CN117736959B

CN117736959B - Engineering strain of zymomonas mobilis, preparation method and application

Info

Publication number: CN117736959B
Application number: CN202410121669.XA
Authority: CN
Inventors: 杨世辉; 王振; 晏雄鹰; 王霞; 何桥宁
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2024-01-26
Filing date: 2024-01-26
Publication date: 2024-05-14
Anticipated expiration: 2044-01-26
Also published as: CN117736959A

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 strain of zymomonas mobilis, preparation method and application

Technical Field

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.

Background

The zymomonas mobilis (Zymomonas mobilis) is taken as a facultative anaerobic gram-negative bacterium for naturally producing ethanol, has a unique ED metabolic pathway and higher sugar fermentation efficiency, and has the characteristics of high ethanol yield, less yield, strong ethanol tolerance, high osmotic pressure resistance, no need of additional oxygen in the fermentation process and other ideal industrial cell factories. Production and fermentation of PHB, 2, 3-butanediol, isobutanol, lactic acid and other products have been realized in zymomonas mobilis at present. In addition, the zymomonas mobilis has higher tolerance to lignocellulose hydrolysate, the production of the cellulose ethanol has been commercialized, and the research on the mechanism related to the tolerance of the inhibitor in the hydrolysate is also mature. In addition, by synthetic biology and metabolic engineering means, zymomonas mobilis can be engineered into chassis cells that utilize lignocellulosic hydrolysates to produce different platform compounds.

Disclosure of Invention

The application discloses an engineering strain of zymomonas mobilis, a preparation method and application thereof. 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.

Based on the above, the embodiment of the application at least discloses the following technical scheme:

In a first aspect, embodiments disclose engineered strains of Zymomonas mobilis. The engineering strain is the sdaA gene of the zymomonas mobilis is knocked out; 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.

In a second aspect, the examples disclose methods of making engineered strains of Zymomonas mobilis. The preparation method comprises the following steps: obtaining zymomonas mobilis ZM4 as an initial 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, said second knockout plasmid knockout the glyA gene of said zymomonas mobilis; and transferring at least one of the first knockout plasmid and the second knockout plasmid into the zymomonas mobilis to obtain the engineering strain.

In a third aspect, the examples disclose a method of preparing L-serine. The method comprises the following steps: obtaining the engineering strain of the first aspect or the engineering strain prepared by the preparation method of the second aspect; fermenting the engineering strain; and harvesting the L-serine from the fermentation product.

In a fourth aspect, embodiments disclose the use of an engineered strain according to the first aspect or an engineered strain produced by the production method according to the second aspect. The application is selected from at least one of the following: synthesizing L-serine; synthesizing cycloserine; chiral resolution of serine; preparing a food additive; or preparing a nutritional supplement.

Drawings

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

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

FIG. 3 is a schematic diagram showing the construction of the over-expression plasmids pEZ-A1, pEZ-A2, pEZ-A3, pEZ-A4, engineering strains S02A1B3, S02A2B3, S02A3B3 and S02A4B3, and fermentation test results of the engineering strains S02A1B3, S02A2B3, S02A3B3 and S02A4B3 provided in the examples.

FIG. 4 is an amino acid sequence comparison of the PGDH enzyme of E.coli and the PGDH enzyme derived from Zymomonas mobilis (A), a schematic diagram of the three-dimensional structure of the PGDH enzyme mutant (B), schematic diagrams of the structures of the overexpression plasmids pEZ-A4, pEZ-A5, pEZ-A6, pEZ-A7, pEZ-A8, pEZ-A9 and pEZ-A10 and the fermentation test results of the corresponding engineering strains S02A4B3, S02A5B3, S02A6B3, S02A7B3, S02A8B3, S02A9B3 and S02A10B3 (C), and a structure diagram (D) of Tc growth rate, glucose consumption and ethanol yield of the engineering strains S02A9B 3.

FIG. 5 is a schematic diagram of the structures of the overexpressing plasmids pEZ-A9 and 39-B4 provided in the examples, and the fermentation test results obtained with the engineering strain S02A9B 4.

FIG. 6 is a schematic structural diagram of the overexpression plasmid 39-B5 and the fermentation test result (A) of the engineering strain S02A9B5, and the Tc growth rate, glucose consumption and ethanol yield structural diagram (B) of the engineering strain S02A9B 5.

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

Fig. 8 is a schematic diagram showing genome structure alignment of ZM4, engineering strain S02 and engineering strain S02 provided in the examples.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

Serine is a neutral aliphatic polar alpha-amino acid, a non-essential amino acid. It plays a vital role in the biological growth process, including one-carbon unit metabolism (one-carbon unit is an important material for nucleotide synthesis), protein synthesis, purine and pyrimidine synthesis, and cell membrane production and processing. L-serine is one of the thirty skeleton compounds which are most attractive in the chemical industry field, and is widely applied to the fields of medical treatment, cosmetics, food, scientific research and the like. The method has wide market and application prospects, and the annual market demand growth rate is estimated to be 7-10%.

The most widely used methods for L-serine production are whole cell catalytic enzyme catalysis and microbial fermentation. Both whole cell catalysis and enzyme catalysis require expensive precursors such as glycine as a substrate, methanol and the like as a carbon compound supplement, are expensive to produce, and can produce environmental pollution. Therefore, the serine produced from renewable raw materials by utilizing microorganisms is more environment-friendly, more efficient and attractive. Biosynthesis of L-serine takes advantage of the intracellular synthesis of the microorganism's own metabolism and is excreted extracellularly via the transporter. However, most microbial fermentation processes for producing L-serine are aerobic and require a large amount of energy to supply oxygen. And the continuous production cost can be increased by introducing oxygen into the fermentation device, and the waste of the effective volume of the bioreactor device can be caused.

