CN118406630B - Genetically engineered bacterium for producing L-threonine and preparation method and fermentation process thereof - Google Patents

Genetically engineered bacterium for producing L-threonine and preparation method and fermentation process thereof Download PDF

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CN118406630B
CN118406630B CN202410833385.3A CN202410833385A CN118406630B CN 118406630 B CN118406630 B CN 118406630B CN 202410833385 A CN202410833385 A CN 202410833385A CN 118406630 B CN118406630 B CN 118406630B
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threonine
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CN118406630A (en
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卢煜
曹国强
李婧瑶
邹德勇
张宇擎
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Harbin Xiangbai Biotechnology Co ltd
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Abstract

A genetically engineered bacterium for producing L-threonine, a preparation method and a fermentation process thereof belong to the technical field of genetic engineering. The invention provides a preparation method of a genetic engineering bacterium for producing L-threonine, which aims to solve the technical problems of large carbon consumption, low conversion rate and byproduct accumulation of strains for fermenting and synthesizing L-threonine in the prior art, obtains the genetic engineering bacterium XY-Ta33 by knocking out ptsI, pyk, pck and ackA genes and introducing glk, galp, xfpk and ppc genes of escherichia coli ACThr, and provides a fermentation process for producing L-threonine by using the engineering bacterium. The genetic engineering bacterium XY-Ta33 and the fermentation process provided by the invention have the advantages that the yield of L-threonine and the sugar acid conversion rate are improved, the production of byproduct acetic acid is reduced, the fermentation production efficiency is improved, and the method is suitable for large-scale industrial production of L-threonine.

Description

Genetically engineered bacterium for producing L-threonine and preparation method and fermentation process thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to a genetic engineering bacterium, a preparation method and a fermentation process thereof.
Background
L-threonine is one of 8 amino acids necessary for growth of humans and animals, plays an important role in growth and development of humans and animals, and is widely used in aspects of feed, food additives, medical products, and the like.
The production methods of L-threonine include protein hydrolysis, chemical synthesis, direct fermentation and enzymatic methods, which are not basically used in industrial production because of various disadvantages; the L-threonine produced by the enzyme method has the characteristics of high specificity, single product and easy refining, but the required enzyme is difficult to obtain, so that the application of the enzyme method is restricted; the direct fermentation method has the advantages of low production cost, resource saving and small environmental pollution, and is a main mode for industrially producing L-threonine at present. However, the strain used in the direct fermentation method in the prior art consumes more carbon, thereby increasing the fermentation cost; the traditional mutagenized fermentation strain grows slowly, the specific site where mutation occurs is difficult to judge, more by-product acetic acid is produced in the fermentation process, the conversion rate is low, and the requirement of large-scale industrial production cannot be met.
In summary, in the art, for the production technology of L-threonine, the improvement of the conversion rate and the reduction of the accumulation of by-product acetic acid have been technical problems which all technicians have been dedicated to solve, and the core technology for the technical problems is the fermentation strain.
Disclosure of Invention
The invention provides a genetically engineered bacterium for producing L-threonine, a preparation method and a fermentation process thereof, and aims to solve the technical problems of large carbon consumption, low conversion rate and byproduct accumulation of strains for fermenting and synthesizing L-threonine in the prior art.
The invention aims at providing a preparation method of genetically engineered bacteria for producing L-valine, which comprises the following steps:
s1: transferring the vector into competent cells of an original strain, and preparing electrotransformation competent cells to obtain a strain to be modified;
S2: inserting glk genes and galp genes into the strain to be modified obtained in the step S1 by utilizing a genome editing tool carrier pGRB, and knocking out ptsI genes to obtain genetically engineered bacteria XY-Ta36;
s3: carrying out knockout of pykA and pykF genes on the genetically engineered bacterium XY-Ta36 obtained in the S2 by utilizing a genome editing tool carrier pGRB to obtain genetically engineered bacterium XY-Ta38;
S4: inserting xfpk genes into the genetically engineered bacterium XY-Ta38 obtained in the step S3 by utilizing a genome editing tool carrier pGRB to obtain genetically engineered bacterium XY-Ta40;
S5: inserting ppc genes and knocking out Pck genes of the genetically engineered bacteria XY-Ta40 obtained in the step S4 by utilizing a genome editing tool carrier pGRB to obtain genetically engineered bacteria XY-Ta57;
s6: carrying out ackA gene knockout on the genetically engineered bacterium XY-Ta57 obtained in the step S5 by utilizing a genome editing tool carrier pGRB to obtain genetically engineered bacterium XY-Ta33;
The starting strain in the S1 is Escherichia coli ACThr1032, the preservation number of ACThr strain is CGMCCN0.16144, the strain is classified and named as Escherichia coli, and the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), the preservation address is North Star, the university of China academy of sciences 3, the Korean area of Beijing, and the preservation date is 2018, 7, 23 days;
the nucleotide sequence of the glk gene is shown as SEQ ID No. 1;
The nucleotide sequence of galp gene is shown in SEQ ID No. 2;
the nucleotide sequence of xfpk gene is shown in SEQ ID No. 6;
the nucleotide sequence of the ppc gene is shown in SEQ ID No. 7.
In a preferred embodiment of the invention, the vector described in S1 is pRed-Cas9.
In a preferred embodiment of the present invention, the nucleotide sequence of the ptsI gene described in S2 is shown in SEQ ID No. 3.
In a preferred embodiment of the present invention, the nucleotide sequence of the pykA gene as set forth in S3 is shown in SEQ ID No.4 and the nucleotide sequence of the pykF gene is shown in SEQ ID No. 5.
In a preferred embodiment of the present invention, the nucleotide sequence of the Pck gene described in S5 is shown in SEQ ID No. 8; the nucleotide sequence of the ackA gene described in S6 is shown in SEQ ID No. 9.
The second object of the invention is to provide a genetically engineered bacterium XY-Ta33, wherein the genetically engineered bacterium XY-Ta33 is obtained by adopting the preparation method.
The invention further aims to provide a fermentation process for producing L-threonine, which comprises inoculating the genetically engineered bacterium XY-Ta33 into a fermentation culture medium, automatically controlling and maintaining pH within 7.0-7.2 by using 25% ammonia water at 37 ℃, and controlling dissolved oxygen to be more than or equal to 30% by adjusting pressure, rotating speed and air quantity to maintain the dissolved oxygen; and after fermentation starts 5h, liquid glucose is added, the concentration of residual sugar in the fermentation tank is maintained to be less than or equal to 1.0 g/L, and fermentation broth containing L-threonine is obtained after fermentation is completed.
