CN117384811A - Genetically engineered strain for producing L-homoserine, construction method and application - Google Patents

Genetically engineered strain for producing L-homoserine, construction method and application Download PDF

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CN117384811A
CN117384811A CN202311317213.2A CN202311317213A CN117384811A CN 117384811 A CN117384811 A CN 117384811A CN 202311317213 A CN202311317213 A CN 202311317213A CN 117384811 A CN117384811 A CN 117384811A
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aspc
primer
homoserine
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张莎莎
邱媛媛
史鲁秋
薛虹宇
李华山
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Nanjing Shengde Chuangying Biotechnology Co ltd
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Abstract

The invention discloses a genetic engineering strain for producing L-homoserine, which takes escherichia coli as a host, and knocks out aspartate aminotransferase gene aspC on a host chromosome to obtain recombinant strain ST11C; the recombinant vector plasmid pA is introduced into the mutant escherichise:Sub>A coli ST11C to obtain se:Sub>A recombinant engineering strain named 11C-A. According to the invention, aspC genes in the engineering bacterise:Sub>A 11-A of the escherichise:Sub>A coli are knocked out, so that the obtained recombinant bacterise:Sub>A 11C-A not only can not reduce the yield and conversion rate of L-homoserine, but also can reduce the yield of L-glutamic acid as se:Sub>A byproduct, reduce the post-treatment difficulty and save the cost.

Description

Genetically engineered strain for producing L-homoserine, construction method and application
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a genetic engineering strain for producing L-homoserine, a construction method and application thereof.
Background
L-homoserine is a naturally occurring nonessential amino acid and is a precursor for the synthesis of the essential amino acids L-threonine, L-methionine and L-isoleucine. L-homoserine is also used as an important platform compound for O-acetylhomoserine, L-azetidine, homoserine lactone, isobutanol, gamma-butyrolactone, etc. Also has the functions of improving plant stress resistance and promoting poultry growth. Based on the basic skeleton of L-type-alpha amino acid of L-homoserine and the diversity of chemical activity of gamma-hydroxyl, the L-homoserine and the derivatives thereof have important application prospects in agriculture, pharmacology, physiology and the like as important functional amino acids.
At present, the methods for producing L-homoserine at home and abroad mainly comprise the following steps: (1) chemical methods; (2) chemical chiral resolution; (3) biological methods. The chemical method for synthesizing L-homoserine mainly adopts L-methionine with relatively high cost as a raw material, adopts methyl iodide or methyl bromide with serious biotoxicity as a methylation reagent, protects amino by nucleophilic attack, and hydrolyzes under the weak alkaline condition to obtain the product. The method has high cost, needs iodide and sulfide generation, and is not friendly to the environment; the chemical chiral resolution method is to separate L-homoserine by utilizing the property difference of non-corresponding isomers after the reaction of the mixed homoserine and chiral reagent. The method has low yield, high reagent cost and large amount of organic solvent, and has great pollution threat to the environment; the biological method mainly comprises two modes of a biological enzyme method and a fermentation method at present. Wherein the biological enzyme method utilizes pyruvic acid and formaldehyde to generate amino acid homoserine under the combined action of aldolase and L-amino acid dehydrogenase. The main problems of the process are still high cost, the need of using toxic raw materials formaldehyde and formic acid, and the need of using expensive coenzyme, etc. In recent years, with rapid development of metabolic engineering and synthetic biology, the fermentative synthesis of L-homoserine has received attention. The microbial fermentation method has the advantages of low cost, mild condition, less environmental pollution and the like. L-homoserine biosynthesis takes oxaloacetic acid as a precursor, and is synthesized by catalysis of aspartokinase, homoserine dehydrogenase and aspartate semialdehyde dehydrogenase, wherein aspartokinase and homoserine dehydrogenase are speed limiting enzymes. L-homoserine is usually not accumulated in the cell but is directly metabolized to L-threonine. In addition, L-lysine competes with L-homoserine for the precursor aspartate semialdehyde. Therefore, the common metabolic engineering strategies of the L-homoserine production strain mainly comprise the steps of enhancing the synthesis of a precursor oxaloacetate, blocking the competition and degradation pathways of L-homoserine and the like.
The L-homoserine synthesis route designed by the invention can theoretically generate 3 molecules of L-homoserine from 2 molecules of glucose, wherein 1 molecule of L-homoserine is synthesized by a glyoxylate pathway, and 2 molecules of L-homoserine is synthesized by a oxaloacetic acid pathway. Oxaloacetate is converted to fumaric acid via malic acid, succinic acid is also converted to fumaric acid by succinate dehydrogenase, and fumaric acid obtained in the 2-way is converted to L-aspartic acid by aspartate lyase, and thus to L-homoserine. By the way design, the reducing force balance can be realized, and the molar conversion rate is 1.5mol/mol, and the mass conversion rate is 99%.
