CN118147034A - Genetically engineered bacterium for high-yield of L-cysteine, construction method and application - Google Patents

Genetically engineered bacterium for high-yield of L-cysteine, construction method and application Download PDF

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CN118147034A
CN118147034A CN202410412745.2A CN202410412745A CN118147034A CN 118147034 A CN118147034 A CN 118147034A CN 202410412745 A CN202410412745 A CN 202410412745A CN 118147034 A CN118147034 A CN 118147034A
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glk
galp
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柳志强
杨辉
张博
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-cysteine, a construction method and application thereof. The invention reduces the conversion of phosphoenolpyruvate to pyruvate by inhibiting the glucose uptake mode mediated by a phosphotransferase system, thereby being beneficial to weakening the flow of carbon metabolism to the pyruvate; through over-expression Glk/GalP mediated glucose uptake system, the problem of glucose uptake weakening caused by inhibition of a phosphotransferase system is enhanced, the assimilation efficiency of glucose is further enhanced, and the carbon metabolism flux of L-cysteine biosynthesis is improved; the expression of the key gene cysE f is driven by utilizing a Trc derivative promoter with stronger expression level, so that the problem of accumulation of L-serine, which is an L-cysteine precursor after the carbon metabolic flow of L-cysteine biosynthesis is enhanced, is solved, the efficient conversion of the precursor to L-cysteine is promoted, and the efficient synthesis of L-cysteine is realized.

Description

Genetically engineered bacterium for high-yield of L-cysteine, construction method and application
Technical Field
The invention relates to the technical field of microbial metabolism engineering, in particular to a genetically engineered bacterium for high-yield L-cysteine, a construction method and application thereof.
Background
L-cysteine is a sulfur-containing amino acid, and is one of the important components in humans and many other organisms. It plays an important role in protein synthesis and is also a key participant in biochemical processes in many organisms. L-cysteine plays a number of important roles in vivo. First, it is one of the essential amino acids of a synthetic protein, involved in the maintenance of the structure and function of the protein. Second, L-cysteine is also a precursor of glutathione, an important antioxidant that helps cells resist oxidative stress and maintain intracellular redox balance. In addition, L-cysteine is involved in other biochemical pathways in the body, such as synthesis of thioredoxin, and the like. L-cysteine has certain application in the medical field due to its important physiological function. For example, it is associated with the occurrence of cardiovascular diseases, and fluctuations and abnormalities in L-cysteine levels may lead to the occurrence of diseases. Therefore, L-cysteine has important commercial application value.
The production method of cysteine mainly comprises reduction method after hair hydrolysis, enzymatic synthesis method, chemical synthesis method, microbial fermentation method, etc. The hair hydrolysis and reduction method mainly uses keratin in hair (such as hair, pig hair and feather stalk) to hydrolyze, and the obtained cystine needs further electrolytic reduction to obtain L-cysteine. Enzymatic synthesis requires the catalytic synthesis of L-cysteine using a specific enzyme. The chemical synthesis method requires multiple steps of reactions, and the required L-cysteine can be obtained after chemical resolution. The microbial fermentation method utilizes microorganisms to ferment and produce L-cysteine, and has a plurality of advantages compared with other methods, such as simplified production steps, high substrate conversion rate and reduced pollution. Therefore, the microbial fermentation method for producing L-cysteine has important research significance.
In E.coli, the synthetic pathways of L-cysteine are largely divided into the carbon metabolic pathway and the sulfur metabolic pathway. In the carbon metabolic pathway, L-cysteine is synthesized from glucose, and 3-phosphoglycerate is produced through a series of reactions. 3-phosphoglycerate generates L-serine which is an important precursor of L-cysteine through three-step reaction. L-serine produces O-acetylserine under the action of serine acetyltransferase. Subsequently, O-acetylserine is reduced by the sulfur-containing molecule through the assimilation sulfur metabolic pathway to produce L-cysteine. In addition, L-serine is also catalyzed by Serine Hydroxymethyltransferase (SHMT) to glycine, which enters the carbon metabolic pathway.