In a preferred embodiment of the invention, the inoculation amount of the genetically engineered bacterium XY-Ta33 in the fermentation process is 20-30%.
In a preferred embodiment of the invention, the fermentation time of the fermentation process is 29 h, the pressure in the fermentation process is 0.05 MPa, the rotating speed is adjusted to be 200-600 rpm, and the air quantity is adjusted to be 0.2-1 m 3/h.
The invention has the beneficial effects that: the invention provides a genetic engineering bacterium for producing L-threonine and a preparation method thereof, wherein the genetic engineering bacterium XY-Ta33 is obtained by knocking out ptsI, pyk, pck and ackA genes and introducing glk, galp, xfpk and ppc genes of escherichia coli ACThr. The genetic engineering bacterium XY-Ta33 provided by the invention improves the accumulation amount of phosphoenolpyruvic acid (PEP) in the process of producing L-threonine, reduces the loss of carbon, promotes fructose-6-phosphate to generate acetyl phosphate (AcP), furthest converts the acetyl phosphate into L-threonine precursor oxaloacetic acid (OAA), simultaneously reduces the synthesis of byproduct acetic acid, further improves the yield and conversion rate of L-threonine, and has good growth capacity compared with a starting strain.
The invention also provides a fermentation process for producing L-threonine by utilizing the genetically engineered bacterium XY-Ta33, the yield of the L-threonine produced by the fermentation process is 133.88 g/L, the yield is improved by 18.6% compared with that of the original strain Escherichia coli ACThr1032, and the conversion rate is improved by 22.6% compared with that of the original strain. Therefore, the genetic engineering bacterium XY-Ta33 provided by the invention is combined with a fermentation process, so that the production of byproduct acetic acid is reduced while the yield and conversion rate of L-threonine are improved, the strain can efficiently produce and accumulate L-threonine, the fermentation production efficiency is improved, and the requirement of large-scale industrial production can be met.
Detailed Description
The reagents, instruments or equipment used in the invention can be purchased through commercial paths unless special specifications are available, the related experimental methods are implemented by using the kit according to the operation of the kit, and the other methods are conventional molecular biology experimental operations unless special specifications are available.
Abbreviations for genes involved in the examples of the present invention:
glk: a glucokinase encoding gene;
galp: galactose/hydrogen ion cotransporter encoding genes;
ptsI: a phosphotransferase I encoding gene;
pyk: a pyruvate kinase I encoding gene;
xfpk: a phosphoketolase encoding gene;
ppc: a phosphoenolpyruvate carboxylase encoding gene;
pck: a phosphoenolpyruvate carboxykinase encoding gene;
ackA: acetate kinase coding gene.
The culture medium is fermented by a 24-deep pore plate tank:
Slant Medium (g/L): sucrose 2.0 g, ammonium chloride 1.0 g, kh 2PO41.5 g,NaHPO43.5 g,MgSO4·7H2 O0.1 g, agar 20 g, ph=7.0-7.2;
Seed medium (g/L): sucrose 40.0 g,（NH4）2SO410.0 g,KH2PO41.0 g,MgSO4·7H2O 0.5 g, yeast extract 2.0 g,MOPS 15.0 g;
Fermentation medium (g/L): sucrose 80.0 g,（NH4）2SO425.0 g,KH2PO42.0 g,MgSO4·7H2O 1.0 g, yeast extract 4.0 g, feSO 4·5H2O 0.5 g,MnSO4·5H2 O0.5 g,MOPS 30.0 g.
The fermentation steps of the 24-deep pore plate tank in the invention are as follows:
(1) Activating strains: taking out glycerol tube strain from a refrigerator at-80 ℃ and naturally melting, dipping 3 rings of the melted strain on a solid slant culture medium by using an inoculating loop in a sterile environment, scribing, and inversely culturing at 36.5 ℃ to 19 h;
(2) And (3) dilution coating: inoculating the cultured slant strain in 50 ml sterilized normal saline with glass beads, scattering thallus by using the glass beads, diluting the bacterial suspension OD to about 0.3-0.4 in a gradient manner to 10 -5 and 10 -6, respectively sucking 0.3 ml of the diluted bacterial liquid, coating the bacterial liquid on a flat plate, vertically culturing the flat plate at 36.5 ℃ for 3 h, reversely and continuously culturing until 24 h is reached, and taking out;
(3) Inoculating: adding 800 mu L of fermentation medium into the deep hole plate before inoculation, using a 10 ul gun head to insert the single colony cultured in the step (2) into the deep hole plate, sealing the deep hole plate by using a sealing plate film, and carrying out shake culture on the single colony at 36.5 ℃ and under the condition of 800 rpm to obtain 18 h;
(4) Detection of pH, OD, acid production, residual sugar and volatilization:
pH: detecting by using pH test paper;
OD: taking 0.2 ml of fermentation liquor, adding 1.8 ml of water, diluting for 20 times to a detection plate, and measuring absorbance by using an enzyme-labeled instrument under the condition of 562 and nm wavelength;
Residual sugar: centrifuging the deep hole plate, diluting the supernatant 100 times, and detecting by using a biosensing analyzer;
Acid production: detecting by chromatography visual method and liquid phase method;
The volatilization amount is as follows: and measuring the volume of the fermentation liquid after the fermentation is finished, and calculating the volatilization amount of the fermentation liquid.
The starting strain used in the invention is escherichia coli ACThr1032, the preservation number of ACThr1032 strain is CGMCCN0.16144, the classification name is ESCHERICHIA COLI, the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), the preservation address is North Star Xiya 1, 3, china academy of sciences of China, and the preservation date is 2018, 7 months and 23 days.