Disclosure of Invention
In order to solve the problems, the invention provides a genetic engineering strain for producing L-homoserine, a construction method and application thereof. According to the invention, aspC genes in the engineering bacterise:Sub>A 11-A of the escherichise:Sub>A coli are knocked out, so that the obtained recombinant bacterise:Sub>A 11C-A can not only ensure that the yield and conversion rate of L-homoserine are not reduced, but also can reduce the yield of L-glutamic acid as se:Sub>A byproduct, reduce the post-treatment difficulty and save the cost.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
The invention provides a genetic engineering strain for producing L-homoserine, which takes escherichia coli as a host, and knocks out an aspartate aminotransferase gene aspC on a host chromosome to obtain recombinant strain ST11C; the recombinant vector plasmid pA is introduced into the mutant escherichise:Sub>A coli ST11C to obtain se:Sub>A recombinant engineering strain named 11C-A.
Further, the host is escherichia coli recombinant engineering strain 11-A, and is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms (China) on the day of 10 and 23 of 2020: CGMCC No.20947.
The invention also provides a construction method of a genetic engineering strain for producing L-homoserine, wherein the genetic engineering strain is obtained by directionally modifying a host Escherichia coli by adopting a CRISPR/Cas9 mediated gene editing technology, and the construction method comprises the following steps:
(1) PCR amplification is carried out by taking pTargetF as a template and using primers pTarget-aspC-F and pTarget-aspC-R, the amplified fragments are digested by DpnI methylase and then are transformed into escherichia coli Fast-T1 competent, positive clones are screened on LB plates containing streptomycin, and sequencing verification is carried out by using the primers pTarget-cexu-F, and the fragments are respectively named as pTarget-aspC after the sequencing is correct;
(2) Performing PCR amplification by using primer pairs aspC-up500-F and aspC-down500-R, aspC-KO-F and aspC-KO-R to obtain two fragments respectively, and performing PCR amplification by using a mixture of the two fragments as a template and using the primer pairs aspC-up500-F and aspC-down500-R to obtain a delta aspC targeting fragment; recovering the obtained targeting fragment delta aspC;
(3) Preparing competent cells from escherichia coli mutant ST11, converting pCas plasmids, coating on an LB plate containing kanamycin, culturing at 30 ℃, and screening positive clones;
(4) Selecting positive clones from the plate in the step (3), preparing electrotransformation competent cells, mixing the electrotransformation competent cells with pTarget-aspC plasmid and targeting fragment delta aspC, placing the electrotransformation competent cells in an electrotransformation cup for electric shock, adding LB liquid culture medium for resuscitation at 30 ℃, coating the electrotransformation competent cells on LB plate containing kanamycin and streptomycin, culturing at 30 ℃, screening positive clones, carrying out PCR (polymerase chain reaction) amplification on aspC-up800-F and aspC-down800-R by using primers, and sequencing and verifying amplified fragments to screen out positive clones;
(5) Inoculating the positive clone obtained in the above to LB liquid medium containing IPTG and kanamycin, culturing at 30 ℃ for overnight to eliminate pTarget-aspC plasmid, streaking the strain after overnight culture on LB solid plate containing kanamycin, culturing at 30 ℃ for overnight to obtain Escherichia coli mutant ST11 delta aspC containing pCas plasmid;
(6) Inoculating the Escherichia coli mutant ST11 delta aspC containing the pCas plasmid into an LB liquid culture medium, culturing overnight at 37 ℃ to eliminate the pCas plasmid, streaking the strain after overnight culture on an LB solid plate, and culturing overnight at 37 ℃ to obtain the Escherichia coli mutant ST11 delta aspC without the plasmid, which is abbreviated as ST11C;
(7) The recombinant vector plasmid pA is introduced into the mutant escherichise:Sub>A coli ST11C to obtain se:Sub>A recombinant engineering strain named 11C-A.
Further, the nucleotide sequence of the primer pTarget-aspC-F in the step (1) is shown as SEQ ID NO.1, and the nucleotide sequence of the primer pTarget-aspC-R is shown as SEQ ID NO. 2; the nucleotide sequence of the primer pTarget-cexu-F is shown as SEQ ID NO. 3;
the PCR amplification system comprises: 5X SF Buffer 10uL dNTP Mix (10 mM each) 1uL, template pTargetF20ng, primer (10 uM) 2uL each, phanta Super-Fidelity DNA Polymerase 1uL, distilled water 34uL, total volume 50uL;
the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min, 1 cycle; denaturation at 95℃for 10 seconds, annealing at 55℃for 20 seconds, elongation at 72℃for 1.5 minutes, 30 cycles; extension at 72℃for 10 min, 1 cycle.
