture solution supplemented with a nitrogen source. 9. Use of the engineered strain according to any one of claims 1 to 4 or the engineered strain obtained by the preparation method according to any one of claims 5 or 6, wherein the use is for synthesizing L-serine.