CN117286087A - Genetically engineered bacterium for high-yield of L-homoserine as well as construction method and application thereof - Google Patents

Genetically engineered bacterium for high-yield of L-homoserine as well as construction method and application thereof Download PDF

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CN117286087A
CN117286087A CN202311267786.9A CN202311267786A CN117286087A CN 117286087 A CN117286087 A CN 117286087A CN 202311267786 A CN202311267786 A CN 202311267786A CN 117286087 A CN117286087 A CN 117286087A
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homoserine
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牛坤
郭梦韶
郑锐
章苗
周海岩
柳志强
郑裕国
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Zhejiang University of Technology ZJUT
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Abstract

The invention relates to the technical field of microbial metabolism engineering, in particular to a genetically engineered bacterium for high-yield L-homoserine, and a construction method and application thereof. When the genetically engineered bacterium HS3 in the invention adopts a fermentation method to produce L-homoserine, the essential amino acid is not required to be added exogenously, the fermentation cost is reduced, the uncertainty of the adding time of the essential amino acid in the fermentation process is solved, the operation difficulty of fermentation regulation is reduced, and the yield of the L-homoserine in a shake flask is increased from 7.25g/L obtained by adding three essential amino acids to 10.88g/L when no exogenously added amino acid is added. The key gene is over-expressed on the basis of the non-auxotroph strain to obtain the strain HS8, the yield of the strain HS8 in shake flask fermentation reaches 14.05g/L, and the final shake flask yield is improved by 93.8%. The engineering strain HS8 is fermented in a 5L fermentation tank for 113h, the concentration of the product reaches 110g/L, the sugar-acid conversion rate is 0.43g/g glucose, and compared with 45.25g/L of the initial strain HS, the yield of the strain HS8 is improved by 139.1%. The L-homoserine yield is obviously improved, and the method has a good application prospect.

Description

Genetically engineered bacterium for high-yield of L-homoserine as well as construction method and application thereof
Technical Field
The invention relates to the technical field of microbial metabolism engineering, in particular to a genetically engineered bacterium for high-yield L-homoserine, and a construction method and application thereof.
Background
Homoserine, also known as 2-amino-4-hydroxybutyric acid, is divided into two isomers of L-homoserine and D-homoserine, is an important non-protein amino acid, is a precursor of essential amino acids such as L-methionine, L-threonine and L-isoleucine, is also an intermediate for synthesizing herbicide L-glufosinate and various C4 compounds (such as gamma-butyrolactone and isobutanol), and has the capability of improving plant resistance to diseases and supporting the growth of young chicks. The efficient conversion of methionine by enzymes is also currently an attractive method for large scale production of methionine. Therefore, homoserine is widely used in the industries of pesticides, feed additives, medicines, chemical industry and the like.
The traditional L-homoserine production method mainly comprises a chemical synthesis method and a biological enzyme catalysis method, but has the problems of complicated post purification steps, long reaction time, high production cost, complex process, environmental pollution and the like, and is not suitable for large-scale production. In recent years, with the development of metabolic engineering synthesis biology, a microbial fermentation method for producing a target chemical by microbial metabolism using inexpensive and environment-friendly biomass as a raw material has been attracting more attention. The microbial fermentation method for producing L-homoserine utilizes the metabolism of microbial cells to convert a substrate into a target product, has the advantages of environmental protection, conforms to the current industrial development trend, but does not reach the industrial production level, so that the microbial fermentation for producing L-homoserine becomes a current research hot spot.
The L-homoserine is first obtained during the production of L-threonine and L-lysine by fermentation of Corynebacterium glutamicum by biological method, and the strain is fermented in a medium containing 80g/L corn steep liquor and 150g/L sucrose for 72 hours, finally obtaining 14.5 g/L-homoserine. In 2021, mu et al designed a redox balance route for glucose fermentation of L-homoserine: since L-aspartic acid lacks the reducing power to produce L-homoserine by reducing the tricarboxylic acid cycle intermediate oxaloacetate, this deficiency can be remedied by activating glyoxylate shunt and driving the flux of fumaric acid to L-aspartic acid, the redox balance route being adjusted for the flux of fumaric acid to L-aspartic acid, followed by an enhancement of the outflow of L-homoserine, 84.1 g/LL-homoserine being obtained in fed-batch fermentation. In 2022, cai et al first obtained an E.coli L-homoserine initial strain by regulating the L-homoserine degradation pathway and enhancing the synthesis procedure. To facilitate the production of L-homoserine, gene expression is enhanced by optimizing the copy number on the chromosome, and the transport system is improved to facilitate the outflow of L-homoserine; subsequently, a strategy for co-utilization of cofactors was proposed and successfully applied to achieving L-homoserine production, and the L-homoserine yield of the finally obtained engineering strain can reach 85.29g/L, which is the highest yield of the plasmid-free, antibiotic-free, inducer-free and non-auxotrophic strains reported so far.
However, the current process of producing L-homoserine and its derivatives by biological method still has some disadvantages, such as low fermentation yield or too low sugar acid conversion rate, which makes it difficult to carry out industrial production. Thus, it remains a challenge to construct a highly efficient microbial cell factory for the production of L-homoserine and its derivatives.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a genetically engineered bacterium for producing L-homoserine with high yield and a construction method thereof, and is applied to fermentation production of L-homoserine, so as to solve the problems that the L-homoserine production strain in the prior art has lower yield in fermentation production of L-homoserine, and necessary amino acid needs to be added in the fermentation process, so that industrial production is difficult to carry out.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
in a first aspect, the invention provides a genetically engineered bacterium for producing L-homoserine at high yield, which is constructed and obtained by the following method:
Ptrc-rhtA ΔiclR ΔptsG ΔgalR Ptrc-metLPtrc-thrA Ptrc-rhtA Ptrc-stra Ptrc-glkPtrc-gltB as starting strain, integrating thrB gene promoted by self-regulated promoter at yjiV gene locus, integrating lysA gene with original initiation codon replaced with GTG initiation codon at ycdN gene locus, integrating metA gene with original initiation codon replaced with GTG initiation codon at in situ point of metA gene in its genome, integrating metB gene with original initiation codon replaced with GTG initiation codon at ydeU gene locus, replacing in situ promoter of pntAB gene with strong promoter, knocking out tdcC gene and sstT gene, and inserting tdcC gene and sstT gene at cttT locus, respectively fbr The gene is obtained to obtain the genetically engineered bacterium for producing the L-homoserine at high yield.