Example 1: preparation of genetically engineered bacterium XY-Ta36
S1: the website (http:// www.rgenome.net/cas-designer /) is used for designing sgRNA, the sgRNA is synthesized by dimerization of an upstream primer 001-ptsI-sg-F (shown as SEQ ID No. 10) and a downstream primer 002-ptsI-sg-R (shown as SEQ ID No. 11), and the primer dimerization reaction system is as follows: upstream primer 10 [ mu ] L and downstream primer 10 [ mu ] L; the reaction procedure is: 95℃5 min, 55℃1 min, 30℃1 min, 22℃infinity; diluting the obtained sgRNA to 20 ng/MuL after dimerization to obtain diluted sgRNA;
S2: PCR amplifying pGRB vector plasmid DNA by using an upstream primer pGRB-F (shown as SEQ ID No. 12) and a downstream primer pGRB-R (shown as SEQ ID No. 13) to obtain a PCR amplified product;
the PCR amplification reaction system comprises: 2.5 mu L of upstream primer, 2.5 mu L, pGRB of downstream primer, 2.5 mu L, PFU of vector plasmid DNA, 25 mu L of enzyme and ddH 2 O18 mu L;
the PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 15 s-58 ℃, annealing at 15 s-72 ℃ for 1 min, circulating for 35 times, thoroughly extending at 72 ℃ for 5 min, and preserving at 4 ℃;
S3: digesting the PCR amplification product obtained in S2 through DpnI, removing circular pGRB plasmid DNA, reacting 1h at 37 ℃, detecting through agarose gel electrophoresis, cutting gel for recovery of correct bands with the size of an electrophoresis detection fragment of 2000 bp for standby, obtaining linearized pGRB vector plasmid DNA, and diluting the linearized pGRB vector plasmid DNA to the concentration of 50 ng/mu L;
S4: ligating the sgRNA obtained in S1 with the linearized pGRB vector plasmid DNA obtained in S3 by using ligase c112 (purchased from vazemy), reacting at 37 ℃ for 30min, transforming competent cells of escherichia coli, ice-bath for 30min, heat-beating for 45S at 42 ℃ and rapidly ice-bath for 2 min, recovering at 37 ℃ for 1h, coating an Amp-resistant plate, culturing at 37 ℃ overnight, selecting a monoclonal strain, directly sequencing, and selecting a colony with correct sequencing result to extract plasmid DNA for standby, thereby obtaining ptsI-sgRNA-pGRB;
S5: primer5 software is used for designing repair template fusion PCR primers, 156-ptsI-up-F (shown as SEQ ID No. 14) and 157-ptsI-up-R (shown as SEQ ID No. 15) are used as primers for amplifying homologous arms at the upstream of ptsI genes, 158-ptsI-down-F (shown as SEQ ID No. 16) and 159-ptsI-down-R (shown as SEQ ID No. 17) are used as primers for amplifying homologous arms at the downstream of ptsI genes, and fragments of homologous arms at the upstream and downstream of ptsI genes are obtained;
Amplifying the glk gene fragment by using 154-galp +glk-pGRB-F2 (shown as SEQ ID No. 18) and 001-glk-R (shown as SEQ ID No. 19) as primers;
amplifying galP gene fragments by using 001-galP-F (shown as SEQ ID No. 20) and 155-galP +glk-pGRB-R (shown as SEQ ID No. 21) as primers;
Connecting the galp gene fragment and the glk gene fragment obtained above between upstream and downstream homology arms of the ptsI gene by fusion PCR through an upstream primer 156-ptsI-up-F (shown as SEQ ID No. 14) and a downstream primer 159-ptsI-down-R (shown as SEQ ID No. 17), detecting through agarose gel electrophoresis, and performing gel cutting recovery on an amplification product with the size of 3500 bp of the electrophoresis detection fragment for standby to obtain galp genes and glk gene repair templates;
s6: transferring the tool vector pRed-Cas9 plasmid into a chemically transformed competent cell of the original strain ACThr1032, and obtaining an electrically transformed competent cell of the ACThr1032-pRed-Cas9 strain by the following steps;
(S6-1) cell culture:
S6-1-1: taking ACThr-pRed-Cas 9 bacterial liquid streaked LB solid plates out of the freezing tube, and standing and culturing overnight at 37 ℃ (12 h-16 h);
S6-1-2: picking single colony from LB solid plate in S6-1-1, inoculating into 5mL LB liquid culture medium, shake culturing overnight at 30deg.C under 220 rpm (12 h-16 h);
S6-1-3: taking ACThr1032-pRed-Cas9 bacterial liquid which is cultured at 500 mu L overnight in S6-1-2, inoculating the bacterial liquid into a 500 mL LB liquid culture medium, and carrying out shake culture under the conditions of 30 ℃ and 220 rpm until OD 660 =0.7-0.9 (about 4-6 h) to obtain ACThr1032-pRed-Cas9 bacterial liquid;
(S6-2) preparation of electrotransformation competent cells:
S6-2-1: placing ACThr1032-pRed-Cas9 bacterial liquid which is qualified in OD 660 detection in (S6-1) on ice for precooling by 20-30 min, transferring to a precooled 50mL centrifuge tube, centrifuging for 10 min under the condition of 4500 rpm at 4 ℃, and discarding the supernatant to obtain ACThr1032-pRed-Cas9 bacterial body; adding 5mL pre-cooled sterile 10% glycerol into the thalli, gently blowing and beating uniformly, then supplementing to 30mL by using 10% glycerol, centrifuging for 10 min under the condition of 4500 rpm at 4 ℃, discarding the supernatant, repeating for 2 times to obtain ACThr1032-pRed-Cas9 thalli precipitate;
s6-2-2: adding 500 mu L of pre-cooled 10% glycerol to the ACThr-pRed-Cas 9 bacterial precipitate obtained in S6-2-1, re-suspending, and sub-packaging into pre-cooled 1.5 mL EP tubes according to the volume of 90 mu L per tube to obtain electric transformation competent cells of ACThr1032-pRed-Cas9 bacterial strain, and immediately using or preserving at-80 ℃;
S7: the electrotransformation competent cells of the ACThr1032-pRed-Cas9 strain obtained in S6 were edited using the ptsI-sgRNA-pGRB obtained in S4 and the repair template obtained in S5 at a loading concentration of ptsI-sgRNA-pGRB: repair template = 1:2, performing electric shock transformation by using a 0.1 cm electric shock cup 1.8 KV, adding an LB liquid culture medium containing IPTG (100 mM) after transformation, inducing 4h at 30 ℃, coating a Spe and Amp resistance plate, culturing 30 h at 30 ℃ and then forming single colonies on the plate, aligning the single colonies on a new culture medium, and picking streaked colonies; PCR verification is carried out by using a primer ptsI-genome identification-F (shown as SEQ ID No. 22) and a downstream primer ptsI-genome identification-R (shown as SEQ ID No. 23), detection is carried out by agarose gel electrophoresis, and the correct band size is 3700 bp by electrophoresis; and performing pGRB plasmid loss by using arabinose to induce the colony verified by PCR, and performing shaking culture to induce pRed-Cas9 plasmid loss at 37 ℃ to obtain ACThr1032 delta ptsI, glk, galp strain, which is called as genetic engineering bacterium XY-Ta36 for short.