In the metabolic engineering process of engineering strains, modifying glucose metabolic pathways of cells to concentrate the collection of carbon metabolism flow to target metabolic pathways has important significance for improving the yield of target metabolites. Glucose is one of the most common carbon sources in microbial fermentation processes. In E.coli, glucose uptake is mainly via two pathways, the phosphotransferase system (PTS) and the Glk/GalP mediated glucose uptake system. PTS is widely found in bacteria, fungi and some archaebacteria. PTS is mainly used for phosphorylating various sugars and derivatives thereof through a phosphate cascade reaction and then transporting the phosphorylated sugars and derivatives thereof into cells, and the phosphorylated sugars and derivatives not only participate in carbon and nitrogen center metabolism, regulate iron and potassium steady states and regulate the toxicity of certain pathogens, but also mediate stress reactions. However, the PTS system is capable of catalyzing the conversion of phosphoenolpyruvate to pyruvate, thereby facilitating the conversion of glucose to pyruvate. This results in a loss of carbon source for L-cysteine which is a precursor of the intermediate metabolite, triphosphate, in the conversion of glucose to pyruvate. The GLK/GalP system is a non-PTS glucose uptake system, and compared with the PTS system, the growth rate and glucose consumption rate of escherichia coli can be changed by precisely controlling the gene expression combination thereof, without depending on phosphoenolpyruvate, but there are many uncertainty factors that need to be continuously tried and improved to achieve optimization and improvement of the production efficiency of L-cysteine by modifying the genome of the strain to achieve effective control of yield and quality.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a genetically engineered bacterium for high-yield L-cysteine and a construction method thereof, and is applied to fermentation production of L-cysteine so as to solve the problems of the prior art that the carbon metabolic flow of an L-cysteine production strain is not concentrated and the L-cysteine yield is lower in the fermentation production process of L-cysteine.
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 high-yield L-cysteine, which is constructed and obtained by the following method:
(1) Taking a strain BW08: 125126 glyA:cysM: nrdH/pTrc99a-cysE f as a starting strain, marking the starting strain as a strain BW11/pE, knocking out a gene ptsG in the genome of the starting strain, and inserting a gene glk and a gene galP into the genome of the starting strain to obtain engineering bacteria BW11 DeltatssG:glk:galP/pE;
(2) The Trc promoter of the cysE f gene on the plasmid pTrc99a-cysE f is replaced by a TrcX promoter to obtain a vector plasmid pEX5, and the vector plasmid pEX5 is introduced into engineering bacteria BW11 delta ptsG glk galP to obtain engineering bacteria BW11 delta ptsG glk galP/pEX5, namely the high-yield L-cysteine genetic engineering bacteria.
In E.coli, L-cysteine biosynthesis is largely divided into the carbon metabolic pathway and the sulfur metabolic pathway. The flux of the carbon metabolic pathway has a significant effect on the production of L-cysteine. Glucose is a common carbon source in metabolic engineering. When glucose is used as a carbon source, E.coli absorbs and utilizes the carbon source mainly through two glucose uptake pathways, a phosphotransferase system and Glk/GalP-mediated glucose uptake system, respectively. Among them, the phosphotransferase system depends on the availability of phosphoenolpyruvate, which is accompanied by the conversion of phosphoenolpyruvate into pyruvate, which leads to a loss of carbon flow for L-cysteine biosynthesis. Therefore, the invention improves the concentration of carbon flow to the L-cysteine pathway by optimizing the glucose uptake mode of the strain, and simultaneously realizes the improvement of the biosynthesis of L-cysteine in escherichia coli. By attenuating glucose uptake mediated by the phosphotransferase system, glk/GalP-mediated glucose uptake is enhanced to reduce the conversion of carbon to the pyruvate pathway and to increase the metabolic flux of L-cysteine biosynthesis. Meanwhile, by increasing the expression of the key gene cysE f, the problem of front volume accumulation generated after the flux of the L-cysteine carbon metabolism pathway is enhanced is solved, the smooth conversion of carbon flow to the L-cysteine is effectively realized, and the escherichia coli genetic engineering strain for high-yield L-cysteine is obtained. The method comprises the following steps:
To increase the metabolic flux of the L-cysteine carbon metabolic stream, the phosphotransferase system is deleted to reduce the conversion of metabolic flux to pyruvate. The present invention redirects carbon metabolic flux to the L-cysteine metabolic pathway of cells by deleting ptsG genes on the genome to inhibit the phosphotransferase system. Inactivation of the phosphotransferase system, although potentially reducing conversion of phosphoenolpyruvate to pyruvate, also results in limited glucose uptake by the strain, and therefore, the invention enhances glk gene expression, enhances Glk/GalP-mediated glucose uptake, and improves L-cysteine biosynthesis. The gene galP is one of the important components of the Glk/GalP-mediated glucose uptake system. To further evaluate the effect of enhancing Glk/GalP-mediated glucose uptake system on L-cysteine production, the gene galP was further over-expressed to enhance L-cysteine production. In order to achieve efficient conversion of L-serine to L-cysteine, it is necessary to further increase the expression level of cysE f which releases the feedback inhibition. To further achieve high expression of cysE f, the Trc promoter (nucleotide sequence shown in SEQ ID NO. 5) driving cysE f expression on the pE plasmid (obtained by cloning cysE f into vector pTrc99 a) was replaced with a higher level TrcX promoter, thus obtaining vector plasmid pEX5, thereby enhancing the expression level of the target gene.
Of these, the starting strain BW08: 125126 glyA:cysM: nrdH/pTrc99a-cysE f is disclosed in patent CN 116836905A.
Preferably, the nucleotide sequence of the gene ptsG is shown as SEQ ID NO.1, the nucleotide sequence of the gene glk is shown as SEQ ID NO.2, and the nucleotide sequence of the gene galP is shown as SEQ ID NO. 3.
Preferably, the nucleotide sequence of the TrcX promoter is shown as SEQ ID NO. 6.
Preferably, the nucleotide sequence of the cysE f gene is shown in SEQ ID NO. 4.
In a second aspect, the invention provides a method for constructing the genetically engineered bacterium for producing L-cysteine at high yield, which takes a strain BW08: 125126 glyA:cysM: nrdH/pTrc99a-cysE f as a starting strain, and is recorded as a strain BW11/pE, and comprises the following steps:
(1) Knocking out the gene ptsG in the genome to obtain engineering bacteria BW11ΔptsG/pE;
(2) Inserting a gene glk into a genome of engineering bacteria BW11ΔptsG/pE to obtain engineering bacteria BW11ΔptsG: glk/pE;
(3) Inserting a gene galP into a genome of engineering bacteria BW11ΔptsG:: glk/pE to obtain engineering bacteria BW11ΔptsG:: glk:: galP/pE;
(4) The Trc promoter of the cysE f gene on the plasmid pTrc99a-cysE f is replaced by a TrcX promoter to obtain a vector plasmid pEX5, and the vector plasmid pEX5 is introduced into engineering bacteria BW11 delta ptsG glk galP to obtain engineering bacteria BW11 delta ptsG glk galP/pEX5, namely the high-yield L-cysteine genetic engineering bacteria.
In a third aspect, the invention provides the genetically engineered bacterium for high-yield L-cysteine or the application of the genetically engineered bacterium for high-yield L-cysteine constructed by the method in the preparation of L-cysteine by microbial fermentation.
Preferably, the genetic engineering strain is inoculated to a fermentation culture medium, and is subjected to fermentation culture at the temperature of 28-37 ℃ and the speed of 150-200rpm, and after fermentation, the supernatant of the fermentation broth is taken, separated and purified to obtain the L-cysteine.
Preferably, the fermentation culture is carried out in a culture medium at a temperature of between 30 and 37 ℃ and at a speed of between 150 and 200rpm for 12 to 24 hours before the fermentation culture is carried out, and then the fermentation culture is inoculated into the fermentation culture medium.