The invention mainly carries out strain transformation around improving the fermentation level of L-homoserine, but an initial strain E.coli W3110 delta metJ delta metI delta metB delta thrB delta metA delta lysA delta lacI is Ptrc-rhtA delta ptsG delta galR Ptrc-metL Ptrc-thrA Ptrc-rhtA Ptrc-stream A Ptrc-glkPtrc-gltB is auxotroph, and key genes thrB, lysA, metA and metB in the synthetic pathway of L-lysine, L-threonine and L-methionine are deleted on a genome, so that the essential amino acids are inevitably required to be added exogenously in the fermentation process, the strain is slow to grow, the fermentation process is not easy to regulate and control, and the additional addition of the required amino acids not only can increase the fermentation cost on the process but also can increase the operation complexity. The invention expresses water by weakening proteinInstead of static knockouts, strains are more amenable to commercial production, such as by using self-regulating promoters or replacing relatively weak initiation codons (e.g., GTG initiation codons). The construction process comprises the following steps: the expression attenuation and the complementation of key genes thrB, lysA, metA and metB for synthesizing essential amino acids threonine, lysine and methionine in the genome of the Chassis bacteria are carried out; then using strong promoter P trc Replacing the in situ promoter of the pyridine nucleotide transhydrogenase gene pntAB; knocking out tdCC gene and sstT gene, and finally respectively inserting 1 anti-feedback inhibition gene thrA into the knocked tdcC and sstT gene sites fbr The method comprises the steps of carrying out a first treatment on the surface of the Finally, the recombinant genetically engineered bacterium of the non-induced, plasmid-free and non-auxotroph high-yield L-homoserine is obtained. The concrete transformation content is as follows:
in the genome of the original strain, firstly, a self-regulating promoter is introduced to dynamically regulate and control the expression of a key gene of byproduct amino acid L-threonine, namely homoserine kinase coding gene thrB, and the gene thrB is integrated on yjiV gene locus; by replacing the original initiation codon of the diaminopimelate decarboxylase encoding gene lysA with a relatively weak GTG initiation codon, the level of translation of the gene is such that protein expression is attenuated and integrated into the ycdN gene locus, which allows the production of lysine that can be utilized by itself; the metA gene with the replaced initiation codon is integrated into the original locus of the metA gene in the genome by replacing the original initiation codon of the metA and metB genes with a relatively weak GTG initiation codon, and the metB gene with the replaced initiation codon is integrated into the ydeU gene locus in the genome, so that methionine capable of being utilized by itself is produced; replacing the in situ promoter of the pyridine nucleotide transhydrogenase gene pntAB with a strong promoter to increase the intracellular NADPH content; the tdcC and sstT genes are knocked out, so that the absorbing capacity of the strain to extracellular L-homoserine is weakened; and inserting a copy number of genes thrA encoding anti-feedback inhibition aspartokinase I and homoserine dehydrogenase I into the knocked tdcC and sstT gene loci respectively fbr 。
The initial strain E.coli W3110 ΔmetJ ΔmetI ΔmetB ΔthrB ΔmetA ΔlysA ΔlacI: ptrc-rhtA ΔiclR ΔptsG Δga lR Ptrc-metL Ptrc-thrA Ptrc-rhtA Ptrc-eamA Ptrc-glk Ptrc-gltB is disclosed in paper Liu P, zhang B, yao Z H, et al multiple design of metabolic network for production of L-homoserine in Escherichia coli [ J ]. Applied and Environmental Microbiology,2020,86 (20).
Preferably, the self-regulating promoter is selected from the group consisting of P fliA Promoter, P fliC Promoter, P flgC One of the promoters.
Preferably, the self-regulating promoter is P fliA A promoter.
Further preferably, the P fliA The nucleotide sequence of the promoter is shown as SEQ ID NO. 1.
Preferably, the strong promoter is P trc The nucleotide sequence of the promoter is shown as SEQ ID NO. 10.
Preferably, the nucleotide sequence of the thrB gene is shown as SEQ ID NO.2, the nucleotide sequence of the lysA gene is shown as SEQ ID NO.4, the nucleotide sequence of the metA gene is shown as SEQ ID NO.6, the nucleotide sequence of the metB gene is shown as SEQ ID NO.7, the nucleotide sequence of the pntAB gene is shown as SEQ ID NO.11, the nucleotide sequence of the tdcC gene is shown as SEQ ID NO.12, the nucleotide sequence of the sstT gene is shown as SEQ ID NO.13, and the nucleotide sequence of the thrA gene is shown as SEQ ID NO.13 fbr The nucleotide sequence of the gene is shown as SEQ ID NO. 14.
In a second aspect, the present invention provides a method for constructing the above-described genetically engineered bacterium for producing L-homoserine, using as a starting strain the strain E.coli W3110. Delta. MetJ. Delta. MetI. Delta. MetB. Delta. ThrB. Delta. MetA. Delta. LysA. Delta. LacI:: ptrc-rhtA. Delta. IclR. Delta. PtsG. Delta. GalR Ptrc-metLPtrc-thrA Ptrc-rhtA Ptrc-eamA Ptrc-glkPtrc-gltB, comprising the steps of:
(1) Using the CRISPR-Cas9 system, the promoter P will be used fliA Integrating the started thrB gene into the yjiV gene locus in the genome of the chassis fungus, and marking the engineering fungus obtained by the method as a strain HS1;
(2) Replacing a raw start codon of lysA gene with a GTG start codon by using a CRISPR-Cas9 system, integrating the raw start codon into a ycdN gene locus in a genome of a strain HS1, and marking the engineering bacterium obtained by the method as a strain HS2;
(3) Replacing the original start codon of the metA gene with a GTG start codon, replacing the original start codon of the metB gene with a GTG start codon by using a CRISPR-Cas9 system, integrating the metA gene and the metB gene with the replaced start codons into the original locus of the metA gene and the ydeU gene locus in the genome of the strain HS2 respectively, and marking the engineering bacteria obtained by the method as the strain HS3;
(4) Replacement of the in situ promoter of the pntAB gene in the HS3 genome of the strain with a strong promoter P using CRISPR-Cas9 system trc The engineering bacteria obtained in this way are designated as strain HS4;
(5) Using a CRISPR-Cas9 system to knock out tdcC genes in a genome of the strain HS4, and marking the engineering bacteria obtained by the method as a strain HS5;
(6) Knocking out sstT genes in a genome of the strain HS5 by using a CRISPR-Cas9 system, and marking the engineering bacteria obtained by the method as a strain HS6;
(7) Inserting thrA at tdcC gene knockout site in strain HS6 genome by CRISPR-Cas9 system fbr The gene, the engineering bacteria obtained by the gene is marked as strain HS7;
(8) Insertion of thrA at sstT gene knockout site in strain HS7 genome using CRISPR-Cas9 system fbr The gene is used for obtaining the genetically engineered bacterium for producing the L-homoserine at high yield.
The nucleotide sequence of the yjiV gene is shown as SEQ ID NO.3, the nucleotide sequence of the ycdN gene is shown as SEQ ID NO.5, the nucleotide sequence of the insertion site gene of the metA gene with the replaced start codon is shown as SEQ ID NO.8, and the nucleotide sequence of the ydeU gene is shown as SEQ ID NO. 9.
In a third aspect, the invention provides the genetically engineered bacterium for producing L-homoserine with high yield or the application of the genetically engineered bacterium for producing L-homoserine with high yield constructed by the method in the fermentation production of L-homoserine.
Preferably, the application is to inoculate the genetically engineered bacterium into a fermentation culture medium, ferment and culture the genetically engineered bacterium at the temperature of 25-35 ℃ and the rpm of 100-200, and separate and purify a culture solution to obtain the L-homoserine.
Preferably, the fermentation medium is composed of: glucose 40g/L, (NH) 4 ) 2 SO 4 16 g/L, yeast extract 4g/L, KH 2 PO 4 1 g/L、MgSO 4 1 g/L、FeSO 4 ·7H 2 O 0.005g/L、MnSO 4 ·7H 2 O 0.005g/L、ZnSO 4 0.005g/L、CaCO 3 25g/L, deionized water as solvent and pH value of 6.8.