The term "engineering strain" refers to a strain obtained by genetically engineering a wild-type strain, for example, by transforming its genome, and transferring an over-expression plasmid into its body. For example, a strain obtained by knocking out or overexpressing a gene in a genome of a wild type Zymomonas mobilis, and transferring an overexpressed plasmid into the genome is called an "engineering strain".

However, in a first aspect, the examples disclose engineered strains of Zymomonas mobilis. The engineering strain is the sdaA gene of the zymomonas mobilis is knocked out; 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.

In some embodiments, the engineered strain is a strain in which the sdaA gene of zymomonas mobilis is knocked out and the glyA gene is knocked out. Engineering strains with the sdaA and glyA genes of zymomonas mobilis knocked out to block two ways of main degradation of L-serine in the strains, thereby realizing accumulation of L-serine.

In some embodiments, the engineered strain is an engineered strain in which the sdaA gene, the glyA gene, and the EceamA gene of zymomonas mobilis are knocked out. In order to enhance extracellular transport and tolerance of Z.mobilis to L-serine, an engineering strain was introduced into the transporter gene EceamA in E.coli, achieving transport and tolerance to L-serine.

In some embodiments, the engineered strain is a strain in which the sdaA gene, the glyA gene, the EceamA gene, the SerA gene, the SerC gene, and the SerB gene are knocked out, or overexpressed. The engineering strain is over-expressed with endogenous gene SerA, serC, serB, which can enhance the synthesis of L-serine.

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

In some embodiments, the method of making comprises transferring the first knockout plasmid and the second knockout plasmid to the zymomonas mobilis to obtain the engineered strain. Thus, engineering strains with the sdaA and glyA genes of the zymomonas mobilis knocked out are obtained, and two ways of main degradation of L-serine in the strains are blocked, so that accumulation of L-serine can be realized.

In some embodiments, the method of preparing further comprises obtaining a third over-expression plasmid that over-expresses EceamA genes. And transferring the third over-expression plasmid into engineering strains with the knocked-out sdaA and glyA genes to obtain engineering strains with the knocked-out sdaA genes, the knocked-out glyA genes and the over-expressed EceamA genes. Thus, the engineering strain can improve the transporting and tolerance capacity to L-serine.

In some embodiments, the method of making further comprises obtaining a fourth overexpression plasmid of the overexpression elements of the SerA gene, the SerC gene, and the SerB gene; and transferring the third and fourth over-expression plasmids into an engineering strain in which the sdaA gene is knocked out, the glyA gene is knocked out, and the EceamA gene is over-expressed. Thus, the engineering strain can over-express SerA, serC, serB and can enhance the synthesis of L-serine.

In some embodiments, the method of making further comprises obtaining a fifth over-expression plasmid that over-expresses the pgk gene, and transferring the fifth over-expression plasmid into an engineered strain in which the sdaA gene is knocked out, the glyA gene is knocked out, the EceamA gene is over-expressed, and the SerA, serC, serB gene is over-expressed. The engineering strain over-expression enhancement precursor related gene pgk obtained in this way can enhance the synthesis of L-serine.

In a third aspect, the examples disclose a method of preparing L-serine. The method comprises the steps of obtaining the engineering strain in the first aspect or the engineering strain prepared by the preparation method in the second aspect; fermenting the engineering strain; and harvesting the L-serine from the fermentation product.

In some embodiments, the method of producing L-serine further comprises fermenting the engineered strain in a culture broth supplemented with a nitrogen source.

In a fourth aspect, embodiments disclose the use of an engineered strain according to the first aspect or an engineered strain produced by the production method according to the second aspect. L-serine can be used as a biochemical reagent and a food additive; nutritional supplements, for example as skin nutritional additives in cosmetics; can be used for biochemical and nutritional research, and can also be used as raw material for synthesizing cycloserine. Thus, embodiments provide an application selected from at least one of: synthesizing L-serine; synthesizing cycloserine; chiral resolution of serine; preparing a food additive; or preparing a nutritional supplement.

Preparation of engineering strains with sdaA and/or glyA knockdown

In some embodiments, the L-serine dehydratase and hydroxymethylserine transferase (SHMT) encoded by the sdaA and glyA genes are knocked out on the Zymomonas mobilis genome using the Zymomonas mobilis endogenous I-F CRISPR-Cas gene editing system to block the degradation of L-serine by Zymomonas mobilis to produce pyruvic acid and glycine. Thus, the engineering strain S01 with the sdaA knocked out (ΔsdaA, sdaA knocked out) and the engineering strain S02 with the glyA knocked out (ΔsdaA ΔglyA, sdaA and glyA knocked out). As shown in FIG. 1, the growth delay period of the engineering strains S01 and S02 is increased by about 3 hours, the final biomass is unchanged, and the accumulation amount of L-serine is increased from 15.3 mg/L to 50.7 mg/L.

In some embodiments, knockout of sdaA is achieved by transfer of the first knockout plasmid into zymomonas mobilis to yield engineered strain S01.

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

In some embodiments, the first knockout plasmid carries the first targeting element and the first donor sequence. The first targeting element comprises two repeat sequences as shown in SEQ ID NO.1 and a first leader sequence as shown in SEQ ID NO.2 located between the two repeat sequences. The first targeting element targets the sdaA gene. The first donor sequence consists 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 comprises two repeat sequences as shown in SEQ ID NO.1 and a second targeting sequence as shown in SEQ ID NO.3 located between the two repeat sequences. The second targeting element targets the glyA gene. The second donor sequence consists of a 600bp sequence upstream of the glyA gene and a 600bp sequence downstream of the sdaA gene.