This example attenuated the phosphoenolpyruvate (PEP) -depleted sugar transport system by knocking out the ptsI gene; and enhancing accumulation of PEP by enhancing a glucose kinase-mediated sugar transport system by expressing galp and glk genes, and increasing accumulation of L-threonine precursor Oxaloacetate (OAA).
In this example, 24 deep-hole plate tank fermentation verification is performed on the obtained genetically engineered bacterium XY-Ta36, and the result is shown in Table 1, wherein the yield of L-threonine synthesized by the original strain and the genetically engineered bacterium XY-Ta36 is similar, the conversion rate of the genetically engineered bacterium XY-Ta36 is 39.01% and lower than that of the original strain, and the reason is that the consumption of PEP is reduced due to the change of a sugar transport system, but the conversion rate of the genetically engineered bacterium XY-Ta36 is not completely converted into L-threonine.
TABLE 1
Example 2: preparation of genetically engineered bacterium XY-Ta38
S1: the web site (http:// www.rgenome.net/cas-designer /) is used to design sgRNA, and the pykA-sgRNA is synthesized by dimerization of the upstream primer 161-pykAsg1-F (shown as SEQ ID No. 24) and the downstream primer 162-pykAsg1-R (shown as SEQ ID No. 25);
Dimerization of the upstream primer 203-pykFsg4-F (shown as SEQ ID No. 26) and the downstream primer 204-pykFsg-R (shown as SEQ ID No. 27) to obtain pykF-sgRNA;
the primer dimerization reaction system is as follows: upstream primer 10 [ mu ] L and downstream primer 10 [ mu ] L; the reaction procedure is: 95℃5min, 55℃1 min, 30℃1 min, 22℃infinity; diluting the obtained sgRNA to 20 ng/MuL after dimerization to obtain diluted pykA-sgRNA and pykF-sgRNA;
S2: the pykA-sgRNA and pykF-sgRNA obtained in S1 were ligated with linearized pGRB vector plasmid DNA obtained in S3 of example 1, respectively, using ligase c112 (purchased from vazemy), the ligation method being described in the description; converting competent cells of escherichia coli after reaction at 37 ℃ for 30min, carrying out ice bath for 30min, carrying out heat shock for 45 s at 42 ℃, carrying out ice bath for 2 min rapidly, recovering at 37 ℃ for 1h, coating an Amp resistance plate, culturing at 37 ℃ overnight, selecting a monoclonal strain for direct sequencing, and selecting a colony with correct sequencing result for extracting plasmid DNA for later use to obtain pykA-sgRNA-pGRB and pykF-sgRNA-pGRB;
S3: PCR primers were designed using primer5 software and 165-pykAup-F (shown as SEQ ID No. 28) and 166-pykAup-R (shown as SEQ ID No. 29) were used as primers to amplify the upstream homology arm of the pykA gene; amplifying the downstream homology arm of the pykA gene by using 167-pykAdown-F (shown as SEQ ID No. 30) and 168-pykAdown-R (shown as SEQ ID No. 31) as primers to obtain an upstream homology arm fragment and a downstream homology arm fragment of the pykA gene;
amplifying the upstream homology arm of the pykF gene by using 175-pykFup-F (shown as SEQ ID No. 32) and 176-pykFup-R (shown as SEQ ID No. 33) as primers; amplifying a downstream homology arm of the pykF gene by using 177-pykFdown-F (shown as SEQ ID No. 34) and 178-pykFdown-R (shown as SEQ ID No. 35) as primers to obtain an upstream homology arm fragment and a downstream homology arm fragment of the pykF gene;
The pykA gene repair template is obtained by fusion PCR amplification of the pykA gene by taking a homologous arm fragment at the upstream and downstream of the pykA as a template and 165-pykAup-F (shown as SEQ ID No. 28) and 168-pykAdown-R (shown as SEQ ID No. 31) as primers;
the pykF gene repair template is obtained by fusion PCR amplification of the pykF gene by taking the upstream and downstream homologous arm fragments of the pykF as templates and 175-pykFup-F (shown as SEQ ID No. 32) and 178-pykFdown-R (shown as SEQ ID No. 35) as primers;
S4: transferring a tool vector pRed-Cas9 plasmid into the chemically transformed competent cells of the XY-Ta36 strain obtained in the S6 of the example 1, and obtaining the electrically transformed competent cells of the XY-Ta36-pRed-Cas9 strain through electric transformation;
S5: the XY-Ta36-pRed-Cas9 strain obtained in S4 was edited using the pykA-sgRNA-pGRB obtained in S2 and the pykA gene repair template obtained in S3 at a loading concentration of pykA-sgRNA-pGRB: repair template = 1:2, performing electric shock transformation by using a 0.1 cm electric shock cup 1.8 KV, adding an LB liquid culture medium containing IPTG (100 mM) after transformation, inducing 4 h at 30 ℃, coating a Spe and Amp resistance plate, culturing 30 h at 30 ℃ and then forming single colonies on the plate, aligning the single colonies on a new culture medium, and picking streaked colonies; the pykA gene was identified by using the upstream primer 169-pykA genome-F (shown as SEQ ID No. 36) and the downstream primer 170-pykA genome-R (shown as SEQ ID No. 37), and detected by agarose gel electrophoresis, the correct band size for the electrophoresis detection was 1384 bp;
s6: the XY-Ta36-pRed-Cas9 strain obtained in S4 was edited using the pykF-sgRNA-pGRB obtained in S2 and the pykF gene repair template obtained in S3 at a loading concentration of pykF-sgRNA-pGRB: repair template = 1:2, performing electric shock transformation by using a 0.1 cm electric shock cup 1.8 KV, adding an LB liquid culture medium containing IPTG (100 mM) after transformation, inducing 4h at 30 ℃, coating a Spe and Amp resistance plate, culturing 30 h at 30 ℃ and then forming single colonies on the plate, aligning the single colonies on a new culture medium, and picking streaked colonies; identifying the pykF gene by using an upstream primer 179-pykF genome-F (shown as SEQ ID No. 38) and a downstream primer 180-pykF genome-R (shown as SEQ ID No. 39), detecting by agarose gel electrophoresis, and detecting by electrophoresis that the correct band size is 1180 bp;
S7: and inducing the bacterial colony which passes the PCR verification in the S5 and the S6 by utilizing arabinose to carry out pGRB plasmid loss, and then inducing pRed-Cas9 plasmid loss by shaking bacteria at 37 ℃ to obtain an XY-Ta36 ΔpykAΔ pykF strain, which is called as a genetically engineered bacterium XY-Ta38 for short.