Compared with the prior art, the invention has the following beneficial effects:
the present invention uses self-regulating promoter P fliA The in-situ promoter of the gene thrB is replaced, so that the synthesis way of threonine is dynamically regulated, the high-efficiency utilization of a carbon source is realized, and the sugar acid conversion rate of L-homoserine synthesis is improved. When the genetically engineered bacterium HS3 in the invention adopts a fermentation method to produce L-homoserine, the essential amino acid is not required to be added exogenously, the fermentation cost is reduced, the uncertainty of the adding time of the essential amino acid in the fermentation process is solved, the operation difficulty of fermentation regulation is reduced, and the yield of the L-homoserine in a shake flask is increased from 7.25g/L obtained by adding three essential amino acids to 10.88g/L when no exogenously added amino acid is added. The key gene is over-expressed on the basis of the non-auxotroph strain to obtain the strain HS8, the yield of the strain HS8 in shake flask fermentation reaches 14.05g/L, and the final shake flask yield is improved by 93.8%. The engineering strain HS8 is fermented in a 5L fermentation tank for 113h, the concentration of the product reaches 110g/L, the sugar-acid conversion rate is 0.43g/g glucose, and compared with 45.25g/L of the initial strain HS, the yield of the strain HS8 is improved by 139.1%. The L-homoserine yield is obviously improved, and the method has a good application prospect.
Drawings
FIG. 1 shows the biomass OD of strains HS1-HS8 600 And a concentration bar graph of L-homoserine.
FIG. 2 shows the biomass OD of the fed-batch fermentation of strain HS in a 5L fermenter 600 And a concentration profile of L-homoserine.
FIG. 3 shows the OD of biomass from the fed-batch fermentation of strain HS8 in a 5-L fermenter 600 And a concentration profile of L-homoserine.
Detailed Description
The invention is further described below with reference to the drawings and specific examples. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified.
The test materials used in the examples below, unless otherwise specified, were all conventional biochemical reagents.
(1) Seed culture: streaking engineering bacteria with high yield of L-homoserine on an LB solid plate, and culturing for 12 hours at 37 ℃; single colony on the flat plate is selected and inoculated in LB liquid culture medium, and cultured for 12 hours at 37 ℃ and 180-200 rpm, the culture is inoculated in new LB liquid culture medium according to the volume concentration of 0.5 percent, and the culture is cultured for 8-10 hours at 37 ℃ and 180-200 rpm, so as to obtain seed liquid;
(2) Shake flask fermentation culture: inoculating the seed solution into a shake flask fermentation medium according to the inoculum size with the volume concentration of 5%, and fermenting and culturing for 48 hours at the temperature of 30-32 ℃ and the rpm of 150-180 rpm to obtain fermentation liquor containing L-homoserine; the fermentation medium consists of: glucose 40g/L, (NH) 4 ) 2 SO 4 16 g/L, yeast extract 4g/L, KH 2 PO 4 1 g/L、MgSO 4 1 g/L、FeSO 4 ·7H 2 O0.005g/L、MnSO 4 ·7H 2 O 0.005g/L、ZnSO 4 0.005 g/L、CaCO 3 25 g/L, deionized water as solvent and pH value of 6.8.
(3) Fermenting and culturing in a 5L tank: streaking the transformed strain HS8 in an LB plate, culturing overnight at 37 ℃ until colonies grow out, picking single colonies with uniform cell morphology, inoculating the single colonies into a test tube filled with 10mL of LB liquid medium, and culturing overnight at 37 ℃ at 180 rpm; inoculating 1mL test tube seed solution into 200mL LB liquid culture medium shake flask at 37deg.C and 180rCulturing pm for 8-10h; the shake flask seed solution was completely inoculated into a 5L fermenter containing 1.8mL of fermentation medium. The formula of the fermentation medium is as follows: glucose 20g/L, (NH 4) 2 SO 4 17 g/L、Yeast extract 4g/L、KH 2 PO 4 1 g/L、MgSO 4 1g/L, 2g/L betaine, 1mL/L metal mixture and 1mL/L defoamer; the formula of the feed medium in the process is as follows: glucose 500g/L, KH 2 PO 4 12.5g/L betaine 2g/L, naHCO 3 10g/L。
LB medium composition: 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride, and the solvent is deionized water. The solid medium requires the addition of agar at a final concentration of 20 g/L.
The Chassis bacteria are recombinant E.coli W3110. DELTA.metJ. DELTA.metI. DELTA.metB. DELTA.thrB. DELTA.metA. DELTA.lysA. DELTA.lacI: ptrc-rhtA ΔiclR ΔptsG ΔgalR Ptrc-metL Ptrc-thrA Ptrc-rhtA Ptrc-amA Ptrc-glk Ptrc-gltB, as disclosed in Liu P, zhang B, yao Z H, et al, multiplex design of metabolic network for production of L-homoserine in Escherichia coli [ J ]. Applied and Environmental Microbiology,2020,86 (20).
Genes and corresponding pathways involved in the strain transformation process are shown in table 1, and primer sequences are shown in table 2:
TABLE 1 Gene involved in Strain engineering and corresponding pathway
Gene name Nucleotide sequence Involves the approach
thrB SEQ ID NO.2 Synthesis of threonine
LysA SEQ ID NO.4 Synthesis of lysine
metA SEQ ID NO.6 Synthesis of O-succinyl-L-homoserine
metB SEQ ID NO.7 Synthesis of methionine
PntAB SEQ ID NO.11 Pyridine nucleotide transhydrogenase
tdcC SEQ ID N0.12 L-threonine internalization
sstT SEQ ID NO.13 L-threonine internalization
thrA fbr SEQ ID NO.14 Anti-feedback inhibition of aspartokinase I and homoserine dehydrogenase I
TABLE 2 primer sequences
Determination of L-homoserine concentration:
sample treatment: the sample concentration was diluted to between 0.1 and 1g/L with ultrapure water.
Standard sample dilution gradient: 0.1g/L, 0.3g/L, 0.5g/L, 1g/L, 1.5g/L, 3g/L.
CNBF (4-chloro-3, 5-dinitrobenzotrifluoride) solution configuration: 0.27g CNBF was dissolved in 10mL acetonitrile (light protection is required).
Boric acid buffer solution preparation: (0.2 mol/L boric acid (HgBO) 3 ) The solution was mixed with 0.05mol/L borax (Na 2 B 4 0 7 )。
Sample reaction conditions: 100uL of each sample was taken, 500uL of boric acid buffer solution and 300uL of CNBF solution were reacted in a metal bath at 60℃and 300rpm for 1 hour, and then the mixture was subjected to film coating (polyvinylidene fluoride, organic film, 0.22 um) for use.
The detection method comprises the following steps: HPLC model: thermo Scientific Utimate the HPLC detection wavelength is 250nm. L-homoserine was isolated using a gradient elution procedure in which mobile phase A was: ultrapure water: acetonitrile: glacial acetic acid: triethylamine=830:170:3:2 (v: v: v: v); mobile phase B component: pure acetonitrile.