Specifically, the genetic engineering strain is inoculated into a 10ml LB culture medium test tube, and cultured for 12-24 hours on a shaking table with the temperature of 30-37 ℃ and the rotating speed of 150-200 rpm. Then inoculating the strain with 1% of volume concentration into 20-50 ml of fermentation culture medium to start fermentation. After 4 to 6 hours of cultivation, 0.1mM IPTG was added. The fermentation temperature is 28-37 ℃, the rotating speed is 150-200rpm, and the fermentation time is 2-4 days. And after fermentation, taking a supernatant of the fermentation liquor, and separating and purifying to obtain the L-cysteine.
Preferably, the fermentation medium composition comprises: glucose 20~30g/L、(NH4)2SO45~10g/L、KH2PO42~5g/L、Na2S2O35~10g/L、 g/L, na 2HPO4 g/15 g/L yeast extract, peptone 1 g/L to 5g/L,1ml/L microelement solution, and deionized water as solvent.
Preferably, the trace element solution comprises :300~500g/L MgSO4·8H2O,2~5g/LMnSO4·8H2O,2~5g/L ZnSO4·7H2O,2~8g/L Fe2SO4, solvent which is deionized water.
Compared with the prior art, the invention has the following beneficial effects:
the invention proposes to promote the production of L-cysteine by improving the glucose uptake pattern of E.coli. By inhibiting the glucose uptake mode mediated by the phosphotransferase system, the conversion of phosphoenolpyruvate to pyruvate is reduced, which helps to attenuate the loss of carbon flow in L-cysteine biosynthesis due to the flow of carbon metabolism towards pyruvate. Through over-expression Glk/GalP mediated glucose uptake system, the problem of glucose uptake weakening caused by inhibition of a phosphotransferase system is enhanced, the glucose assimilation efficiency is further enhanced, and the carbon metabolism flux of L-cysteine biosynthesis is improved. Then, the expression of the key gene cysE f is driven by using a Trc derivative promoter with stronger expression level, so that the problem of L-serine accumulation, which is an L-cysteine precursor after the carbon metabolic flow of the L-cysteine biosynthesis is enhanced, is solved, and the efficient conversion of the precursor into L-cysteine is promoted. The invention successfully constructs a genetic engineering strain for high-yield L-cysteine through reasonable metabolic engineering transformation, and realizes the efficient synthesis of the L-cysteine.
Drawings
FIG. 1 shows the L-cysteine yields and OD 600 for the strains BW11/pE and BW11ΔptsG/pE.
FIG. 2 shows the L-cysteine yield and OD 600 of the strain BW11ΔptsG:: glk/pE.
FIG. 3 shows the L-cysteine yield and OD 600 of the strain BW11ΔptsG:: glk:: galP/pE.
FIG. 4 shows the L-serine yield and OD 600 of the strain BW11ΔptsG:: glk:: galP/pE.
FIG. 5 shows the L-cysteine yield and OD 600 of the strain BW11ΔptsG:: glk:: galP/pEX 5.
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.
In the following examples, the final concentration of kanamycin in the medium was 50mg/L, the final concentration of spectinomycin in the medium was 50mg/L, and the final concentration of ampicillin in the medium was 100mg/L. The starting strain BW08::125126glyA:: cysM:: nrdH/pTrc99a-cysE f is disclosed in patent CN116836905A from the institute of biotechnology for the synthesis of university of Zhejiang.
Example 1 determination of L-cysteine yield in fermentation broth
(1) 1ML of the bacterial liquid was centrifuged at 12000rpm for 1 minute in a 2mL EP tube, and the supernatant and the pellet were separated. The supernatant was used for detection of L-cysteine, L-serine and other metabolites.
(2) 0.27G CNBF g was weighed out and dissolved in 10mL acetonitrile as solution I; the mother liquor is 0.2M boric acid solution and 0.05M borax solution, and the standard buffer solution with pH=9.0 is prepared by mixing 4:1 volumes and is named as solution II. The sample is diluted to 0-5 g/L concentration, mixed according to the proportion of 100 mu L of the sample, 300 mu L of the I solution and 500 mu L of the II solution, and reacted for 0.5-1 hour at the temperature of 40-60 ℃ and the rpm of 500-1000 in a constant temperature oscillator. And filling the sample into a liquid phase bottle through a film to be tested.