In the embodiment, 24 deep-hole plate tank fermentation verification is carried out on the obtained genetically engineered bacterium XY-Ta38, and the result is shown in Table 2, and the growth of the genetically engineered bacterium XY-Ta38 in a fermentation medium is affected, so that L-threonine is not synthesized; in this example, the pyk gene was knocked out, resulting in complete interruption of pyruvate synthesis and failure to produce acetyl-CoA, a precursor of tricarboxylic acid cycle, thereby failing to normally grow the genetically engineered bacterium XY-Ta38 in the fermentation medium.
TABLE 2
Example 3: preparation of genetically engineered bacterium XY-Ta40
S1: the website (http:// www.rgenome.net/cas-designer /) is used for designing the sgRNA, and the sgRNA is synthesized by dimerization of an upstream primer 110-yfaS-sg1-F (shown as SEQ ID No. 40) and a downstream primer 111-yfaS-sg1-R (shown as SEQ ID No. 41);
The primer dimerization reaction system is as follows: upstream primer 10 [ mu ] L and downstream primer 10 [ mu ] L; the reaction procedure is: 95℃5min, 55℃1 min, 30℃1 min, 22℃infinity; diluting the obtained sgRNA to 20 ng/MuL after dimerization to obtain diluted sgRNA;
S2: ligating the sgRNA obtained in S1 with the linearized pGRB vector plasmid DNA obtained in S3 of example 1 using ligase c112 (purchased from vazemy), the ligation method is described in the specification, reacting 30 min and then transforming E.coli competent cells at 37 ℃, ice-bath 30 min, heat-shocking 45S at 42 ℃ and rapidly ice-bath 2 min, recovering 1h at 37 ℃, coating Amp-resistant plates, culturing overnight at 37 ℃, selecting monoclonal strains for direct sequencing, and selecting colonies with correct sequencing results for extracting plasmid DNA for later use to obtain yfaS-sgRNA-pGRB;
S3: designing PCR primers by using primer5 software, and amplifying an upstream homology arm of the xfpk gene by using 102-yfaS-up-F (shown as SEQ ID No. 42) and 205-yfas-xfpk-up-R (shown as SEQ ID No. 43) as primers; the downstream homology arm of yfaS gene is amplified by PCR with 208-yfas-xfpk-down-F (shown as SEQ ID No. 44) and 105-yfaS-down-R (shown as SEQ ID No. 45) as primers to obtain xfpk gene and yfaS gene upstream and downstream homology arm fragments;
the gene fragment xfpk is obtained by PCR amplification of xfpk gene with 206-xfpk-yfaS-F (shown as SEQ ID No. 46) and 207-xfpk-yfaS-R (shown as SEQ ID No. 47) as primers;
Taking the upstream and downstream homologous arm fragments of yfaS gene as templates, and fusing the obtained xfpk gene fragments with the upstream and downstream homologous arm fragments of yfaS gene by fusion PCR by using an upstream primer 102-yfaS-up-F (shown as SEQ ID No. 42) and a downstream primer 105-yfaS-down-R (shown as SEQ ID No. 45) to obtain repair templates of xfpk and yfaS genes;
s4: transferring the tool vector pRed-Cas9 plasmid into the chemically transformed competent cells of the XY-Ta38 strain obtained in S7 of example 2, and obtaining the electrically transformed competent cells of the XY-Ta38-pRed-Cas9 strain by electrotransformation;
S5: editing the repair templates obtained in yfaS-sgRNA-pGRB and S3 obtained in S2, and the XY-Ta38-pRed-Cas9 strain obtained in S4, wherein the loading concentration is yfaS-sgRNA-pGRB: repair template = 1:2, performing electric shock transformation by using a 0.1 cm electric shock cup 1.8 KV, adding an LB liquid culture medium containing IPTG (100 mM) after transformation, inducing 4h at 30 ℃, coating a Spe and Amp resistance plate, culturing 30 h at 30 ℃ and then forming single colonies on the plate, aligning the single colonies on a new culture medium, and picking streaked colonies; identifying xfpk gene by using upstream primer 130-yfaS genome-F (shown as SEQ ID No. 48) and downstream primer 131-yfaS genome-R (shown as SEQ ID No. 49), detecting by agarose gel electrophoresis, and detecting by electrophoresis that the correct band size is 1390 bp; and performing pGRB plasmid loss by using arabinose to induce the colony verified by PCR, and performing shaking culture to induce pRed-Cas9 plasmid loss at 37 ℃ to obtain XY-Ta38 Δ yfaS: xfpk strain, which is called as genetic engineering bacterium XY-Ta40 for short.
In the embodiment, the exogenous xfpk gene is introduced to catalyze fructose-6-phosphate to generate acetyl phosphate (AcP), and further a precursor acetyl coenzyme A required by tricarboxylic acid circulation is generated to supplement the phenomenon of abnormal strain growth caused by deletion of pyruvic acid, so that the genetically engineered bacterium XY-Ta40 is recovered to normally grow in a fermentation medium.
In this example, 24 deep-hole plate tank fermentation verification is performed on the obtained genetically engineered bacterium XY-Ta40, and the result is shown in Table 3, the genetically engineered bacterium XY-Ta40 grows normally in a fermentation medium, the yield of synthesized L-threonine is slightly lower than that of the original strain, but the acid production rate and the sugar acid conversion rate of the genetically engineered bacterium XY-Ta40 are lower than those of the original strain, and the reason is that PEP accumulated in the early stage is not completely converted into L-threonine precursor OAA.