Example 1 construction of genetically engineered bacterium (HS 8) producing high levels of L-homoserine:
1. construction of Strain HS1
The initial strain E.coli W3110. DELTA. MetJ. DELTA. MetI. DELTA. MetB. DELTA. ThrB. DELTA. MetA. DELTA. LysA. DELTA. LacI:: ptrc-rhtA. DELTA. IclR. DELTA. PtsG. DELTA. GalR Ptrc-metLPtrc-thrA Ptrc-rhtA Ptrc-eamA Ptrc-glkPtrc-gltB was designated as strain HS, since the initial strain HS would encode the key gene th for threonine synthesisrB knockout, the inevitable need of additional threonine addition during strain fermentation increases production cost and operational complexity, so that the critical gene thrB of the initial strain HS is complemented back and P is used fliA The promoter dynamically regulates and controls threonine synthesis, so that carbon flow can flow to the synthesis of target products more.
1 copy of thrB gene is inserted into the chassis fungus HS genome by CRISPR-Cas9 system, and the specific operation is as follows:
(1) Connector fragment U yjiV -P fliA -thrB-D yjiV
The E.coli W3110 genome was used as a template, and the upstream and downstream homology arms of the gene yjiV were amplified using primers yjiV-P1 and yjiV-P2, yjiV-P3 and yjiV-P4, respectively, and primers thrB-F and thrB-R, P fliA -F and P fliA R amplification of thrB and promoter P, respectively fliA The PCR product was detected by 1.0% agarose gel electrophoresis and then treated with DpnI at 37℃for 1 hour, and finally the purified fragment was recovered by using the Clean Up kit, and the purified fragment was fused with primers yjiV-P1 and yjiV-P4 to give a ligation fragment U by PCR yjiV -P fliA -thrB-D yjiV The method comprises the steps of carrying out a first treatment on the surface of the The PCR conditions were as follows: 95 ℃ for 5min; repeating 30 cycles at 95℃for 30s,58℃for 30s, and 72℃for 1.5 min; the extension was continued for 10min at 72 ℃.
(2) Constructing a pTarget-thrB (yjiV) -sg vector, using a pTarget plasmid as a template, using yjiV-F and yjiV-R as primers, amplifying sgRNA capable of expressing a targeted gene yjiV, constructing a pTarget-thrB (yjiV) -sg mutation vector, and linearizing the pTarget-yjiV plasmid by using pTarget-line-F and pTarget-line-R as primers; the PCR conditions were as follows: 95 ℃ for 5min; repeating 30 cycles at 95℃for 30s,58℃for 30s, and 72℃for 2.5 min; the extension was continued for 10min at 72 ℃.
(3) Construction of pTarget-P fliA thrB (yjiV) plasmid
The ligation fragment U in step (1) is ligated yjiV -P fliA -thrB-D yjiV And the linearized mutation vector pTarget-thrB (yjiV) -sg in step (2) is cloned in one step by the following reaction procedure: 30min at 37 ℃; the cloned product is transformed into E.coli DH5 alpha receptor bacteria and coated onThe cells were incubated on LB solid plates containing spectinomycin hydrochloride resistance at a final concentration of 50mg/L at 37℃for 12 hours. Selecting positive clone strain, transferring into LB liquid medium containing final concentration of 50mg/L spectinomycin hydrochloride resistance, culturing at 37deg.C for 12 hr, and obtaining pTarget-P by plasmid extraction kit fliA thrB (yjiV) plasmid.
(4) Construction of Strain HS1
Taking HS as a starting strain, and successfully replacing thrB genes to yjiV gene loci by using CRISPR/Cas9 gene editing technology; pTarget-P fliA The electrotransformation of the thrB (yjiV) plasmid into the HS strain containing the pCas9 vector was performed as follows: HS strain transformed with pCas9 vector was inoculated in 10ml LB liquid medium (containing 50mg/L kanamycin and 10mM L-arabinose), cultured overnight at 30℃at 180rpm as seed liquid; 1mL of the seed solution was aspirated from the tube and added to a 250mL shaking flask containing 50mL of LB medium, and cultured at 30℃and 180rpm to OD 600 The value is about 0.6; transferring the bacterial liquid from the shake flask into a precooled 50mL centrifuge tube, centrifuging at 4 ℃ and 5000rpm for 5min, and discarding the supernatant; adding 40mL of sterile water precooled in advance, gently blowing to make the thalli suspend, centrifuging at 4 ℃ and 5000rpm for 5min, and discarding the supernatant; repeating the previous step; adding 40mL of pre-chilled 10% (v: v) glycerol solution, gently blowing to make the thalli suspended, centrifuging at 4 ℃ and 5000rpm for 5min, and discarding the supernatant; 1mL of a pre-chilled 10% (v: v) glycerol solution was added and the suspension was gently blown.
Placing the prepared electrotransformation competent cells on ice, adding 5 mu L of plasmid after melting, adding the competent cells mixed with the plasmid into a pre-cooled 2mm electric shock cup, adjusting the working parameters of an electrotransformation instrument to 2500V, 25 mu F and 200 omega for electric shock, rapidly adding 800mL of LB culture medium, and culturing for 3h at 30 ℃ and 180 rpm; the bacterial liquid after the electrotransformation was spread on LB plates containing 50mg/L kanamycin and 50mg/L spectinomycin hydrochloride resistance, and cultured at 30℃for 24 hours. Selecting single colony as template, PCR with primer thrB-VF and primer thrB-VR, and the PCR product has 1800bp DNA band in 1.0% agarose gel, confirming that successful P insertion is obtained fliA Positive clone strain HS1 of thrB.
Picking the strain which is verified to be correct into LB culture medium containing 50mg/L kanamycin and 5mM IPTG (isopropyl thiogalactoside) for culturing at 30 ℃ for 12 hours to eliminate pTarget plasmid, picking single colony which is successfully verified to eliminate pTarget plasmid into a test tube filled with 10mL LB culture medium, culturing at 42 ℃ overnight to eliminate pCas plasmid (pCas plasmid is temperature sensitive and is easy to lose when cultured at more than 37 ℃); the resulting plasmid-free strain was designated HS1.
2. Construction of Strain HS2
Similarly, the critical gene lysA for synthesizing the essential amino acid lysine is also complemented, further reducing the cost of fermentation, while the use of the relatively weak initiation codon GTG reduces the expression level of the protein.
1 copy of lysA gene was inserted into the chassis fungus HS genome by CRISPR-Cas9 system, and the specific procedures were as follows:
(1) Connector fragment U ycdN -lysA-D ycdN
Using E.coli W3110 genome as template, amplifying upstream and downstream homology arms of ycdN gene with primers ycdN-P1 and ycdN-P2, ycdN-P3 and ycdN-P4, amplifying lysA gene fragment with primers lysA-F and lysA-R, detecting PCR product with 1.0% agarose gel electrophoresis, treating with DpnI at 37deg.C for 1h, recovering purified fragment with clear Up kit, obtaining connecting fragment U by fusing PCR with purified fragment with primers ycdN-P1 and ycdN-P4 ycdN -(GTG)lysA-D ycdN The method comprises the steps of carrying out a first treatment on the surface of the The PCR conditions were as follows: 95 ℃ for 5min; repeating 30 cycles at 95℃for 30s,58℃for 30s, and 72℃for 1.5 min; the extension was continued for 10min at 72 ℃.