(3) The instrument is a Siemens flight UPLC ultra-high pressure liquid chromatograph. The chromatographic column was a C18 column (4.6X105 mm,5 μm); the ultraviolet detector detects the wavelength of 260nm; the sample injection amount is 10 mu L; column temperature is 30 ℃; the flow rate is 0.8mL/min; the mobile phase used was AB two phases, phase A neat acetonitrile, phase B50 mM HAc-NaAc buffer: acetonitrile: triethylamine = 82.8:17:0.2, ph=4.9. The gradient elution procedure is shown in table 1.
TABLE 1 gradient elution procedure
EXAMPLE 2 construction of vector pE overexpressing cysE f Gene
In order to achieve accumulation of L-cysteine, it is necessary to overexpress cysE f which releases the feedback inhibition. To achieve overexpression of cysE f, cysE f was cloned into the vector pTrc99a and expression was driven by the Trc promoter on pTrc99 a.
(1) The pTrc99a plasmid was used as a template (99 aline-F and 99 aline-R), and amplification was performed by PCR to obtain a linearized vector. The E.coli BW25113 genome is used as a template (primers cysE-F and cysE-R), and a gene cysE fragment is obtained by amplification. The PCR product was digested with DpnI. All PCR products were detected by 1.0% agarose gel electrophoresis and PCR fragments were purified. The linearized vector was ligated with the gene fragment cysE, transformed into E.coli DH 5. Alpha. And plated on ampicillin resistant plates according to the instructions of the one-step cloning kit (One step clonekit, vazyme Biotech, nanjing, china), single colonies were picked and verified by colony PCR (primers 99a-VF and 99 a-VR), and the pTrc99a-cysE plasmid was obtained by sequencing verification.
(2) The plasmid pTrc99a-cysE is used as a template, primers T167A-F and T167A-R, G245S-F and G245S-R are used for PCR, the primers are transformed into E.coli DH5 alpha, the primers are coated on an ampicillin resistance plate, single colony is picked up and verified by colony PCR (primers 99a-VF and 99 a-VR), and the plasmid containing pE of cysE mutant cysE f is obtained by sequencing verification.
TABLE 2 example 2 primers
Primer name Sequence (5 '-3')
cysE-F tttcacacaggaaacagaccatgtcgtgtgaagaactggaaattg
cysE-R tctcatccgccaaaacagccttagatcccatccccatactcaaatg
99aline-F ggctgttttggcggatgagag
99aline-R ggtctgtttcctgtgtgaaattg
T167A-F gttggtgaagcggcggtgattgaaaacgac
T167A-R tcaccgccgcttcaccaacgacgatgcctg
G245S-F tattgtcagcaaaccagacagcgataagcc
G245S-R ctggtttgctgacaatacgagccggaacgc
99a-VF gtttgacagcttatcatcgactgc
99a-VR agaccgcttctgcgttctg
Example 3 construction of genetically engineered strain BW11ΔptsG/pE for L-cysteine production to increase the metabolic flux of the L-cysteine carbon metabolic stream, the phosphotransferase system was deleted to reduce the conversion of metabolic flux to pyruvate. The present invention redirects carbon metabolic flux to the L-cysteine metabolic pathway of cells by deleting ptsG genes on the genome to inhibit the phosphotransferase system.
(1) The gRNA was subjected to site-directed mutagenesis by PCR amplification (primers pT-ptsG-F and pT-ptsG-R) using the pTarget plasmid (ADDGENE PLASMID # 62226) as template. The PCR product was digested with DpnI. Digestion products are transferred to E.coli DH 5. Alpha. And spread on a spectinomycin plate, single colonies are picked for sequencing verification (primers pT-VF and pT-VR), and the mutated pTarget-ptsG plasmid is selected.
(2) The E.coli BW25113 genome is used as a template, and the upstream and downstream 500bp of the gene ptsG is obtained through PCR amplification by using primers ptsG-Up-F and ptsG-Up-R, ptsG-Down-F and ptsG-Down-R. The two DNA fragments were fused by fusion PCR to obtain the fragment Donor-ptsG.