TABLE 3 Table 3
Example 4: preparation of genetically engineered bacterium XY-Ta57
S1: the web site (http:// www.rgenome.net/cas-designer /) is used for designing sgRNA, and pck-sgRNA is synthesized by dimerization of an upstream primer 124-pck-sg 2-F (shown as SEQ ID No. 50) and a downstream primer 125-pck-sg 2-R (shown as SEQ ID No. 51);
Dimerization of the upstream primer 135-yjivsg2-F (shown as SEQ ID No. 52) and the downstream primer 136-yjivsg2-R (shown as SEQ ID No. 53) to form yjiv-sgRNA;
The primer dimerization reaction system is as follows: upstream primer 10 [ mu ] L and downstream primer 10 [ mu ] L; the reaction procedure is: 95℃5min, 55℃1 min, 30℃1 min, 22℃infinity; diluting the obtained sgRNA to 20 ng/MuL after dimerization, and obtaining pck-sgRNA and yjiv-sgRNA obtained after dilution;
S2: ligating the pck-sgRNA and yjiv-sgRNA obtained in S1 with the linearized pGRB vector plasmid DNA obtained in S3 of example 1, respectively, using ligase c112 (purchased from vazemy), reacting at 37 ℃ for 30 min, then transforming competent cells of E.coli, ice-bath for 30 min, heat-shocking for 45S at 42 ℃, rapidly ice-bath for 2 min, resuscitating for 1h at 37 ℃ and then coating Amp-resistant plates, culturing overnight at 37 ℃, selecting monoclonal strains for direct sequencing, and selecting colonies with correct sequencing results for extracting plasmid DNA for later use to obtain pck-sgRNA-pGRB and yjiv-sgRNA-pGRB;
S3: designing PCR primers by using primer5 software, and amplifying an upstream homology arm of a pck gene by using 126-pckup-F (shown as SEQ ID No. 54) and 127-pckup-R (shown as SEQ ID No. 55) as primers; 128-pckdown-F (shown as SEQ ID No. 56) and 129-pckdown-R (shown as SEQ ID No. 57) are used as primers, and a pck gene downstream homology arm fragment is obtained by PCR amplification of the pck gene downstream homology arm;
Amplifying the upstream homology arm of the yjiv gene by PCR with 137-yjivup-F (shown as SEQ ID No. 58) and 138-yjivup-R (shown as SEQ ID No. 59) as primers; 141-yjivdown-F (shown as SEQ ID No. 60) and 142-yjivdown-R (shown as SEQ ID No. 61) are used as primers, and a downstream homology arm of the yjiv gene is amplified by PCR to obtain an upstream homology arm fragment and a downstream homology arm fragment of the yjiv gene;
the ppc gene fragment is obtained by PCR amplification of the ppc gene with 139-ppc-F (shown as SEQ ID No. 62) and 140-ppc-R (shown as SEQ ID No. 63) as primers;
the upstream and downstream homology arm fragments of the pck gene are used as templates, and the upstream primer 126-pckup-F (shown as SEQ ID No. 54) and the downstream primer 129-pckdown-R (shown as SEQ ID No. 57) are used for amplifying the upstream and downstream homology arms of the pck gene through fusion PCR to obtain a repair template of the upstream and downstream homology arms of the pck gene;
The ppc gene fragment obtained above is connected between upstream and downstream homologous arms of yjiv genes by fusion PCR by using an upstream primer 137-yjivup-F (shown as SEQ ID No. 58) and a downstream primer 142-yjivdown-R (shown as SEQ ID No. 61), so as to obtain a ppc gene repair template;
S4: transferring a tool vector pRed-Cas9 plasmid into the chemically transformed competent cells of the XY-Ta40 strain obtained in S5 of example 3, and obtaining the electrically transformed competent cells of the XY-Ta40-pRed-Cas9 strain through electric transformation;
S5: editing the XY-Ta40-pRed-Cas9 strain obtained in S4 by using the pck-sgRNA-pGRB obtained in S2 and a pck gene upstream and downstream homology arm repair template obtained in S3, wherein the loading concentration is pck-sgRNA-pGRB: repair template = 1:2, performing electric shock transformation by using a 0.1 cm electric shock cup 1.8 KV, adding an LB liquid culture medium containing IPTG (100 mM) after transformation, inducing 4h at 30 ℃, coating a Spe and Amp resistance plate, culturing 30 h at 30 ℃ and then forming single colonies on the plate, aligning the single colonies on a new culture medium, and picking streaked colonies; identifying pck gene by using an upstream primer 150-pck genome-F (shown as SEQ ID No. 64) and a downstream primer 151-pck genome-R (shown as SEQ ID No. 65), detecting by agarose gel electrophoresis, wherein the correct band size detected by electrophoresis is 1372 bp;
S6: editing the XY-Ta40-pRed-Cas9 strain obtained in S4 by using yjiv-sgRNA-pGRB obtained in S2 and the ppc gene repair template obtained in S3, wherein the loading concentration is yjiv-sgRNA-pGRB: repair template = 1:2, performing electric shock transformation by using a 0.1 cm electric shock cup 1.8 KV, adding an LB liquid culture medium containing IPTG (100 mM) after transformation, inducing 4 h at 30 ℃, coating a Spe and Amp resistance plate, culturing 30 h at 30 ℃ and then forming single colonies on the plate, aligning the single colonies on a new culture medium, and picking streaked colonies; identifying yjiv gene by using upstream primer yjiv genome-F (shown as SEQ ID No. 66) and downstream primer yjiv genome-R (shown as SEQ ID No. 67), detecting by agarose gel electrophoresis, detecting the correct band size by electrophoresis to 1139 bp, and preparing electrotransformation competent cells;
s7: and inducing the bacterial colony of the S5 and the S6 through PCR to carry out pGRB plasmid loss, and inducing pRed-Cas9 plasmid loss by shaking at 37 ℃ to obtain the XY-Ta40 Δ pck, namely ppc bacterial strain, namely gene engineering bacterium XY-Ta57.
In the embodiment, the pck gene is knocked out and the ppc gene is inserted, so that the capability of transforming the strain XY-Ta57 PEP into L-threonine precursor OAA is enhanced, a large amount of PEP accumulated in early transformation is transformed into L-threonine precursor OAA, and the yield and the conversion rate of L-threonine are improved.
In the embodiment, 24 deep-hole plate tank fermentation verification is carried out on the obtained genetically engineered bacterium XY-Ta57, the result is shown in Table 4, the yield of L-threonine synthesized by the genetically engineered bacterium XY-Ta57 is 10.67 g/L which is higher than 9.66 g/L of the original strain, and the sugar acid conversion rate of the genetically engineered bacterium XY-Ta57 is 49.02% which is far higher than 39.01% of the original strain.