(2) Construction of pTarget-lysA (ycdN) -sg vector
Amplifying sgRNA capable of expressing a target gene ycdN by taking pTarget plasmid as a template and taking ycdN-F and ycdN-R as primers to construct a pTarget-lysA (ycdN) -sg mutation vector, and linearizing the pTarget-ycdN plasmid by taking pTarget-line-F and pTarget-line-R as primers; the PCR conditions were as follows: 95 ℃ for 5min; repeating 30 cycles at 95℃for 30s,58℃for 30s, and 72℃for 2.5 min; the extension was continued for 10min at 72 ℃.
(3) Construction of pTarget- (GTG) lysA (ycdN) plasmid
Will step by stepThe linker fragment U in step (1) ycdN -lysA-D ycdN And (2) carrying out one-step cloning on the linearization mutation vector pTarget-lysA (ycdN) -sg in the step (2), wherein the reaction procedure is as follows: 30min at 37 ℃; the cloned product was transformed into E.coli DH 5. Alpha. Receptor bacteria, spread on LB solid plates containing spectinomycin hydrochloride resistance at a final concentration of 50mg/L, and incubated at 37℃for 12h. Selecting positive clone strains, transferring the positive clone strains into LB liquid medium containing spectinomycin hydrochloride resistance with the final concentration of 50mg/L, culturing for 12 hours at 37 ℃, and obtaining pTarget-lysA (ycdN) plasmids by using a plasmid extraction kit; finally, the cloning product was transformed into E.coli DH 5. Alpha. Receptor bacteria using the primers lysA (G) -F and the start codon of the lysA (G) -R mutant gene lysA, spread on LB solid plates with a final concentration of 50mg/L spectinomycin hydrochloride resistance, and incubated at 37℃for 12h. The positive clone strain was picked up and transferred to LB liquid medium containing spectinomycin hydrochloride resistance at a final concentration of 50mg/L, cultured at 37℃for 12 hours, and pTarget- (GTG) lysA (ycdN) plasmid was obtained using a plasmid extraction kit.
(4) Construction of Strain HS2
Successfully replacing lysA gene to ycdN gene locus by using CRISPR/Cas9 gene editing technology; the pTarget- (GTG) lysA (ycdN) plasmid was electrotransformed into HS1 strain containing pCas9 vector, and the procedure was as follows: HS1 strain transformed with pCas9 vector was inoculated in 10ml LB liquid medium (containing 50mg/L kanamycin and 10mM L-arabinose), cultured overnight at 30℃at 180rpm as seed liquid; 1mL of the seed solution was aspirated from the tube and added to a 250mL shaking flask containing 50mL of LB medium, and cultured at 30℃and 180rpm to OD 600 The value is about 0.6; transferring the bacterial liquid from the shake flask into a precooled 50mL centrifuge tube, centrifuging at 4 ℃ and 5000rpm for 5min, and discarding the supernatant; adding 40mL of sterile water precooled in advance, gently blowing to make the thalli suspend, centrifuging at 4 ℃ and 5000rpm for 5min, and discarding the supernatant; repeating the previous step; adding 40mL of pre-chilled 10% (v: v) glycerol solution, gently blowing to make the thalli suspended, centrifuging at 4 ℃ and 5000rpm for 5min, and discarding the supernatant; 1mL of a pre-chilled 10% (v: v) glycerol solution was added and the suspension was gently blown.
Placing the prepared electrotransformation competent cells on ice, adding 5 mu L of plasmid after melting, adding the competent cells mixed with the plasmid into a pre-cooled 2mm electric shock cup, adjusting the working parameters of an electrotransformation instrument to 2500V, 25 mu F and 200 omega for electric shock, rapidly adding 800mL of LB culture medium, and culturing for 3h at 30 ℃ and 180 rpm; the bacterial liquid after the electrotransformation was spread on LB plates containing 50mg/L kanamycin and 50mg/L spectinomycin hydrochloride resistance, and cultured at 30℃for 24 hours. Single colonies were picked as templates and PCR was performed with primers lysA-VF and lysA-VR, and the PCR product was subjected to a DNA band of 2000bp in a 1.0% agarose gel, confirming that a positive clone strain HS2 of successfully inserted (GTG) lysA was obtained.
Picking the strain which is verified to be correct into LB culture medium containing 50mg/L kanamycin and 5mM IPTG (isopropyl thiogalactoside) for culturing at 30 ℃ for 12 hours to eliminate pTarget plasmid, picking single colony which is successfully verified to eliminate pTarget plasmid into a test tube filled with 10mL LB culture medium, culturing at 42 ℃ overnight to eliminate pCas plasmid (pCas plasmid is temperature sensitive and is easy to lose when cultured at more than 37 ℃); the resulting plasmid-free strain was designated HS2.
3. Construction of Strain HS3
To further optimize the fermentation conditions, the critical genes metA and metB for synthetic methionine were also complemented and non-auxotrophic strains were constructed without exogenously added amino acids. The specific operation is as follows:
the process of the complementation of the key gene metA is as follows:
(1) Connector fragment U Original source -metA-D Original source
Using E.coli W3110 genome as template, amplifying upstream homology arm and downstream homology arm at original locus of gene metA by using primer metA-P1 and original metA-P2, original metA-P3 and original metA-P4, amplifying gene fragment of metA by using primer metA-F and metA-R, detecting PCR product by 1.0% agarose gel electrophoresis, treating with DpnI at 37 ℃ for 1h, recovering purified fragment by Clean Up kit, obtaining ligation fragment U by adopting step 1 Original source -metA-D Original source 。
(2) Construction of pTarget-metA (pro) -sg vector
The pTarget-metA (original) -sg mutation vector is constructed by amplifying sgRNA capable of expressing the original site of target gene metA by using pTarget plasmid as template and original metA-F and original metA-R as primers, and then linearizing pTarget-original metA plasmid by using pTarget-line-F and pTarget-line-R as primers.
(3) Construction of pTarget- (GTG) metA (original) plasmid
The ligation fragment U in step (1) is ligated Original source -metA-D Original source And (2) cloning the linearization mutation vector pTarget-metA (original) -sg in one step, and obtaining pTarget-metA (original) plasmid by adopting a method of the step one; finally, using the primers metA (G) -F and the initiation codon of metA (G) -R mutant gene metA, pTarget- (GTG) metA (original) plasmid was obtained by the same procedure as in step one.
(4) Construction of strains
Successfully replacing the metA gene to the metA primary gene locus by using CRISPR/Cas9 gene editing technology; the pTarget- (GTG) metA (original) plasmid was electrotransformed into the above strain containing pCas9 vector, and the procedure was followed by PCR with primers metA-VF and metA-VR, the PCR product was in the presence of 1600bp DNA band in 1.0% agarose gel, confirming that a positive clone strain was obtained with successful insertion (GTG) metA.
On the basis of the complementation of metA, the process of the complementation of the key gene metB is as follows:
(1) Connector fragment U ydeU -metB-D ydeU
The E.coli W3110 genome was used as a template, the upstream and downstream homology arms of the gene ydeU were amplified using primers ydeU-P1 and ydeU-P2, ydeU-P3 and ydeU-P4, respectively, the metB gene fragment was amplified using primers metB-F and metB-R, the PCR product was detected by 1.0% agarose gel electrophoresis, treated with DpnI at 37℃for 1h, and finally the purified fragment was recovered using the Clean Up kit, and the ligation fragment U was obtained using step 1 ydeU -metB-D ydeU 。
(2) Construction of pTarget-metB (ydeU) -sg vector
The pTarget-metB (ydeU) -sg mutation vector is constructed by amplifying sgRNA capable of expressing targeted target gene ydeU by using pTarget plasmid as a template and ydeU-F and ydeU-R as primers, and then linearizing the pTarget-ydeU plasmid by using pTarget-line-F and pTarget-line-R as primers.