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

1. Preparation of first and second knockout plasmids

1) First, second guide sequence

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

2) First and second targeting plasmids

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

In some embodiments, the targeting primer sequence (grsdaA-F, SEQ ID NO. 4; grsdaA-R (SEQ ID NO. 5) is designed based on the first targeting sequence, the cleavage of the targeting site by the nucleotidase is guided, the targeting annealing is followed by ligation onto the base plasmid (pEZ Asp), and the first targeting plasmid is obtained by selection.

Some first targeting plasmid construction methods specifically include:

Cleavage of the vector pEZ Asp with restriction enzyme BsaI gives linearized pEZ Asp; then, the primer (grsdaA-F, grsdaA-R) of the first guide sequence is annealed according to the system shown in the table 1 (an annealing system comprises 1 [ mu ] L grsdaA-F (10 [ mu ] M), 1 [ mu ] L grsdaA-R (10 [ mu ] M) and 8 [ mu ] L ddH2O in terms of 10 [ mu ] L), denatured at 95 ℃ for 5min, and cooled to room temperature for standby; ligation of the annealed product (i.e.the first leader sequence) with linearized pEZ Asp using T4 ligase was carried out for 3-6h at 22℃in the system shown in Table 2; the connection product is transferred into an escherichia coli clone strain DH5 alpha by adopting a general chemical conversion method in the field to construct plasmids; single colonies were picked using spectinomycin plates and verified by colony PCR using pEZ A-F (shown as SEQ ID NO. 6) and pEZ A-R (shown as SEQ ID NO. 7) primers, respectively. The band size was verified by sequencing consistent with the expected. Wherein the T4 reaction system comprises 20-40ng linearization pEZ Asp,2 [ mu ] L first leader sequence, 0.5 [ mu ] L T ligase, 1 [ mu ] L Buffer and the rest ddH ₂ O in terms of 10 [ mu ] L. The reaction program of colony PCR is 3min at the temperature of pre-denaturation 98 ℃ and 1 cycle; denaturation at 98℃for 10s, annealing at 55℃for 10s, extension at 72℃for 30 cycles at 10s/kb depending on fragment length; extending at 72 ℃ for 2min,1 cycle; store 2min at 12℃for 1 cycle.

In some examples of this embodiment, the spectinomycin knockout plasmid vector (pEZ Asp) is a coding gene for spectinomycin inserted on pEZ a as a marker gene. Wherein pEZ a is obtained by reference to "Yang S,Mohagheghi A,Franden M A,et al.Metabolic engineering of Zymomonas mobilis for 2,3-butanediolproduction from lignocellulosic biomass sugars[J].Biotechnol Biofuels,2016,9(1):189.". For pEZ a of different coding genes (e.g. resistance genes) reference can be made to the construction and application of the "plasmid pUC19-CM-D [ J ]. The method disclosed in the agricultural science of Anhui, 2010, stage 19" in which different marker genes are inserted.

In some embodiments, a second targeting element comprising a second targeting sequence is inserted into the base plasmid to yield a second targeting plasmid, the first targeting plasmid targeting 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 targeting sequence of the glyA gene is ligated to a spectinomycin knockout plasmid vector (pEZ Asp) containing a CRISPR-IF expression unit to yield a second targeting plasmid. The preparation process of the second targeting plasmid is the same as that of the first targeting plasmid, and a detailed description is omitted. Wherein,

3) Construction of first knockout plasmid (pL 2R-sdaA) and second knockout plasmid (pL 2R-glyA)

In some embodiments, the preparation step of the first knockout plasmid comprises:

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

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

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

4) The reverse-expanded first targeting plasmid and first donor fragment were Gibson assembled and ligated in a 1:3 ratio and the ligation product was transferred to E.coli competent cells. Single colonies were picked using spectinomycin plates, verified by colony PCR with pEZ A-F (shown as SEQ ID NO. 6) and pEZ A-R (shown as SEQ ID NO. 7) primers, respectively, and the band sizes were verified by sequencing to be consistent with expectations. The first knockout plasmid was extracted and isolated from the verified positive clone.

The preparation method of the second knockout plasmid (pL 2R-glyA) is the same as that of the first knockout plasmid. The sequences involved are: glyAUS-F, SEQ ID NO. 17. glyAUS-R is shown as SEQ ID NO. 18. glyADS-F is shown in SEQ ID NO. 19. glyADS-R is shown as SEQ ID NO. 20. A second donor fragment, shown in SEQ ID NO.21, is made up of a glyA upstream sequence linked to a glyA downstream sequence.

2. Electrotransformation of first and second knockout plasmids

Mu.g of the first knockout plasmid or the second knockout plasmid was added to 50. Mu.L of competent ZM4 competent S01, and after mixing, the mixture was added to a 0.1cm electric cup, and electric transfer was performed according to the procedure of 1800V, 25. Mu.F, 200Ω. After the electrotransfer is completed, transferring the strain into 1mL RM culture medium, standing and culturing for 4-6 hours in a 30 ℃ incubator, then taking 200 mu L of strain to uniformly coat on a 100 mu g/mL spectinomycin resistance plate, and culturing for 2-3 days in the 30 ℃ incubator in an inverted manner.

3. Screening of engineering strains S01/S02

After colonies grow out, colony PCR detection is carried out on the engineering strains by using verification primers SDAACHECK-F (shown as SEQ ID NO. 22) and SDAACHECK-R (shown as SEQ ID NO. 23), the strains with the strip sizes consistent with expectations are verified by sequencing, and the correct strains are preserved for standby, and are named as S01.

The screening of S02 strain is the same as that of S01, wherein the primers for screening are: GLYACHECK-F, SEQ ID NO. 24; GLYACHECK-R, SEQ ID NO. 25.