TABLE 4 Table 4
Example 5: preparation of genetically engineered bacterium XY-Ta33
S1: the web site (http:// www.rgenome.net/cas-designer /) is used for designing the sgRNA, and the sgRNA is synthesized by dimerization of an upstream primer 191-ackAsg2-F (shown as SEQ ID No. 68) and a downstream primer 192-ackAsg2-R (shown as SEQ ID No. 69);
The primer dimerization reaction system is as follows: upstream primer 10 [ mu ] L and downstream primer 10 [ mu ] L; the reaction procedure is: 95℃5min, 55℃1 min, 30℃1 min, 22℃infinity; diluting the obtained sgRNA to 20 ng/MuL after dimerization to obtain diluted sgRNA;
S2: ligating the sgRNA obtained in S1 and the linearized pGRB vector plasmid DNA obtained in S3 of example 1 using ligase c112 (purchased from vazemy), the ligation method is described in the specification, converting E.coli competent cells after reaction at 37 ℃ for 30 min, ice-bath for 30 min, heat-shocking for 45S at 42 ℃ and rapidly ice-bath for 2 min, recovering for 1 h at 37 ℃ and then coating an Amp-resistant plate, culturing overnight at 37 ℃, selecting a monoclonal strain for direct sequencing, and selecting a colony with correct sequencing result for extracting plasmid DNA for standby to obtain ackA-sgRNA-pGRB;
S3: PCR primers were designed using primer5 software, and 193-ackAup-F (shown as SEQ ID No. 70) and 194-ackAup-R (shown as SEQ ID No. 71) were used as primers to amplify the upstream homology arm of the ackA gene by PCR; 195-ackAdown-F (shown as SEQ ID No. 72) and 196-ackAdown-R (shown as SEQ ID No. 73) are used as primers, and the upstream and downstream homology arm fragments of the ackA gene are obtained by PCR amplification of the downstream homology arm of the ackA gene;
The upstream and downstream homology arm fragments of the ackA gene are used as templates, and the upstream primer 193-ackAup-F (shown as SEQ ID No. 70) and the downstream primer 196-ackAdown-R (shown as SEQ ID No. 73) are used for amplifying the upstream and downstream homology arm fragments of the ackA gene by fusion PCR to obtain an upstream and downstream homology arm repair template of the ackA gene;
S4: transferring the tool vector pRed-Cas9 plasmid into the chemically transformed competent cells of the XY-Ta57 strain obtained in S7 of example 4, and obtaining the electrically transformed competent cells of the XY-Ta57-pRed-Cas9 strain by electrotransformation;
S5: editing the repair templates obtained in S2, namely ackA-sgRNA-pGRB and S3, and the XY-Ta57-pRed-Cas9 strain obtained in S4, wherein the loading concentration is ackA-sgRNA-pGRB: repair template = 1:2, performing electric shock transformation by using a 0.1 cm electric shock cup 1.8 KV, adding an LB liquid culture medium containing IPTG (100 mM) after transformation, inducing 4 h at 30 ℃, coating a Spe and Amp resistance plate, culturing 30 h at 30 ℃ and then forming single colonies on the plate, aligning the single colonies on a new culture medium, and picking streaked colonies; identifying the ackA gene by using an upstream primer ackA genome-F (shown as SEQ ID No. 74) and a downstream primer ackA genome-R (shown as SEQ ID No. 75), detecting by agarose gel electrophoresis, wherein the correct band size detected by the electrophoresis is 1269 bp; and performing pGRB plasmid loss by using arabinose to induce the colony verified by PCR, and performing shaking induction on pRed-Cas9 plasmid loss at 37 ℃ to obtain an XY-Ta57 Δ ackA strain, which is called as a genetically engineered bacterium XY-Ta33 for short.
According to the embodiment, on the premise of keeping higher L-threonine yield and higher conversion rate, the reaction process of synthesizing acetic acid by Acp is weakened by knocking out the ackA gene, the accumulation of byproduct acetic acid is reduced, and meanwhile, acp generated by introducing exogenous xfpk genes is ensured to be completely used for synthesizing acetyl-CoA, so that the conversion rate of the genetically engineered bacterium XY-Ta33 is further improved, the OD value is close to that of a starting strain, and the genetically engineered bacterium XY-Ta33 with good growth state is obtained.
In the embodiment, 24 deep-hole plate tank fermentation verification is carried out on the obtained genetically engineered bacterium XY-Ta33, the result is shown in Table 5, the yield of L-threonine synthesized by the genetically engineered bacterium XY-Ta53 is 10.64 g/L which is higher than 9.06 g/L of the original strain, and the sugar acid conversion rate 48.29% of the genetically engineered bacterium XY-Ta33 is far higher than 34.08% of the original strain.
TABLE 5
Example 6: fermentation process for producing L-threonine
S1: taking out a glycerol tube filled with the genetically engineered bacterium XY-Ta33 obtained in the example 5 from a refrigerator at the temperature of minus 80 ℃, naturally melting, dipping a 3-ring into a solid slant culture medium (g/L) by using an inoculating loop, streaking on glucose 2.0 g, ammonium chloride 1.0 g, KH 2PO41.5 g、NaHPO43.5 g、MgSO4·7H2 O0.1 g and agar 20 g, and reversely culturing 19h at the temperature of 36.5 ℃ to obtain the XY-Ta33 slant strain;
S2: inoculating the cultured XY-Ta33 inclined plane strain in the step S1 into 50ml sterilized normal saline filled with glass beads, scattering thalli by using the glass beads, diluting the concentration gradient to 10 -5 and 10 -6 by using the OD 660 of the XY-Ta33 genetically engineered strain suspension of 0.3-0.4, respectively sucking the XY-Ta33 genetically engineered strain suspensions with different concentrations of 0.3-ml to coat the flat plate, then placing the flat plate in a condition of 36.5 ℃ for normal culture for 3h, and reversely and continuously culturing to 24h to obtain the activated genetically engineered strain XY-Ta33;
s3: transferring the colony of the activated genetically engineered bacterium XY-Ta33 obtained in the step S2 into a seed tank filled with a seed culture medium (the seed culture medium (g/L): glucose 40.0 g、（NH4）2SO410.0 g、KH2PO41.0 g、MgSO4·7H2O 0.5 g、 yeast extract 2.0 g and MOPS 15.0 g), culturing for 7-8 h, and obtaining the cultured genetically engineered bacterium XY-Ta33 when the genetically engineered bacterium XY-Ta33 OD 660 reaches 0.7;
s4: inoculating the genetically engineered bacterium XY-Ta33 cultured in the step S3 into a fermentation medium (the fermentation medium (g/L): glucose 80.0 g、（NH4）2SO425.0 g、KH2PO42.0 g、MgSO4·7H2O 1.0 g、 yeast extract 4.0 g, feSO 4·5H2O 0.5 g、MnSO4·5H2 O0.5 g and MOPS 30.0 g), wherein the inoculation amount of the genetically engineered bacterium XY-Ta33 is 30%; at 37 ℃, 25% ammonia water is utilized to automatically control and maintain pH=7.2, the pressure is regulated to 0.05 MPa, the rotating speed is regulated to 400 rpm, the air quantity is regulated to 0.2-1 m 3/h, and the dissolved oxygen is controlled to be more than or equal to 30%; after fermentation starts 5h, liquid glucose is added, the concentration of residual sugar in a fermentation tank is maintained to be less than or equal to 1.0 g/L, the fermentation time is 29 and h, fermentation liquor containing L-threonine is obtained, and different strains are repeated for 3 times.