(3) Construction of pTarget- (GTG) metB (ydeU) plasmid
The ligation fragment U in step (1) is ligated ydeU -metB-D ydeU And (2) cloning the linearization mutation vector pTarget-metB (ydeU) -sg in one step, and obtaining pTarget-metB (ydeU) plasmid by adopting the method in the first step; finally, using the primers metB (G) -F and the initiation codon of metB (G) -R mutant gene metB, pTarget- (GTG) metB (ydeU) plasmid was obtained by the same procedure as in step one.
(4) Construction of Strain HS3
Successfully replacing metB gene to ydeU gene locus by CRISPR/Cas9 gene editing technology; the pTarget- (GTG) metB (ydeU) plasmid was electrotransformed into the above strain containing pCas9 vector, and the procedure was followed by PCR with primers metB-VF and metB-VR using the procedure of step one, and the PCR product was found to have a DNA band of 2000bp in 1.0% agarose gel, confirming that the positive clone strain HS3 was obtained with successful insertion (GTG) metB.
4. Construction of Strain HS4
The pntAB gene codes for pyridine nucleotide transhydrogenase and NADP is converted into NADP + Conversion to NADPH, to further increase the intracellular content of NADPH, P is used trc The promoter replaces the original promoter of the pntAB gene to achieve the purpose of enhancing the expression of the pntAB gene.
Editing the promoter sequence of the pntAB gene in the HS3 strain genome by CRISPR-Cas9 system, the specific procedure is as follows:
(1) Connector fragment U pntAB -P trc -D pntAB
The E.coli W3110 genome was used as a template, primers pntAB-P1 and pntAB-P2, pntAB-P3 and pntAB-P4 were used to amplify the upstream and downstream homology arms of the pntAB gene, and primers pntAB-P1 and pntAB-P4 were used to perform fusion PCR, and the ligation fragment U was obtained in step 1 pntAB -P trc -D pntAB 。
(2) Constructing a pTarget-pntAB-sg vector, using a pTarget plasmid as a template, pntAB-F and pntAB-R as primers, amplifying sgRNA capable of expressing a targeted target gene pntAB, constructing a pTarget-pntAB-sg mutation vector, and linearizing the pTarget-pntAB plasmid by using the pTarget-line-F and the pTarget-line-R as primers.
(3) Construction of pTarget-P trc Plasmid pntAB
The ligation fragment U in step (1) is ligated pntAB -P trc -D pntAB And (2) carrying out one-step cloning on the linearization mutation vector pTarget-pntAB-sg in the step (2), and obtaining pTarget-P by adopting a method of the step one trc The pntAB plasmid.
(4) Construction of Strain HS4
Substitution of the original promoter of the gene pntAB with strong P using CRISPR/Cas9 gene editing technique trc A promoter; pTarget-P trc The plasmid pntAB was electrotransformed into HS3 strain containing pCas9 vector, the procedure was a step one, PCR was performed with primers pntAB-VF and pntAB-VR, and the PCR product was in the presence of 1300bp DNA band in 1.0% agarose gel, confirming that positive clone HS4 was obtained, which successfully replaced the pntAB original promoter.
5. Construction of Strain HS5
In order to prevent the accumulation of the target product in the cell in a large amount, the inhibition of the product occurs, the amino acid product must be transported out of the cell rapidly, the modification of the target product transport system can effectively increase the yield of the target product, and it is found that the threonine transport system is regulated by genes tdcC and sstT, and the key genes involved in the threonine transport system may be equally effective on homoserine.
The tdcC gene in the genome of the HS4 strain is knocked out by the CRISPR-Cas9 system, and the specific operation is as follows:
(1) Connector fragment U tdcC -D tdcC
The E.coli W3110 genome was used as a template, the upstream and downstream homology arms of the gene tdcC were amplified using primers tdcC-P1 and tdcC-P2, tdcC-P3 and tdcC-P4, respectively, and fusion PCR was performed using primers tdcC-P1 and tdcC-P4, and the ligation fragment U was obtained in step 1 tdcC -D tdcC 。
(2) Construction of pTarget-tdcC-sg vector the pTarget plasmid is used as template, tdcC-F and tdcC-R are used as primer, the sgRNA capable of expressing target gene tdcC is amplified, the pTarget-tdcC-sg mutation vector is constructed, and then the pTarget-tdcC plasmid is linearized by using pTarget-line-F and pTarget-line-R as primer.
(3) Construction of pTarget-tdcC plasmid
The ligation fragment U in step (1) is ligated tdcC -D tdcC And (2) carrying out one-step cloning on the linearization mutation vector pTarget-tdcC-sg in the step (2), and obtaining the pTarget-tdcC plasmid by adopting a method of the step one.
(4) Construction of Strain HS5
Knocking out the gene tdcC by using CRISPR/Cas9 gene editing technology; the pTarget-tdcC plasmid was electrotransformed into HS3 strain containing pCas9 vector, the procedure adopted step one method, PCR was performed with primers tdcC-VF and tdcC-VR, and the 1300bp DNA band was present in 1.0% agarose gel in the PCR product, confirming that positive clone strain HS5 was obtained with successful tdcC knockout.
6. Construction of Strain HS6 the same knockout of the sstT gene was performed, and the sstT gene in the HS5 strain genome was knocked out by CRISPR-Cas9 system, as follows:
(1) Connector fragment U sstT -D sstT
The E.coli W3110 genome was used as a template, the primers sstT-P1 and sstT-P2, sstT-P3 and sstT-P4 were used to amplify the upstream and downstream homology arms of the gene sstT, and fusion PCR was performed using the primers sstT-P1 and sstT-P4, and the ligation fragment U was obtained in step 1 sstT -D sstT 。
(2) Constructing a pTarget-sstT-sg vector, using a pTarget plasmid as a template, using sstT-F and sstT-R as primers, amplifying sgRNA capable of expressing a target gene sstT, constructing a pTarget-sstT-sg mutation vector, and linearizing the pTarget-sstT plasmid by using pTarget-line-F and pTarget-line-R as primers.
(3) Construction of pTarget-sstT plasmid
The ligation fragment U in step (1) is ligated sstT -D sstT And (2) carrying out one-step cloning on the linearization mutation vector pTarget-sstT-sg in the step (2), and obtaining the pTarget-sstT plasmid by adopting a method in the step one.
(4) Construction of Strain HS6
Knocking out the gene sstT by using a CRISPR/Cas9 gene editing technology; the pTarget-sstT plasmid is electrically transformed into HS3 strain containing pCas9 vector, the operation steps adopt a method of step one, PCR is carried out by using primers sstT-VF and primers sstT-VR, 1300bp DNA band exists in 1.0% agarose gel of PCR product, and positive clone strain HS6 with successful knockout of sstT is confirmed.
7. Construction of Strain HS7
In order to increase the carbon flux from L-aspartic acid to L-homoserine, one copy of thrA gene is introduced into the tdcC locus of the genome, but the thrA gene is subjected to feedback inhibition by L-threonine and L-isoleucine, so that the anti-feedback inhibition thrA is obtained through site-directed mutagenesis fbr And (3) a gene.