4. Fermentation test of engineering strains S01 and S02

(1) Testing the growth and fermentation performance of engineering strains:

The L-serine fermentation performance of the engineering strains S01 and S02 described in the above examples at different initial glucose concentrations was tested. Engineering strains S01 and S02 were inoculated into 50mL Erlenmeyer flasks containing 80% of RMG5, and 5 g/L of glutamic acid hydrochloride was added thereto, and cultured in a shaker at 30℃at a shaking speed of 100 rpm. Samples at various time points were centrifuged and supernatants (12000 rpm,2 min) were taken as test samples during fermentation by HPLC.

The RMG5 enrichment medium contained 50g/L glucose, 10g/L yeast extract, and 2g/L KH ₂PO₄.

(2) Detection of fermentation supernatants

1) Detection of glucose, ethanol and acetic acid

Adopts Shimadzu commercial company Agilent 1100 series high performance liquid chromatograph (LC-20 AD); the detector is a differential refraction detector (RID-10A); the chromatographic column is an organic acid chromatographic column (Bio-Rad Aminex HPX-87H,300 mm. Times.7.8 mm); the pool temperature is 40 ℃, and the column temperature box temperature is 60 ℃; the flow rate of sulfuric acid with the mobile phase of 5 mM is 0.5 mL/min, the initial flow rate is set to be 0.2 mL/min when the instrument is operated, and the flow rate gradually increases to 0.5 mL/min at 0.1 mL/min after the column pressure is stabilized; the sample loading was 20. Mu.L. Configuration of mobile phase: taking 1.41 mL chromatographic grade concentrated sulfuric acid into a blue cap bottle of 5L, fixing the volume to 5L by ultrapure water, uniformly mixing, and filtering by using a water phase filter membrane with the aperture of 0.45 mu m. And subpackaging the filtered mobile phase into a 1L mobile phase blue cap bottle for ultrasonic degassing for 20-30 min. And the product can be used after being restored to room temperature.

2) Detection of L-serine

L-serine cannot be detected directly and needs to be reacted with O-phthalimidone (OPA) to form a stable light absorbing OPA-L-serine derivative before HPLC injection detection. The derivative has strong ultraviolet absorptivity, and can be detected and quantitatively analyzed by an ultraviolet detector in an HPLC system. L-serine assays were analyzed using SPD-20A through Agilent Advance bio AAA amino acid analysis columns (Agilent, DE, USA) and pre-column derivatization was performed using Agilent amino acid analysis packages. Mobile phase a was 10mM Na ₂HPO₄ and 10mM Na ₂B₄O₇ controlled PH at 8.2. Mobile phase B was 45% methanol and 45% acetonitrile. The column temperature was controlled at 50℃and the flow rate was 1.5 ml/min. The mobile phase ratio procedure 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 end the procedure for 20 min.

Detection of D-serine was determined by pre-column derivatization with 260mM N-isobutyryl-L-cysteine (IBLC reagent) and 170 mM O-phenylenediamine ketone (OPA) by Agilent Advance bio AAA amino acid analysis column reverse phase detection. The product obtained by detection does not contain D-serine. The mobile phase used was phase a as follows: 50 mM sodium acetate (pH 6.0) phase B: 45% methanol 45% acetonitrile. The column temperature was controlled at 30℃and the flow rate was 0.7 ml/min. The mobile phase ratio procedure was: the procedure was ended with 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.

(2) Results

As shown in FIG. 1, the growth delay period of the engineering strains S01 and S02 is increased by about 3 hours, the final biomass is unchanged, and the accumulation amount of L-serine is increased from 15.3 mg/L to 50.7 mg/L.

Preparation of engineering strains incorporating transport proteins

Among the transport proteins which have been found to be used for the outward transport of L-serine are EceamA in E.coli (NC-000913.3) and CgthrE (NC-022040.1) and Cg0580 (NC-021352.1) in C.glutamicum. In addition, the zymomonas mobilis has no gene encoding the L-serine transporter and no homologous similar gene is found.

Based on this, examples were amplified from E.coli and C.glutamicum genomes to Cgthre, cg0580 and EceamA, respectively, and the Ptet promoters were used to construct over-expression plasmids 39p-B1, 39p-B2 and 39p-B3, which were electrotransformed into S02 strain, respectively, to give engineering strain S02B1 over-expressing CgthrE, engineering strain S02B2 over-expressing Cg0580 and engineering strain S02B3 over-expressing EceamA, respectively, in that order.

As shown in FIG. 2, it was found through the tolerance test that the over-expressed EceamA engineering strain S02B3 had the highest growth and L-serine accumulation in the 12 g/L RMG5 added.

In some embodiments, the method for constructing the engineering strain S02B3 of the over-expression EceamA comprises the following steps:

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

The E.coli genome was PCR amplified using primers (EceamA-F, SEQ ID NO. 26; eceamA-R, SEQ ID NO. 27) to give a EceamA sequence. The Zymomonas mobilis ZM genome was PCR amplified using primers (Ptet-F, shown as SEQ ID NO. 29; ptet-R, shown as SEQ ID NO. 30) to give a Ptet strong promoter sequence (shown as SEQ ID NO. 28).

Reverse amplification of vector pEZ A with primers 15Afk-F (shown as SEQ ID NO. 15) and 15Afk-R (shown as SEQ ID NO. 16) gave linearized pEZ A.