Further, the nucleotide sequence of the primer aspC-KO-F in the step (2) is shown as SEQ ID NO. 4; the nucleotide sequence of the primer aspC-KO-R is shown as SEQ ID NO. 5; the nucleotide sequence of the primer aspC-up500-F is shown as SEQ ID NO. 6; the nucleotide sequence of the primer aspC-Down500-R is shown as SEQ ID NO. 7;
the PCR amplification system comprises: 5X SF Buffer 10uL dNTP Mix (10 mM each) 1uL, template pTargetF20ng, primer (10 uM) 2uL each, phanta Super-Fidelity DNA Polymerase 1uL, distilled water 34uL, total volume 50uL;
the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min, 1 cycle; denaturation at 95℃for 10 seconds, annealing at 55℃for 20 seconds, elongation at 72℃for 1.5 minutes, 30 cycles; extension at 72℃for 10 min, 1 cycle.
Further, the nucleotide sequence of the forward primer aspC-up800-F in the step (4) is shown as SEQ ID NO. 8; the nucleotide sequence of the primer aspC-down800-R is shown as SEQ ID NO. 9;
the PCR amplification system comprises: 5X SF Buffer 10uL dNTP Mix (10 mM each) 1uL, template pTargetF20ng, primer (10 uM) 2uL each, phanta Super-Fidelity DNA Polymerase 1uL, distilled water 34uL, total volume 50uL;
the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min, 1 cycle; denaturation at 95℃for 10 seconds, annealing at 55℃for 20 seconds, elongation at 72℃for 1.5 minutes, 30 cycles; extension at 72℃for 10 min, 1 cycle.
In still another aspect, the present invention provides a method for producing L-homoserine, which comprises activating the genetically engineered bacterium, inoculating the activated genetically engineered bacterium into a fermentation medium, and preparing L-homoserine by biological fermentation.
Further, the proper conditions are that at the temperature of 37 ℃, the initial air flux is 2vvm, the stirring speed is 300rpm, the dissolved oxygen concentration is set to 100%, the air flux is regulated to 3vvm in the process of bacterial growth, meanwhile, the stirring speed is correlated with DO value to control the dissolved oxygen concentration to be always more than 30%, when the initial glucose in the fermentation culture medium is consumed, the feed culture medium is started, the pH is controlled to 7.0 by adopting ammonia water in the fermentation process, and when the bacterial density reaches the absorbance OD of 600nm 600 At 30g/L, L-arabinose was added to the fermentation medium to induce protein expression at a final concentration of 2g/L, and when the feed medium was exhausted, the fermentation was ended, and L-homoserine was collected from the fermentation product.
Further, the fermentation medium comprises the following components: 1-5g/L of citric acid, 1-20g/L of monopotassium phosphate, 1-5g/L of nitrogen source, 150uL/L of polyether defoamer, 5-30g/L of glucose and MgSO 4 ·7H 2 O 0.3-1g/L，VB 1 5-10mg/L, 0.1-1g/L of lysine, 0.1-1g/L of methionine, 0.1-1g/L of isoleucine, 0.1-1g/L of threonine and 1-10mL/L of trace inorganic salt I, and the pH value is 7.0+/-0.5;
the trace inorganic salt I comprises the following components: 840mg/L of EDTA was used,CoCl 2 ·6H 2 O 250mg/L，MnCl 2 ·4H 2 O1500mg/L，CuCl 2 ·2H 2 O 150mg/L，H 3 BO 3 300mg/L，Na 2 MoO 4 ·2H 2 O 250mg/L，Zn(CH 3 COO) 2 ·2H 2 1300mg/L of O and 10g/L of ferric citrate; the nitrogen source is selected from one or more of ammonium chloride, ammonium acetate, ammonium sulfate and ammonium phosphate.
Further, the feed medium comprises the following components: glucose 100-800g/L MgSO 4 ·7H 2 1-5g/L of O, 1-10g/L of lysine, 1-10g/L of methionine, 1-10g/L of isoleucine, 1-10g/L of threonine and 1-10mL/L of trace inorganic salt II;
the trace inorganic salt II comprises the following components: EDTA 1300mg/L, coCl 2 ·6H 2 O 400mg/L，MnCl 2 ·4H 2 O2350mg/L，CuCl 2 ·2H 2 O 250mg/L，H 3 BO 3 500mg/L，Na 2 MoO 4 ·2H 2 O 400mg/L，Zn(CH 3 COO) 2 ·2H 2 1600mg/L of O and 4g/L of ferric citrate.
The invention has the following expected effects and advantages: the invention constructs the mutant escherichise:Sub>A coli recombinant engineering strain 11C-A capable of efficiently producing L-homoserine by taking escherichise:Sub>A coli as se:Sub>A starting strain through se:Sub>A genetic engineering technology.