Editing thrA encoding anti-feedback inhibition in HS6 genome by CRISPR-Cas9 system fbr The specific operation of the gene is as follows:
(1) Connector fragment U tdcC -thrA-D tdcC
The E.coli W3110 genome was used as a template, the upstream and downstream homology arms of the gene tdcC were amplified using primers tdcC-P1 and tdcC-P2, tdcC-P3 and tdcC-P4, respectively, the thrA gene fragment was amplified using primers thrA-F1 and thrA-R1, the PCR product was detected by 1.0% agarose gel electrophoresis and then treated with DpnI at 37℃for 1h, and finally the purified fragment was recovered using the Clean Up kit, and the ligation fragment U was obtained using step 1 tdcC -thrA-D tdcC 。
(2) Construction of pTarget-thrA (tdcC) -sg vector
The pTarget-thrA (tdcC) -sg mutant vector is constructed by amplifying sgRNA capable of expressing target gene tdcC by taking pTarget plasmid as a template and taking tdcC-F and tdcC-R as primers, and then the pTarget-tdcC plasmid is linearized by taking pTarget-line-F and pTarget-line-R as primers.
(3) Construction of pTarget-thrA fbr (tdcC) plasmid
The ligation fragment U in step (1) is ligated tdcC -thrA fbr -D tdcC And (2) carrying out one-step cloning on the linearization mutation vector pTarget-thrA (tdcC) -sg in the step (2), and obtaining the pTarget-thrA (tdcC) plasmid by adopting the method in the step one; finally, the 1024 th base of thrA (G) -F and thrA (G) -R mutant gene thrA is used, and the pT is obtained by the method of the first steparget-thrA fbr (tdcC) plasmid.
(4) Construction of Strain HS7
thrA using CRISPR/Cas9 gene editing technique fbr Successful gene replacement to tdcC gene locus; pTarget-thrA fbr The (tdcC) plasmid was electrotransformed into HS6 strain containing pCas9 vector, and the procedure was a step one method, using the primer thrA fbr VF and primer thrA fbr PCR was performed by VR, and the presence of a 2000bp DNA band in 1.0% agarose gel of the PCR product confirmed that the successful insertion of the gene thrA was obtained fbr Is a positive clone of strain HS7.
8. Construction of Strain HS8
Further increasing the carbon flux of L-aspartic acid to L-homoserine by reintroducing a copy of thrA at the sstT site of the genome fbr And (3) a gene.
Editing thrA encoding anti-feedback in HS7 genome by CRISPR-Cas9 system fbr The specific operation of the gene is as follows: (1) Connector fragment U sstT -thrA fbr -D sstT
Using E.coli W3110 genome as template, using primers sstT-P1 and sstT-P2, sstT-P3 and sstT-P4 to amplify upstream and downstream homology arms of gene sstT; pTarget-thrA constructed in strain HS7 fbr The (tdcC) plasmid was used as a template to amplify thrA using primers thrA-F2 and thrA-R2 fbr The PCR product was detected by 1.0% agarose gel electrophoresis and then treated with DpnI at 37℃for 1h, and finally the purified fragment was recovered by using the Clean Up kit, and the ligation fragment U was obtained by using step 1 sstT -thrA fbr -D sstT 。
(2) Construction of pTarget-thrA (sstT) -sg vector
The pTarget-thrA (sstT) -sg mutation vector is constructed by amplifying sgRNA capable of expressing targeted target gene sstT by taking pTarget plasmid as a template and sstT-F and sstT-R as primers, and then linearizing the pTarget-sstT plasmid by taking pTarget-line-F and pTarget-line-R as primers.
(3) Construction of pTarget-thrA fbr (sstT) plasmid
The ligation fragment U in step (1) is ligated sstT -thrA fbr -D sstT And (2) cloning the linearization mutation vector pTarget-thrA (sstT) -sg in one step, and obtaining pTarget-thrA by adopting the method in one step fbr (sstT) plasmid.
(4) Construction of Strain HS8
thrA using CRISPR/Cas9 gene editing technique fbr Successful gene replacement to sstT gene locus; pTarget-thrA fbr The (sstT) plasmid was electrotransformed into HS6 strain containing pCas9 vector, and the procedure was followed in step one using the primer thrA fbr VF2 and primer thrA fbr PCR was performed by VR, and the presence of a 2000bp DNA band in 1.0% agarose gel of the PCR product confirmed that the successful insertion of the gene thrA was obtained fbr Is a positive clone of strain HS8.
Example 2 shaking flask fermentation experiments with strains HS1-HS8
Fermentation experiments were performed on Chaetomium HS and the strains constructed in example 1 above (HS 1, HS2, HS3, HS4, HS5, HS6, HS7, HS 8) in shake flasks to compare the ability to produce L-homoserine between the genotype strains.
Firstly, single colony is selected to 10mL LB liquid culture medium, and is cultured for 10 hours in a constant-temperature shaking incubator at 37 ℃ and 200rpm to be used as seed liquid for shake flask fermentation. Inoculated in 500mL shake flasks containing 20mL of fermentation medium at an inoculum size of 5%, then added with 0.5g of sterile CaCO 3 . The cells were incubated in a thermostatic shaker at 30℃and 180rpm for 48h, and three replicates were set for each genotype strain. After the fermentation process was completed, 2mL of the fermentation broth was removed in a shaking flask and centrifuged at 12000rpm for 2min at room temperature in a 2mL centrifuge tube, wherein the supernatant was transferred to a fresh 1.5mL centrifuge tube, and then 2mL of ultrapure water was added to resuspend the remaining cells (containing CaCO) 3 ) The mixture was centrifuged at 12000rpm for 2min at room temperature, and the supernatant was discarded. The pellet was resuspended again and the supernatant was discarded by centrifugation. Finally, 1.6mL of ultrapure water is added to resuspend the precipitate, and 400 mu L of acetic acid is added to thoroughly mix and dissolve CaCO therein 3 The CaCO is then treated with 3 The dissolved bacterial liquid was diluted 20 times with ultrapure water, and its OD was measured by a spectrophotometer 600 Value, in order to guarantee the accuracy of measurement, three times of measurement are performed on each sample to reduce the operationError. Biomass OD of strains HS1 to HS8 600 And L-homoserine are shown in FIG. 1.
As can be seen from FIG. 1, the yield of L-homoserine in shake flasks was increased from 7.25g/L obtained by adding three essential amino acids to 10.88g/L without addition of exogenous amino acids, compared with the original strain HS of recombinant genetically engineered bacterium HS 3; compared with the original strain HS, the recombinant escherichia coli HS8 has the advantages that the capacity of producing L-homoserine is greatly improved, the yield is improved from 7.25g/L to 14.05g/L in shake flask fermentation, and the 93.79% is improved.
LB medium composition: 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride, and the solvent is deionized water. The solid medium requires the addition of agar at a final concentration of 20 g/L.
Shake flask fermentation medium composition: glucose 40g/L, (NH) 4 ) 2 SO 4 16 g/L, yeast extract 4g/L, KH 2 PO 4 1g/L、MgSO 4 1 g/L、FeSO 4 ·7H 2 O 0.005g/L、MnSO 4 ·7H 2 O 0.005g/L、ZnSO 4 0.005 g/L、CaCO 3 25g/L, deionized water as solvent, and pH 6.8.