These PCR amplification reactions contained 10. Mu.M of each of the upstream and downstream primers in 20. Mu.L, 10. Mu. L PRIMERSTAR DNA Polymerase (Takara), 5 to 10ng of template and the balance ddH2O. The PCR amplification procedure was set as follows: pre-denaturation at 98 ℃ 2 min; denaturation at 98℃for 10 s, annealing at 55℃for 10 s, extension at 72℃ (set according to fragment length of 10 s/kb) for a total of 30 cycles; maintaining the temperature at 72 ℃ after the cyclic reaction is finished at 5 min; the product was stored at-20 ℃ after purification.

The obtained fragment and linearization pEZ A are mixed according to the ratio of 3:1, and after the preparation according to the following table reaction system is completed, the mixture is kept stand on ice for 5 minutes, and then E.coli competent cells are added, and the conversion is carried out by adopting a general method. Single colonies were picked using spectinomycin resistant plates and verified by colony PCR with primer pairs 15A-fwd (shown as SEQ ID NO. 31) and 15A-rev (shown as SEQ ID NO. 32) 1, respectively (PCR amplification procedure set to 98℃for 3 min pre-denaturation, 98℃for 10 s denaturation, 55℃for 10 s annealing, 72℃for 80 s extension, 30 cycles total), and band size was verified to be consistent with expected by sequencing. I.e., the third over-expression plasmid 39p-B3 of EceamA was extracted and isolated from the positive clone that was verified to be correct. The ligation reaction system of EceamA gene fragment and plasmid pEZ A contained 0.12 pM EceamA gene fragment, 0.04 pM plasmid pEZ A, 0.5. Mu.L 10 XBuffer 4 (Thermo), 0.5 UT5 Exonuclease and the balance ddH ₂ O in 5. Mu.L.

2. Obtaining engineering strain S02B3 of over-expression EceamA

Competent cells of Zymomonas mobilis S02 obtained in the above example were prepared, frozen strains were taken out from a-80℃refrigerator, 100. Mu.L was inoculated into a frozen tube containing 1 mL of RMG5, and cultured at 30℃in an incubator to activate the strains. After the culture is turbid, transferring the culture to a 250 mL blue cap bottle filled with 200 mL of RMG5 liquid culture medium, enabling the initial OD 600nm to be within a range of 0.025-0.3, standing and culturing in a 30 ℃ incubator, collecting thalli at normal temperature of 100 rpm when the OD 600nm exceeds 0.3, then washing with sterile water for 1 time and 10% glycerol for two times, finally slowly re-suspending the thalli with 1-2 mL of 10% glycerol, and sub-packaging 55 mu L of the thalli into 1.5 mL EP tubes.

1 Mg of the above third over-expression plasmid 39p-B3 was added to a competent electrocuvette and to a 1.5 mL EP tube containing 55. Mu.L of competent cells, and transferred to a1 mm electrocuvette after gentle mixing. Program setting of the electric converter: 200. omega, capacitance: 25. and [ mu ] F, voltage: 1.6 KV. And placing the electric rotating cup into an electric rotating instrument for electric rotating, immediately adding 1mL of RMG5 liquid culture medium after electric rotating, uniformly mixing, transferring into a sterile EP tube, sealing by using a sealing film, and incubating in a constant temperature incubator at 30 ℃ for 4-6 hours. 100 μl of the bacterial liquid was applied uniformly to RMG5+Spe plates (100 μg/mL spectinomycin). Sealing the plate with sealing film, and culturing in an incubator at 30deg.C. Colony PCR verification, after single colony grows on the plate, single colony is subjected to PCR verification by using 15A-fwd/15A-rev primer. The PCR system and PCR procedure were the same as described above for colony PCR. The correct positive clones obtained were glycerol-protected after activation in medium with rmg5+spe.

In some embodiments, the construction method of the over-expression plasmid 39p-B2 of CgthrE and the over-expression plasmid 39p-B1 of Cg0580 are identical to 39p-B1, respectively. The primer involved therein: cgthrE-F, SEQ ID NO. 33; cgthrE-R, SEQ ID NO. 34; cg0580-F, SEQ ID NO. 35; cg0580-R, SEQ ID NO. 36.

Engineering strain for over-expressing SerA1, serC and SerB genes

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

1. Fourth over-expression plasmid (pEZ-A1-4) and construction of corresponding engineering strain

In some embodiments, the three genes SerA1, serC, serB that enhance the L-serine synthesis pathway are controlled using the strong promoters 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. Four plasmids pEZ-A1, pEZ-A2, pEZ-A3 and pEZ-A4 are respectively constructed and transferred into competent cells of the S02B3 strain, and four engineering strains S02A1B3, S02A2B3, S02A3B3 and S02A4B3 are constructed. pEZ-A1, pEZ-A2, pEZ-A3, pEZ-A4, the specific preparation is referred to the preparation of 39 p-B3.

Wherein, pEZ-A1 plasmid is an over-expression plasmid obtained by inserting an expression element of Pgap, serA1 and RBS, serC, RBS, serB connected in sequence into a vector pEZ A. The pEZ-A2 plasmid is an over-expression plasmid obtained by inserting expression elements, which are sequentially connected with Peno, sera1 and RBS, serC, RBS, serB, into a vector pEZ A. The pEZ-A3 plasmid is an over-expression plasmid obtained by inserting an expression element in which P_ZMO1980, sera1, RBS, serC, RBS, serB are sequentially connected into a vector pEZ A. The pEZ-A4 plasmid is an over-expression plasmid obtained by inserting expression elements in which Ptet, sera1, RBS, serC, RBS, serB are sequentially connected into a vector pEZ A.

Wherein the related primers are as follows: serA1-F, SEQ ID NO. 40; serA1-R, SEQ ID NO. 41; serC-F, SEQ ID NO. 42; serC-R, SEQ ID NO. 43; serB-F, SEQ ID NO. 44; serB-R, SEQ ID NO. 45.