The invention obtains the engineering strain with high L-homoserine yield through genetic engineering improvement, the conversion rate is not reduced compared with that of the starting strain 11-A, and the yield of the byproduct L-glutamic acid is lower, thereby being more beneficial to separation and purification. The extraction cost is reduced by continuously optimizing and upgrading the strain and the manufacturing process, and the production cost is further reduced.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Drawings
FIG. 1 is se:Sub>A graph showing the content of L-homoserine and L-glutamic acid as by-products of E.coli 11C-A during fermentation.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purposes and the effects of the present invention easy to understand, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
The Escherichia coli engineering bacteria 11-A used in the embodiment of the invention are preserved in China general microbiological culture Collection center (CGMCC) with the address of 1 st Xielu No.3 of North Chen in the Korean region of Beijing at the year of 2020, and the preservation number is CGMCC No.20947, and the Escherichia coli is classified and named as Escherichia coli. The strain can ferment and produce L-homoserine in an inorganic salt culture medium. The strain has been applied for patent on 11/13 th 2020, and the application number is 202011017812. X.
In the examples of the present invention, the coding sequence of the asparate aminotransferase encoding Gene aspC is shown as Gene ID 945553 (consisting of 1191 nucleotides) and encodes asparate aminotransferase as shown in accession number NP-415448 (consisting of 396 amino acid residues).
Example 1: construction of E.coli mutant ST11C
The E.coli mutant ST11C is a mutant of E.coli ST11C obtained by knocking out the aspartate aminotransferase gene (aspC) of E.coli ST11 by CRISPR/Cas9 technology (Jiang Y, chen B, duan C, sun B, yang J, yang S: multigene editing in the Escherichia coli genome via the CRISPR-Cas9system.appl Environ Microbiol 2015, 81:2506-2514.), and is abbreviated as ST11C in this application, and the genotype thereof is E.coli ST 11. DELTA aspC.
The specific construction steps of the escherichia coli mutant ST11C are as follows:
(1) Preparation of electrotransformation competent cells: the pCas plasmid (Jiang Y, chen B, duan C, sun B, yang J, yang S: multigene editing in the Escherichia coli genome via the CRISPR-Cas9system.appl Environ Microbiol 2015, 81:2506-2514.) was transformed into E.coli ST11 by chemical transformation, positive clones were selected by culturing at 30℃on LB plates containing kanamycin (kanamycin concentration: 50 ug/mL), and after culturing at 30℃to an OD600 of about 0.6 in LB liquid medium containing 2g/L arabinose, electrically competent cells were prepared.
(2) Construction of pTarget plasmid: the site of N20 knockout was selected using the website https:// crispy. Using pTargetF (Jiang Y, chen B, duan C, sun B, yang J, yang S: multigene editing in the Escherichia coli genome via the CRISPR-Cas9system.Appl Environ Microbiol 2015, 81:2506-2514.) as a template, PCR amplification was performed with primer pairs pTarget-aspC-F and pTarget-aspC-R, respectively, to obtain fragments of about 2100bp in size.
The PCR amplification system is as follows: 5X SF Buffer 10uL, dNTP Mix (10 mM each) 1uL, template pTargetF20ng, primer (10 uM) 2uL each, phanta Super-Fidelity DNA Polymerase (Nanjinopran Biotechnology Co., ltd., catalog P501) 1uL, distilled water 34uL and total volume 50uL.
The amplification conditions were: pre-denaturation at 95 ℃ for 2 min (1 cycle); denaturation at 95℃for 10 seconds, annealing at 55℃for 20 seconds, elongation at 72℃for 1.5 minutes (30 cycles); extension at 72℃for 10 min (1 cycle).
After about 3h of reaction with DpnI methylase, E.coli Fast-T1 competence was transformed directly by chemical transformation, positive clones were screened on LB plates containing streptomycin (streptomycin concentration 50 ug/mL) and verified by sequencing with the primer pTarget-cexu-F. The primer sequences used for the respective sequences designated pTarget-aspC after correct sequencing were as follows (the sequence of N20 is underlined):
pTarget-aspC-F SEQ ID NO.1 5’-agatgagacgggcaaaacccgttttagagctagaaatagc-3’
pTarget-aspC-R SEQ ID NO.2 5’-gggttttgcccgtctcatctactagtattatacctagg ac-3’
pTarget-cexu-F SEQ ID NO.3 5’-ctttcctgcgttatcccctg-3’
(3) Amplifying the targeting fragment: PCR amplification is carried out by using primer pairs aspC-up500-F and aspC-KO-R, aspC-KO-F and aspC-down500-R respectively, so as to obtain fragments with the sizes of about 500bp and 500bp respectively; PCR amplification was performed using a mixture of the two fragments as templates and using primer pairs aspC-up500-F and aspC-down500-R to obtain a ΔaspC targeting fragment of about 1000 bp. The targeting fragment Δaspc was recovered.
The PCR amplification system is as follows: 5X SF Buffer 10uL dNTP Mix (10 mM each) 1uL, 5-20ng template, 2uL each of primer (10 uM), 1uL of Phanta Super-Fidelity DNA Polymerase (product catalog P501, nanjinopran Biotech Co., ltd.), 34uL of distilled water, and a total volume of 50uL.