Example 3 non-induced, plasmid-free, non-auxotrophic strains HS and HS85L fermenter fermentation verification fed-batch fermentation in a 5L fermenter, FIGS. 2 and 3 show the fermentation process of strains HS and HS8, respectively, including residual sugar, biomass (OD 600 ) And the change of L-homoserine production with time. As can be seen from FIG. 3, the initial 20g/L glucose in the broth was substantially consumed at 13h, and when glucose was consumed, the pH of the broth was increased, and when pH was greater than 6.80, automatic feeding was turned on, and as feed medium was added, the pH was gradually decreased and maintained at 6.80, so that the glucose concentration in the medium was maintained at a low level at all times<3 g/L), and the inhibition effect of high-concentration glucose on the cell growth of the strain is avoided. The highest yield of L-homoserine reached a level of 110g/L at the end of fermentation, with a sugar acid conversion of 0.43g/g glucose, and an increase of 139.1% in strain HS8 compared to 45.25g/L of the initial strain HS (FIG. 2).
The genetic engineering bacteria HS8 does not need to add essential amino acid in the fermentation process, the fermentation cost is reduced, the fermentation regulation and control are relatively simple, a certain foundation is laid for the industrial production of L-homoserine, and a certain metabolism transformation thought is provided for the fermentation production of amino acid.

Claims (10)

1. The genetically engineered bacterium for producing L-homoserine with high yield is characterized by being constructed and obtained by the following method:
Ptrc-rhtA ΔiclR ΔptsG ΔgalR Ptrc-metLPtrc-thrA Ptrc-rhtA Ptrc-stra Ptrc-glkPtrc-gltB as starting strain, integrating thrB gene promoted by self-regulated promoter at yjiV gene locus, integrating lysA gene with original initiation codon replaced with GTG initiation codon at ycdN gene locus, integrating metA gene with original initiation codon replaced with GTG initiation codon at in situ point of metA gene in its genome, integrating metB gene with original initiation codon replaced with GTG initiation codon at ydeU gene locus, replacing in situ promoter of pntAB gene with strong promoter, knocking out tdcC gene and sstT gene, and inserting tdcC gene and sstT gene at cttT locus, respectively fbr The gene is obtained to obtain the genetically engineered bacterium for producing the L-homoserine at high yield.
2. The genetically engineered bacterium for high production of L-homoserine according to claim 1, wherein the self-regulating promoter is selected from the group consisting of P fliA Promoter, P fliC Promoter, P flgC One of the promoters.
3. The genetically engineered bacterium for high production of L-homoserine according to claim 1, wherein the self-regulating promoter is P fliA A promoter.
4. The genetically engineered bacterium for high production of L-homoserine according to claim 2 or 3, wherein the P fliA The nucleotide sequence of the promoter is shown as SEQ ID NO. 1.
5. As claimed inThe genetically engineered bacterium capable of producing L-homoserine at high yield as described in 1, wherein the strong promoter is P trc The nucleotide sequence of the promoter is shown as SEQ ID NO. 10.
6. The genetically engineered bacterium of claim 1 or 5, wherein the thrB gene has a nucleotide sequence shown in SEQ ID NO.2, the lysA gene has a nucleotide sequence shown in SEQ ID NO.4, the metA gene has a nucleotide sequence shown in SEQ ID NO.6, the metB gene has a nucleotide sequence shown in SEQ ID NO.7, the pntAB gene has a nucleotide sequence shown in SEQ ID NO.11, the tdcC gene has a nucleotide sequence shown in SEQ ID NO.12, the sstT gene has a nucleotide sequence shown in SEQ ID NO.13, and the thrA gene has a nucleotide sequence shown in SEQ ID NO.13 fbr The nucleotide sequence of the gene is shown as SEQ ID NO. 14.
7. A method for constructing a genetically engineered bacterium which produces L-homoserine as claimed in any one of claims 1 to 6, characterized in that the strain E.coli W3110. DELTA.metI.DELTA.metB.DELTA.thrB.DELTA.metA.DELTA.lysA.DELTA.lacI:: ptrc-rhtA.DELTA.iclR.DELTA.ptsG.DELTA.gal R Ptrc-metL Ptrc-thrA Ptrc-rhtA Ptrc-stream A Ptrc-glk Ptrc-gltB is used as starting strain, comprising the steps of:
(1) Using the CRISPR-Cas9 system, the promoter P will be used fliA Integrating the started thrB gene into the yjiV gene locus in the genome of the chassis fungus, and marking the engineering fungus obtained by the method as a strain HS1;
(2) Replacing a raw start codon of lysA gene with a GTG start codon by using a CRISPR-Cas9 system, integrating the raw start codon into a ycdN gene locus in a genome of a strain HS1, and marking the engineering bacterium obtained by the method as a strain HS2;
(3) Replacing the original start codon of the metA gene with a GTG start codon, replacing the original start codon of the metB gene with a GTG start codon by using a CRISPR-Cas9 system, integrating the metA gene and the metB gene with the replaced start codons into the original locus of the metA gene and the ydeU gene locus in the genome of the strain HS2 respectively, and marking the engineering bacteria obtained by the method as the strain HS3;
(4) Replacement of the in situ promoter of the pntAB gene in the HS3 genome of the strain with a strong promoter P using CRISPR-Cas9 system trc The engineering bacteria obtained in this way are designated as strain HS4;
(5) Using a CRISPR-Cas9 system to knock out tdcC genes in a genome of the strain HS4, and marking the engineering bacteria obtained by the method as a strain HS5;
(6) Knocking out sstT genes in a genome of the strain HS5 by using a CRISPR-Cas9 system, and marking the engineering bacteria obtained by the method as a strain HS6;
(7) Inserting thrA at tdcC gene knockout site in strain HS6 genome by CRISPR-Cas9 system fbr The gene, the engineering bacteria obtained by the gene is marked as strain HS7;
(8) Insertion of thrA at sstT gene knockout site in strain HS7 genome using CRISPR-Cas9 system fbr The gene is used for obtaining the genetically engineered bacterium for producing the L-homoserine at high yield.
8. Use of the genetically engineered bacterium of high-yield L-homoserine according to any one of claims 1 to 6 or the genetically engineered bacterium of high-yield L-homoserine constructed by the method of claim 7 for fermentative production of L-homoserine.
9. The use according to claim 8, wherein the genetically engineered bacterium is inoculated into a fermentation medium, and the fermentation medium is subjected to fermentation culture at 25-35 ℃ and 100-200 rpm, and the culture solution is separated and purified to obtain L-homoserine.
10. The use according to claim 9, wherein the fermentation medium consists of: glucose 40g/L, (NH) 4 ) 2 SO 4 16 g/L, yeast extract 4g/L, KH 2 PO 4 1 g/L、MgSO 4 1 g/L、FeSO 4 ·7H 2 O 0.005g/L、MnSO 4 ·7H 2 O 0.005g/L、ZnSO 4 0.005 g/L、CaCO 3 25 g/L, deionized water as solvent and pH value of 6.8.
CN202311267786.9A 2023-09-28 2023-09-28 Genetically engineered bacterium for high-yield of L-homoserine as well as construction method and application thereof Pending CN117286087A (en)

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