Taking the construction of S02A4B3 as an example, the construction method comprises the following steps: endogenous serA1, serC and serB genes were amplified from the ZM4 genome using primers and a reverse amplification vector was performed using the pEZ A plasmid containing the Ptet promoter as a template. Finally, the three gene fragments are subjected to overlap and connected into a large fragment, and finally are connected with a vector, transformed into competent cells of escherichia coli DH5 alpha, screened by using a spectinomycin resistance (100 mug/mL) plate, picked into single colonies, and verified by PCR with proper primers. The correct transformant pEZ-A4 was obtained. Then electrotransformation is carried out to the competence of the S02B3 strain, so as to obtain the S02A4B3 engineering strain.

As shown in FIG. 3, fermentation tests of four engineering strains S02A1B3, S02A2B3, S02A3B3 and S02A4B3 prepared show that the engineering strain S02A4B3 has the highest L-serine yield of 260.3 mg/L.

2. Fourth over-expression plasmid (pEZ-A5-10) and construction of corresponding engineering strain

The pEZ-A5 plasmid is an over-expression plasmid obtained by inserting expression elements, which are sequentially connected with Ptet, blSera1 and RBS, serC, RBS, serB, into a vector pEZ A. BlSerA1 is Bacillus licheniformis DW2 (Bacillus licheniformis) endogenous SerA, and the sequence is shown as SEQ ID No. 58. The related primers are as follows: blserA 1A-F, SEQ ID NO. 46; blserA 1A-R, SEQ ID NO. 47.

The pEZ-A6 plasmid is an over-expression plasmid obtained by inserting expression elements in which Ptet, bssSerA 1, RBS, serC, RBS, serB are connected in sequence into a vector pEZ A. BsSerA1 is Bacillus subtilis 168 (Bacillus subtilis) endogenous SerA, and has a sequence shown in SEQ ID No. 59. The related primers are as follows: bsserA 1A-F, SEQ ID NO. 48; bsserA 1A-R, SEQ ID NO. 49.

The pEZ-A7 plasmid is an over-expression plasmid obtained by inserting Ptet, ecSerAmut, RBS, serC, RBS, serB-sequentially-connected expression elements into a vector pEZ A. EcSerAmut the primer referred to by ESCHERICHIA COLI reference "Mundhada, H., Schneider, K., Christensen, H.B., Nielsen, A.T., 2016. Engineering of high yield production of L-serine in Escherichia coli. Biotechnol. Bioeng. 113(4), 807-816." is EcserA1mut1-F, shown in SEQ ID NO. 50; ecserA.mu.t1-R, SEQ ID NO. 51.

The pEZ-A8 plasmid is an over-expression plasmid obtained by inserting Ptet, cgSerAmut, RBS, serC, RBS, serB-sequentially-connected expression elements into a vector pEZ A. CgSerAmut is derived from Corynebacterium glutamicum, reference "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 Corynebacterium glutamicum. Microb. Cell Fact. 19(1), 115." relates to a primer CgSerAmut-F, shown in SEQ ID NO. 52; cgSerAmut-R, SEQ ID NO. 53.

In some embodiments, the directed mutation as shown in FIG. 4A, i.e., mutation of amino acids 475, 477, 495 to alanine, is performed by the phosphoglycerate dehydrogenase PGDH encoding gene derived from ZM4 of Zymomonas mobilis, resulting in mutated sequences ZmserAmut (shown in SEQ ID NO. 54) and ZmserAmut2 (shown in SEQ ID NO. 55), and the phosphoglycerate dehydrogenase PGDH of molecular structure as shown in FIG. 4B. Thus, both mutants ZmSerAmut and ZmSerAmut2 were able to release the feedback inhibition by L-serine of the wild-type gene SerA. The ZmserAmut and ZmserAmut sequences were synthesized by the Optimago company, thereby constructing the overexpressing plasmids pEZ, pEZ10 of ZmSerAmut and ZmSerAmut.

The pEZ-A9 plasmid is an over-expression plasmid obtained by inserting the expression elements of Ptet, zmSerAmut and RBS, serC, RBS, serB connected in sequence into a vector pEZ A. The related primers are as follows: zmSerAmut 1A-F, SEQ ID NO. 60. ZmSerAmut 1A-R, SEQ ID NO. 61.

The pEZ-A10 plasmid is an over-expression plasmid obtained by inserting the expression elements of Ptet, zmSerAmut and RBS, serC, RBS, serB connected in sequence into a vector pEZ A. The related primers are as follows: zmSerAmut2-F, SEQ ID NO. 62. ZmSerAmut2-R, SEQ ID NO. 63.

PEZ-A4, pEZ-A5, pEZ-A6, pEZ-A7, pEZ-A8, pEZ-A9 and pEZ-A10 are respectively transferred into engineering strains S02B3 to obtain engineering strains S02A4B3, S02A5B3, S02A6B3, S02A7B3, S02A8B3, S02A9B3 and S02A10B3 in sequence. These engineering strains were subjected to fermentation tests, as can be seen in FIGS. 4C and 4D, wherein the engineering strain S02A9B3 had the highest L-serine yield (536.7 mg/L) which was the highest, and had an ethanol yield of 20.4 g/L.

The construction method of the over-expression plasmids pEZ-A1, pEZ-A2, pEZ-A3, pEZ-A4, pEZ-A5, pEZ-A6, pEZ-A7, pEZ-A8, pEZ-A9 and pEZ-A10 can be specifically referred to the preparation process of 39 p-B3.

Enhancement of SerB expression using a strong promoter and increasing copy number

As shown in FIG. 5, the EcSerA sequence with the strong promoter Ptet and the SerB sequence with the strong promoter Pgap were synthesized based on the above examples, and inserted into the vector pEZ A to obtain the over-expression plasmid 39p-B4, and the specific preparation procedure is referred to as 39p-B3 preparation procedure.