The amplification conditions were: pre-denaturation at 95 ℃ for 2 min (1 cycle); denaturation at 95℃for 10 seconds, annealing at 55℃for 20 seconds, extension at 72℃for 0.5-2 min (30 sec/kb) (30 cycles); extension at 72℃for 10 min (1 cycle).
The primer sequences used were as follows:
(4) Electric conversion: 200ng of pTarget-aspC plasmid, 400ng of targeting fragment ΔaspC were mixed with 100. Mu.L of electrotransformation competent cells prepared in step (1), placed in a 2mm electrorotating cup, shocked at 2.5kV, resuscitated by adding 1mL of LB liquid medium at 30℃and plated on LB plates containing kanamycin and streptomycin (kanamycin concentration is 50ug/mL, streptomycin concentration is 50 ug/mL), cultured at 30℃and positive clones were selected. PCR amplification was performed with the primer pairs aspC-up800-F and aspC-down800-R, and the amplified fragments were sequenced and verified.
The PCR amplification system comprises: green Taq Mix 10uL (Nanjinovain Biotech Co., ltd., product catalog P131), primers (10 uM) each 0.8uL, distilled water 8.4uL, template bacteria liquid 0.2uL, and total volume 20uL;
the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 3 min (1 cycle); denaturation at 95℃for 15 sec, annealing at 55℃for 15 sec, extension at 72℃for 1-5 min (60 sec/kb) (30 cycles); extension at 72℃for 5 min (1 cycle).
(5) Elimination of the pTarget plasmid: positive clones, which were sequenced to verify correct, were inoculated in LB liquid medium containing 0.1mM IPTG and kanamycin and cultured overnight at 30℃to eliminate pTarget plasmid. The strain after overnight culture was streaked on LB solid plates containing kanamycin, and cultured overnight at 30℃to obtain the Escherichia coli mutant ST 11. DELTA. AspC containing pCas plasmid.
(6) The pCas plasmid was eliminated: the E.coli mutant ST 11. DELTA. AspC containing pCas plasmid, which was confirmed to be correct by sequencing, was inoculated in LB liquid medium and cultured overnight at 37℃to eliminate pCas plasmid. The strain after overnight culture is streaked on LB solid plate, and cultured overnight at 37 ℃ to obtain the plasmid-free escherichia coli mutant ST11 delta aspC, 11C for short.
Primer sequences used for verification and sequencing were as follows:
aspC-up800-F SEQ ID NO.8 5’-tgctacctatcgtaactcca-3’
aspC-down800-R SEQ ID NO.9 5’-aatctggtgtgaacaaaccc-3’
example 2: construction of engineering Strain 11C-A with high L-homoserine production
The expression vector pA was transformed into E.coli mutant ST11C by chemical transformation, and positive clones were selected on LB plates containing kanamycin (kanamycin concentration: 50 ug/ml), and the resulting clone strain was designated 11C-A.
Example 3: high density fermentation of Strain 11C-A
The fermentation medium consists of: 1.7g/L of citric acid, 14g/L of monopotassium phosphate, 4g/L of diammonium phosphate, 150uL/L of polyether defoamer, 20g/L of glucose and MgSO 4 ·7H 2 O 0.6g/L，VB 1 9mg/L, 0.4g/L of lysine, 0.2g/L of methionine, 0.2g/L of isoleucine, 0.3g/L of threonine, 10mL/L of trace inorganic salt, and pH 7.0;
the feed medium consists of: glucose 600g/L, mgSO 4 ·7H 2 O2 g/L, lysine 4g/L, methionine 2g/L, isoleucine 2g/L, threonine 3g/L, and trace inorganic salt II 10mL/L.
The trace inorganic salt I comprises the following components: EDTA 840mg/L, coCl 2 ·6H 2 O 250mg/L，MnCl 2 ·4H 2 O 1500mg/L，CuCl 2 ·2H 2 O 150mg/L，H 3 BO 3 300mg/L，Na 2 MoO 4 ·2H 2 O 250mg/L，Zn(CH 3 COO) 2 ·2H 2 O1300 mg/L, ferric citrate 10g/L.
The trace inorganic salt II comprises the following components: EDTA 1300mg/L, coCl 2 ·6H 2 O 400mg/L，MnCl 2 ·4H 2 O 2350mg/L，CuCl 2 ·2H 2 O 250mg/L，H 3 BO 3 500mg/L，Na 2 MoO 4 ·2H 2 O 400mg/L，Zn(CH 3 COO) 2 ·2H 2 1600mg/L of O and 4g/L of ferric citrate.