In the embodiment, 10L tanks of fermentation verification is carried out on the genetically engineered bacterium XY-Ta33, the result is shown in a table 6, the yield of L-threonine is 108.99 g/L and the conversion rate is 55.31% under the fermentation process provided by the embodiment of the invention of an original strain Escherichia coli ACThr 1032; the fermentation process provided by the embodiment improves the yield of L-threonine synthesized by the genetic engineering bacteria XY-Ta33 to 133.88 g/L, and the conversion rate is 67.79%; compared with the original strain, the genetically engineered bacterium XY-Ta33 provided by the invention has the advantages that the L-threonine yield is improved by 18.6%, and the conversion rate is improved by 22.6%.
Therefore, the genetic engineering bacterium XY-Ta33 and the fermentation process provided by the invention can improve the yield and the conversion rate of L-threonine, and simultaneously reduce the production of byproduct acetic acid, so that the strain can efficiently produce and accumulate L-threonine, the fermentation production efficiency is improved, and the requirement of large-scale industrial production can be met.
Table 6.10L results of tank fermentation verification
In conclusion, the invention provides the genetic engineering bacterium XY-Ta33, and the genetic engineering bacterium XY-Ta33 has good growth state in a fermentation medium by accumulating substances required by L-threonine synthesis, reducing carbon loss and reducing accumulation of byproduct acetic acid, so that higher L-threonine yield and higher conversion rate are obtained.
The details of the present invention which are not described in detail in the present specification are known to those skilled in the art. While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.

Claims (9)

1. A method for preparing genetically engineered bacteria for producing L-threonine, which is characterized by comprising the following steps:
s1: transferring the vector into competent cells of an original strain, and preparing electrotransformation competent cells to obtain a strain to be modified;
S2: inserting glk genes and galp genes into the strain to be modified obtained in the step S1 by utilizing a genome editing tool carrier pGRB, and knocking out ptsI genes to obtain genetically engineered bacteria XY-Ta36;
s3: carrying out knockout of pykA and pykF genes on the genetically engineered bacterium XY-Ta36 obtained in the S2 by utilizing a genome editing tool carrier pGRB to obtain genetically engineered bacterium XY-Ta38;
S4: inserting xfpk genes into the genetically engineered bacterium XY-Ta38 obtained in the step S3 by utilizing a genome editing tool carrier pGRB to obtain genetically engineered bacterium XY-Ta40;
S5: inserting ppc genes and knocking out Pck genes of the genetically engineered bacteria XY-Ta40 obtained in the step S4 by utilizing a genome editing tool carrier pGRB to obtain genetically engineered bacteria XY-Ta57;
s6: carrying out ackA gene knockout on the genetically engineered bacterium XY-Ta57 obtained in the step S5 by utilizing a genome editing tool carrier pGRB to obtain genetically engineered bacterium XY-Ta33;
The starting strain in S1 is Escherichia coli ACThr1032, the preservation number of ACThr strain is CGMCC NO.16144, the classification is ESCHERICHIA COLI, the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), the preservation address is North Star, the national institute of China academy of sciences 3, the Korean area North Star, and the preservation date is 2018, 7, 23 days;
the nucleotide sequence of the glk gene is shown as SEQ ID No. 1;
The nucleotide sequence of galp gene is shown in SEQ ID No. 2;
the nucleotide sequence of xfpk gene is shown in SEQ ID No. 6;
the nucleotide sequence of the ppc gene is shown in SEQ ID No. 7.
2. The method of claim 1, wherein the vector in S1 is pRed-Cas9.
3. The preparation method according to claim 1, wherein the nucleotide sequence of ptsI gene in S2 is shown in SEQ ID No. 3.
4. The method of claim 1, wherein the nucleotide sequence of the pykA gene in S3 is shown in SEQ ID No.4 and the nucleotide sequence of the pykF gene is shown in SEQ ID No. 5.
5. The method according to claim 1, wherein the nucleotide sequence of the Pck gene in S5 is shown in SEQ ID No. 8; the nucleotide sequence of the ackA gene in S6 is shown in SEQ ID No. 9.
6. A genetically engineered bacterium XY-Ta33, wherein the genetically engineered bacterium XY-Ta33 is obtained by the preparation method of any one of claims 1 to 5.
7. A fermentation process for producing L-threonine is characterized in that the fermentation process is characterized in that genetically engineered bacterium XY-Ta33 in claim 6 is inoculated into a fermentation culture medium, then the pH is automatically controlled and maintained within the range of 7.0-7.2 by using 25% ammonia water at 37 ℃, and dissolved oxygen is controlled to be more than or equal to 30% by adjusting pressure, rotating speed and air quantity; and after fermentation starts 5h, liquid glucose is added, the concentration of residual sugar in the fermentation tank is maintained to be less than or equal to 1.0 g/L, and fermentation broth containing L-threonine is obtained after fermentation is completed.
8. The fermentation process of claim 7, wherein the inoculation amount of the genetically engineered bacterium XY-Ta33 in the fermentation process is 20-30%.
9. The fermentation process of claim 7, wherein the fermentation time of the fermentation process is 29 h, the pressure in the fermentation process is 0.05 MPa, the rotation speed is adjusted to be 200-600 rpm, and the air quantity is adjusted to be 0.2-1 m 3/h.
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