In some embodiments, the over-expression plasmid 39p-B4 and the over-expression plasmid pEZ-A9 are simultaneously transferred into the engineering strain S02B4, resulting in the engineering strain S02A4B4-1. As shown in FIG. 5, the engineering strain S02A4B3-1 has a yield of 625.6 mg/L of L-serine after fermentation test.

Overexpression of pgk to enhance the precursor 3-phosphoglycerate

Phosphoglycerate kinase (encoded by pgk, AVZ 41530.1) may catalyze glyceraldehyde 3-phosphate to 3-phosphoglycerate. Overexpression of pgk can enhance carbon flow to 3-phosphoglycerate.

Based on this, as shown in FIG. 6, the example uses Pgk-F (shown in SEQ ID NO. 56) and Pgk-R (shown in SEQ ID NO. 57) to amplify a pgk sequence from ZM4 genome of Zymomonas mobilis and inserts it into over-expression plasmid 39p-B4 to obtain a fifth over-expression plasmid (39 p-B5), the specific preparation procedure being referred to the preparation procedure of 39 p-B3.

In some embodiments, the over-expression plasmid 39p-B5 is electrotransformed into an engineered strain S02A9B5 in the competence of the S02A9 strain. The L-serine yield was measured and shown in FIG. 6A, and was 687.6 mg/L. The consumption of glucose, the production of ethanol and the production of acetic acid are shown in FIG. 6B. Wherein the yield of acetic acid is reduced from 1.08 g/L to 0.528 g/L and the yield of ethanol is 20.4 g/L.

Supplementing nitrogen source to enhance L-serine production

It is very necessary to supplement the nitrogen source during the production of the amino acid. The nitrogen source is one of the nutrients necessary for the growth of microorganisms, and the supplementation of sufficient nitrogen source can promote the growth and propagation of the strain, thereby increasing the yield of amino acids. Different nitrogen sources may cause different metabolic pathways to be activated or inhibited, thereby affecting amino acid synthesis and accumulation.

In some embodiments, a RMG5 medium containing 5 g/L of the nitrogen source glutamate hydrochloride and a RMG5 medium containing 5 g/L of ammonium sulfate are provided. The engineering strain S02A9B5 is inoculated into the two culture media for fermentation test, so that the growth condition of the strain is improved (shown in figure 7), and the L-serine yield is further improved to 855.6 mg/L.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims

1. An engineering strain, wherein the engineering strain is a strain in which the sdaA gene of ZM4 of zymomonas mobilis (Zymomonas mobilis) 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 (Zymomonas mobilis) ZM4 are knocked out.

3. An engineered strain, which is a strain in which the sdaA gene of zymomonas mobilis (Zymomonas mobilis) ZM4 is knocked out, the glyA gene is knocked out, and the EceamA gene is overexpressed.

4. An engineered strain which is a strain in which the sdaA gene, the glyA gene, the EceamA gene, the SerA gene, the SerC gene, and the SerB gene of zymomonas mobilis (Zymomonas mobilis) ZM4 are knocked out.

5. A method of preparing an engineered strain comprising:

obtaining zymomonas mobilis (Zymomonas mobilis) ZM4 as a starting strain;

obtaining a first knockout plasmid pL2R-sdaA of a targeted 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

Transferring the first knockout plasmid pL2R-sdaA into the starting strain, or transferring the first knockout plasmid pL2R-sdaA and the second knockout plasmid pL2R-glyA into the starting strain simultaneously to obtain the engineering strain;

Wherein, the construction steps of the first knockout plasmid pL2R-sdaA comprise:

constructing a first targeting plasmid;

amplifying an upstream sequence of sdaA by using primers sdaAUS-F and sdaAUS-R, amplifying a downstream sequence of sdaA by using primers sdaADS-F and sdaADS-R, wherein sdaAUS-F is shown as SEQ ID NO.10, sdaAUS-R is shown as SEQ ID NO.11, sdaADS-F is shown as SEQ ID NO.12, and sdaADS-R is shown as SEQ ID NO. 13;

ligating the sdaA upstream sequence with the sdaA downstream sequence by Overlap PCR to obtain a first donor fragment as shown in SEQ ID No. 14;

Inverse PCR amplification of the first targeting plasmid by using primers 15Afk-F and 15Afk-R to obtain a reverse amplification fragment of the first targeting plasmid, wherein 15Afk-F is shown as SEQ ID NO.15, and 15Afk-R is shown as SEQ ID NO. 16; and

Assembling and ligating the reverse-expanded fragment of the first targeting plasmid and the first donor fragment according to a ratio of 1:3, and transferring the ligation product into competent cells of escherichia coli; screening by using a spectinomycin plate, picking single colonies, verifying by colony PCR by using pEZ A-F and pEZ A-R primers respectively, and verifying by sequencing that the size of the bands is consistent with the expectations; extracting and separating from the verified positive clone to obtain a first knockout plasmid pL2R-sdaA, wherein pEZ A-F are shown as SEQ ID NO.6, and pEZ A-R are shown as SEQ ID NO. 7;

Wherein the constructing step of the first targeting plasmid comprises the following steps: synthesizing primers grsdaA-F and grsdaA-R, and guiding the cleavage of the target site by the nucleotidase; annealing the grsdaA-F and the grsdaA-R, then connecting to a basic plasmid pEZ Asp, and obtaining the first targeting plasmid through screening; grsdaA-F is shown in SEQ ID NO.4, grsdaA-R is shown in SEQ ID NO. 5;

wherein, the construction steps of the second knockout plasmid pL2R-glyA comprise:

Constructing a second targeting plasmid;

Amplifying a glyA upstream sequence by using primers glyAUS-F and glyAUS-R, amplifying a glyA downstream sequence by using primers glyADS-F and glyADS-R, wherein glyAUS-F is shown as SEQ ID NO.17, glyAUS-R is shown as SEQ ID NO.18, glyADS-F is shown as SEQ ID NO.19, and glyADS-R is shown as SEQ ID NO. 20;

ligating said glyA upstream sequence to said glyA downstream sequence via Overlap PCR to provide a second donor fragment as set forth in SEQ ID NO. 21;

inverse PCR amplification of said second targeting plasmid using said primer 15Afk-F and said primer 15Afk-R to obtain a reverse amplified fragment of said second targeting plasmid;

Assembling and ligating the reverse-expanded fragment of the second targeting plasmid with the second donor fragment in a ratio of 1:3, and transferring the ligation product into E.coli competent cells; screening by using a spectinomycin plate, picking single colonies, respectively carrying out colony PCR verification by using pEZ A-F and pEZ A-R as primer pairs, and verifying by sequencing that the sizes of the strips are consistent with expectations; extracting and separating from the verified positive clone to obtain a second knockout plasmid pL2R-glyA;

Wherein the construction step of the second targeting plasmid comprises the following steps: synthesizing primers grglyA-F and grglyA-R, and guiding the cleavage of the target site by the nucleotidase; annealing the grglyA-F and the grglyA-R, then connecting to a basic plasmid pEZ Asp, and obtaining the second targeting plasmid through screening; grglyA-F is shown as SEQ ID NO.8, grglyA-R is shown as SEQ ID NO. 9.

6. A method of preparing an engineered strain comprising:

obtaining zymomonas mobilis (Zymomonas mobilis) ZM4 as a 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 a third over-expression plasmid 39p-B3 over-expressing EceamA genes;

transferring the first knockout plasmid pL2R-sdaA into the starting strain, or transferring the first knockout plasmid pL2R-sdaA and the second knockout plasmid pL2R-glyA into the starting strain respectively, or transferring the first knockout plasmid pL2R-sdaA, the second knockout plasmid pL2R-glyA and the third overexpression plasmid 39p-B3 into the starting strain respectively; and

So as to obtain the engineering strain;

constructing a first targeting plasmid;

inverse PCR amplification of the first targeting plasmid using primers 15Afk-F and 15Afk-R to obtain a reverse amplified fragment of the first targeting plasmid, wherein 15Afk-F is shown as SEQ ID NO.15, and 15Afk-R is shown as SEQ ID NO. 16; and

Constructing a second targeting plasmid;

Amplification of glyA upstream sequence using primers glyAUS-F and glyAUS-R; amplifying a glyA downstream sequence by using the primers glyADS-F and glyADS-R; glyAUS-F is shown as SEQ ID NO.17, glyAUS-R is shown as SEQ ID NO.18, glyADS-F is shown as SEQ ID NO.19, and glyADS-R is shown as SEQ ID NO. 20;

Wherein the construction step of the second targeting plasmid comprises the following steps: synthesizing primers grglyA-F and grglyA-R, and guiding the cleavage of the target site by the nucleotidase; annealing the grglyA-F and the grglyA-R, then connecting to a basic plasmid pEZ Asp, and obtaining the second targeting plasmid through screening; grglyA-F is shown as SEQ ID NO.8, grglyA-R is shown as SEQ ID NO. 9;

Wherein the construction step of the third over-expression plasmid 39p-B3 comprises:

amplifying the escherichia coli genome by utilizing primers EceamA-F and EceamA-R through PCR to obtain a EceamA sequence; amplifying Zymomonas mobilis ZM genome by using a primer Ptet-F and Ptet-R PCR to obtain Ptet strong promoter sequence shown as SEQ ID NO.28, wherein EceamA-F is shown as SEQ ID NO.26, eceamA-R is shown as SEQ ID NO.27, ptet-F is shown as SEQ ID NO.29, and Ptet-R is shown as SEQ ID NO. 30;

connecting the EceamA sequence with the Ptet sequence through Overlap PCR to obtain a connection sequence of Ptet and EceamA;

reverse amplifying the vector pEZ A with primers 15Afk-F and 15Afk-R to give linearized pEZ A;

Mixing the Ptet and EceamA connecting sequence with linearization pEZ A according to the ratio of 3:1, standing on ice for 5min after the preparation according to the reaction system shown in the following table, adding E.coli competent cells, and transforming by adopting a general method;

Screening was performed using a spectinomycin resistant plate, single colonies were picked, and the PCR amplification procedure using primer pairs 15A-fwd and 15A-rev, respectively, was set as follows: pre-denaturation at 98 ℃ 3 min; denaturation at 98℃for 10 s, annealing at 55℃for 10 s, extension at 72℃for 80 s cycles, verification by colony PCR, verification by sequencing of the band size consistent with expectations; namely extracting and separating the third over-expression plasmid 39p-B3 from positive clones which are verified to be correct; 15A-fwd is shown as SEQ ID NO.31, and 15A-rev is shown as SEQ ID NO. 32.

7. A method of preparing L-serine, comprising:

obtaining an engineering strain according to any one of claims 1 to 4 or an engineering strain prepared by the preparation method according to any one of claims 5 or 6;

fermenting the engineering strain; and

Harvesting said L-serine from said fermentation product.

8. The method of claim 7, further comprising fermenting the engineered strain in a culture broth supplemented with a nitrogen source.

9. The engineered strain of any one of claims 1 to 4 or the engineered strain produced by the production method of any one of claims 5 or 6, wherein the engineered strain is produced by synthesis of L-serine.