Those skilled in the art can adjust the above components to a certain extent according to practical situations, and this embodiment only provides a specific implementation scheme. As an alternative embodiment of the present example, the fermentation medium may comprise component contents that are replaced by any value within the following ranges: 1-5g/L of citric acid, 1-20g/L of monopotassium phosphate, 1-5g/L of nitrogen source, 5-30g/L of glucose and MgSO 4 ·7H 2 O 0.3-1g/L，VB 1 5-10mg/L, 0.1-1g/L of lysine, 0.1-1g/L of methionine, 0.1-1g/L of isoleucine, 0.1-1g/L of threonine, 1-10mL/L of trace inorganic salt, and pH 7.0+/-0.5. The nitrogen source is an inorganic nitrogen-containing compound and can be selected from one or more of ammonium chloride, ammonium acetate, ammonium sulfate and ammonium phosphate. The trace inorganic salt is selected from one or more of soluble ferric salt, cobalt salt, cupric salt, zinc salt, manganese salt and molybdate. The feed medium comprises components in amounts that can be replaced by any value within the following ranges: glucose 100-800g/L MgSO 4 ·7H 2 1-5g/L of O, 1-10g/L of lysine, 1-10g/L of methionine, 1-10g/L of isoleucine, 1-10g/L of threonine and 1-10ml/L of trace inorganic salt II.
Seed liquid culture: the 250mL Erlenmeyer flask was filled with 100mL LB and sterilized at 121℃for 20min. And cooling, inoculating the glycerol bacterise:Sub>A 11C-A preserved at-80 ℃ for 7h at the culture temperature of 37 ℃ and the rotation speed of se:Sub>A shaking table of 200rpm, and inoculating the fermentation medium. The person skilled in the art can adjust the above conditions to a certain extent according to the actual situation, and the achievement of the purpose of the invention is not affected. This example provides only one specific implementation, as an alternative to this example, the culture conditions may be replaced with any value within the following ranges: the culture temperature is 25-42 ℃, the rotation speed of the shaking table is 100-300rpm, and the culture time is 6-8h.
Inoculating a fermentation tank: as a preferred embodiment of this example, the volume of the fermentation medium in the 5L fermenter was 2.5L, the seed solution was inoculated with 5% (V/V) after sterilization, and the initial concentration of glucose was 20g/L. The temperature is 37 ℃, the initial air flux is 2vvm, the stirring speed is 300rpm, the dissolved oxygen concentration at the moment is set to be 100%, the air flux is regulated to be 3vvm in the growth process of the thalli, and meanwhile, the stirring speed is related to the DO value to control the dissolved oxygen concentration to be always more than 30%. When the initial glucose in the fermentation medium is consumed, the feed medium is turned on. Ammonia is adopted in the fermentation process to control the pH value to 7.0. When the cell density reached absorbance (OD 600) of 600nm was 30, L-arabinose was added to the fermentation medium to induce protein expression at a final concentration of 2g/L, and the fermentation was ended when the feed medium was depleted. The person skilled in the art can adjust the above conditions to a certain extent according to the actual situation, and the achievement of the purpose of the invention is not affected.
The analysis method comprises the following steps: agilent (Agilent-1200) high performance liquid chromatography was used to determine the components in the fermentation broth. The detection method of L-homoserine comprises the following steps: the sample is diluted properly and then is derivatized by using 2, 4-Dinitrofluorobenzene (DNFB), 50uL of 10g/L DNFB acetonitrile solution and 100uL 0.5M NaHCO3 solution are added into 100uL of the sample, the mixture is fully and uniformly mixed, the mixture is subjected to light-shielding reaction for 1h at 60 ℃, 750uL 0.01M KH2PO4 solution is added after cooling, the mixture is uniformly mixed, and the mixture is filtered by using a 0.22um filter membrane and then is subjected to high performance liquid chromatography detection. The chromatographic column is ZORBAX Eclipse XDB-C18 column (4.6X105 mm,5um; agilent), the column temperature is 30deg.C, the mobile phase is 35% acetonitrile formic acid (thousandth) water solution, the flow rate is 1mL/min, and the detection wavelength is 360nm.
And (3) calculating results: in the transformation solution, the yield of L-homoserine reaches 110.8g/L, and 0.92g/L of glutamic acid is generated. The L-homoserine conversion rate of the whole fermentation stage can reach 0.59g L-homoserine/g glucose, and the ratio of the impurity glutamic acid to the product L-homoserine is 1:120, the calculation formula is 0.92/110.8 (see Table 1 below).
Comparative example 1:
patent No. CN116286566A was obtained by knocking out thrB, lpxM, poxB, metA, iclR, lysA, ompT, galR, ptsG, thrA, and expressing glk, zglf, rhtA, rhtB, asd, ppc and aspA (E.coli BW25113 ΔptsG:: glk, ΔgalR::: zglf, ΔompT::: ppc, ΔldhA:::: rhtA, ΔlpxM::: rhtB, Δpfb::: asd, ΔpoxB:: aspA, ΔiclR, ΔlysA, ΔthrB) on the chromosomal DNA in multiple copies to obtain an engineering strain 11A of L-homoserine, which was fermented to obtain 97.45 g/L-L homoserine and produced in a homoserine to produce L-homoserine at a homoserine 3/L-homoserine impurity ratio of 1/L-homoserine, and 1. 30, the calculation formula is 3.25/97.45, the L-homoserine conversion rate of the whole fermentation stage is 0.567-g L-homoserine/g glucose, the yield of the L-homoserine is obviously lower than 110.8g/L of the strain of the invention, the yield of the L-homoserine is obviously higher than the content of glutamic acid in the strain of the invention (see table 1), and the strain of the invention has fewer impurities.
TABLE 1 comparison of homoserine and glutamic acid yields in different strains
While the invention has been described with respect to preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and that any such changes and modifications as described in the above embodiments are intended to be within the scope of the invention.

Claims (10)

1. A genetic engineering strain for producing L-homoserine is characterized in that the genetic engineering strain takes escherichia coli as a host, and aspartate aminotransferase gene aspC on a host chromosome is knocked out to obtain recombinant strain ST11C; the recombinant vector plasmid pA is introduced into the mutant escherichise:Sub>A coli ST11C to obtain se:Sub>A recombinant engineering strain named 11C-A.
2. The genetically engineered strain of claim 1, wherein the host is escherichia coli recombinant engineered strain 11-a deposited at the chinese microbiological bacterial strain deposit management committee general microbiological center at month 23 of 2020 under accession number: CGMCC No.20947.
3. The construction method of the genetic engineering strain for producing L-homoserine is characterized in that the genetic engineering strain is obtained by directionally modifying a host Escherichia coli by adopting a CRISPR/Cas9 mediated gene editing technology, and comprises the following steps:
(1) PCR amplification is carried out by taking pTargetF as a template and using primers pTarget-aspC-F and pTarget-aspC-R, the amplified fragments are digested by DpnI methylase and then are transformed into escherichia coli Fast-T1 competent, positive clones are screened on LB plates containing streptomycin, and sequencing verification is carried out by using the primers pTarget-cexu-F, and the fragments are respectively named as pTarget-aspC after the sequencing is correct;
(2) Performing PCR amplification by using primer pairs aspC-up500-F and aspC-down500-R, aspC-KO-F and aspC-KO-R to obtain two fragments respectively, and performing PCR amplification by using a mixture of the two fragments as a template and using the primer pairs aspC-up500-F and aspC-down500-R to obtain a delta aspC targeting fragment; recovering the obtained targeting fragment delta aspC;
(3) Preparing competent cells from escherichia coli mutant ST11, converting pCas plasmids, coating on an LB plate containing kanamycin, culturing at 30 ℃, and screening positive clones;
(4) Selecting positive clones from the plate in the step (3), preparing electrotransformation competent cells, mixing the electrotransformation competent cells with pTarget-aspC plasmid and targeting fragment delta aspC, placing the electrotransformation competent cells in an electrotransformation cup for electric shock, adding LB liquid culture medium for resuscitation at 30 ℃, coating the electrotransformation competent cells on LB plate containing kanamycin and streptomycin, culturing at 30 ℃, screening positive clones, carrying out PCR (polymerase chain reaction) amplification on aspC-up800-F and aspC-down800-R by using primers, and sequencing and verifying amplified fragments to screen out positive clones;
(5) Inoculating the positive clone obtained in the above to LB liquid medium containing IPTG and kanamycin, culturing at 30 ℃ for overnight to eliminate pTarget-aspC plasmid, streaking the strain after overnight culture on LB solid plate containing kanamycin, culturing at 30 ℃ for overnight to obtain Escherichia coli mutant ST11 delta aspC containing pCas plasmid;
(6) Inoculating the Escherichia coli mutant ST11 delta aspC containing the pCas plasmid into an LB liquid culture medium, culturing overnight at 37 ℃ to eliminate the pCas plasmid, streaking the strain after overnight culture on an LB solid plate, and culturing overnight at 37 ℃ to obtain the Escherichia coli mutant ST11 delta aspC without the plasmid, which is abbreviated as ST11C;
(7) The recombinant vector plasmid pA is introduced into the mutant escherichise:Sub>A coli ST11C to obtain se:Sub>A recombinant engineering strain named 11C-A.
4. The construction method according to claim 3, wherein the nucleotide sequence of the primer pTarget-aspC-F in the step (1) is shown in SEQ ID NO.1, and the nucleotide sequence of the primer pTarget-aspC-R is shown in SEQ ID NO. 2; the nucleotide sequence of the primer pTarget-cexu-F is shown as SEQ ID NO. 3;
the PCR amplification system comprises: 5X SF Buffer 10uL dNTP Mix (10 mM each) 1uL, template pTargetF20ng, primer (10 uM) 2uL each, phanta Super-Fidelity DNA Polymerase 1uL, distilled water 34uL, total volume 50uL;
the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min, 1 cycle; denaturation at 95℃for 10 seconds, annealing at 55℃for 20 seconds, elongation at 72℃for 1.5 minutes, 30 cycles; extension at 72℃for 10 min, 1 cycle.
5. The construction method according to claim 3, wherein the nucleotide sequence of the primer aspC-KO-F in step (2) is shown in SEQ ID NO. 4; the nucleotide sequence of the primer aspC-KO-R is shown as SEQ ID NO. 5; the nucleotide sequence of the primer aspC-up500-F is shown as SEQ ID NO. 6; the nucleotide sequence of the primer aspC-Down500-R is shown as SEQ ID NO. 7;
the PCR amplification system comprises: 5X SF Buffer 10uL dNTP Mix (10 mM each) 1uL, template pTargetF20ng, primer (10 uM) 2uL each, phanta Super-Fidelity DNA Polymerase 1uL, distilled water 34uL, total volume 50uL;
the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min, 1 cycle; denaturation at 95℃for 10 seconds, annealing at 55℃for 20 seconds, elongation at 72℃for 1.5 minutes, 30 cycles; extension at 72℃for 10 min, 1 cycle.
6. The method of construction according to claim 3, wherein the nucleotide sequence of the forward primer aspC-up800-F in step (4) is shown in SEQ ID NO. 8; the nucleotide sequence of the primer aspC-down800-R is shown as SEQ ID NO. 9;
the PCR amplification system comprises: 5X SF Buffer 10uL dNTP Mix (10 mM each) 1uL, template pTargetF20ng, primer (10 uM) 2uL each, phanta Super-Fidelity DNA Polymerase 1uL, distilled water 34uL, total volume 50uL;
the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min, 1 cycle; denaturation at 95℃for 10 seconds, annealing at 55℃for 20 seconds, elongation at 72℃for 1.5 minutes, 30 cycles; extension at 72℃for 10 min, 1 cycle.
7. A process for producing L-homoserine, which comprises activating the genetically engineered bacterium of any one of claims 1 to 6 under appropriate conditions, inoculating the activated genetically engineered bacterium into a fermentation medium, and preparing L-homoserine by a biological fermentation method.
8. The method according to claim 7, wherein the proper conditions are that at a temperature of 37 ℃, an initial air flux of 2vvm and a stirring rotation speed of 300rpm, the dissolved oxygen concentration is set to 100%, the air flux is adjusted to 3vvm during the growth of the cells, the stirring rotation speed is correlated with DO value to control the dissolved oxygen concentration to be always more than 30%, when the initial glucose in the fermentation medium is consumed, the feed medium is started, the pH is controlled to 7.0 by ammonia water during the fermentation, and when the cell density reaches the absorbance OD of 600nm 600 At 30g/L, L-arabinose was added to the fermentation medium to induce protein expression at a final concentration of 2g/L, and when the feed medium was exhausted, the fermentation was ended, and L-homoserine was collected from the fermentation product.
9. The method of claim 8, wherein the fermentation medium composition is: 1-5g/L of citric acid, 1-20g/L of monopotassium phosphate, 1-5g/L of nitrogen source, 150uL/L of polyether defoamer, 5-30g/L of glucose and MgSO 4 ·7H 2 O 0.3-1g/L，VB 1 5-10mg/L, 0.1-1g/L of lysine, 0.1-1g/L of methionine, 0.1-1g/L of isoleucine, 0.1-1g/L of threonine and 1-10mL/L of trace inorganic salt I, and the pH value is 7.0+/-0.5;
the trace inorganic salt I comprises the following components: EDTA 840mg/L, coCl 2 ·6H 2 O 250mg/L，MnCl 2 ·4H 2 O1500mg/L，CuCl 2 ·2H 2 O 150mg/L，H 3 BO 3 300mg/L，Na 2 MoO 4 ·2H 2 O 250mg/L，Zn(CH 3 COO) 2 ·2H 2 1300mg/L of O and 10g/L of ferric citrate; the nitrogen source is selected from one or more of ammonium chloride, ammonium acetate, ammonium sulfate and ammonium phosphate.
10. The method of claim 8, wherein the feed medium composition is: glucose 100-800g/L MgSO 4 ·7H 2 1-5g/L of O, 1-10g/L of lysine, 1-10g/L of methionine, 1-10g/L of isoleucine, 1-10g/L of threonine and 1-10mL/L of trace inorganic salt II;
the trace inorganic saltII is composed of: EDTA 1300mg/L, coCl 2 ·6H 2 O 400mg/L，MnCl 2 ·4H 2 O2350mg/L，CuCl 2 ·2H 2 O 250mg/L，H 3 BO 3 500mg/L，Na 2 MoO 4 ·2H 2 O 400mg/L，Zn(CH 3 COO) 2 ·2H 2 1600mg/L of O and 4g/L of ferric citrate.
CN202311317213.2A 2023-10-12 2023-10-12 Genetically engineered strain for producing L-homoserine, construction method and application Pending CN117384811A (en)

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