(3) The strain BW11 was prepared to a transformation competent, pCas plasmid (ADDGENE PLASMID # 62225) was transformed into BW11 transformation competent by chemical transformation, and applied to a kanamycin resistance plate to give strain BW11/pCas.
(4) Strain BW11/pCas was made electrotransport competent. The plasmid pTarget-ptsG and the fragment Donor-ptsG are electrically transferred to BW11/pCas to be electrically transferred and then coated on a kanamycin and spectinomycin double-resistance plate, single bacterial colonies are selected for PCR verification (primers ptsG-VF and ptsG-VR), and successfully edited bacterial strains are screened to obtain the bacterial strain BW11ΔptsG. The primers are shown in Table 3.
(5) Positive single colonies were picked up and inoculated into LB tubes containing 1mM IPTG and 0.05mg/L kanamycin, incubated overnight at 30℃and streaked on LB plates containing 0.05mg/L kanamycin, incubated for 24h at 30℃and single colonies were picked up and streaked on LB plates containing 0.05mg/L spectinomycin and failed to successfully eliminate the pTarget-ptsG plasmid on LB plates containing 0.05mg/L spectinomycin. Single colonies successfully eliminated by pTarget-ptsG plasmid were picked up in LB tubes, incubated overnight at 37℃and streaked on LB plates with the next day bacterial solution, incubated for 12h at 37℃and single colonies streaked on LB plates containing 0.05mg/L kanamycin were picked up and single colonies not successfully eliminated on LB plates containing 0.05mg/L kanamycin, which pCas plasmid was finally obtained without plasmid BW11ΔptsG.
(6) The strain BW11ΔptsG was prepared into a chemo-competent cell, and the pE plasmid constructed in example 2 was transformed into BW11ΔptsG competent cell by a chemical transformation method to obtain BW11ΔptsG/pE strain.
(7) The strain BW11ΔptsG/pE was inoculated into 10mL of LB medium and cultured overnight at 30-37℃and 150-200 rpm. 1mL of the preculture is inoculated into a 500mL shaking flask containing 20-50 mL of fermentation medium, and after 4-6 hours of culture, 0.1mM of IPTG is added to ferment for 2-4 days. The fermentation medium comprises the following components: glucose 20~30g/L、(NH4)2SO45~10g/L、KH2PO42~5g/L、Na2S2O35~10g/L、 g/L, na 2HPO4 g/15 g/L yeast extract, peptone 1 g/L to 5g/L,1ml/L microelement solution, deionized water as solvent, and natural pH value. The trace element solution comprises :300~500g/L MgSO4·8H2O,2~5g/L MnSO4·8H2O,2~5g/L ZnSO4·7H2O,2~8g/L Fe2SO4, of deionized water as solvent. The fermentation broth was tested according to example 1 and the OD 600 and L-cysteine content of the supernatant of the fermentation broth are shown in FIG. 1.
As can be seen from the results in FIG. 1, the L-cysteine yield of the strain BW11ΔptsG/pE was 0.55g/L, and the OD 600 value of the cells was 10.45. The L-cysteine yield was reduced by 49% compared to the control strain BW11/pE (1.07 g/L). This is mainly caused by the inhibition of growth, suggesting that merely weakening the phosphotransferase system may affect cell growth, and thus further improvement of glucose uptake function is required.
TABLE 3 example 3 primers
Primer name Sequence (5 '-3')
ptsG-Up-F atcggttactggtggaaactgactc
ptsG-Up-R gtcttacggaaattgagagtgctcctgagtatggg
ptsG-Down-F actctcaatttccgtaagacgttggggagactaag
ptsG-Down-R gtggatgggacagtcagtaaaggg
pT-ptsG-F ggtaaatcgctgatgctgcgttttagagctagaaatagc
pT-ptsG-R cagcatcagcgatttaccgactagtattatacctaggactgagc
ptsG-VF agaaacggcgggtaaattactg
ptsGVR atagccgtctgaccaccac
EXAMPLE 4 Effect of over-expression of the glk Gene on L-cysteine production
Inactivation of the phosphotransferase system, although potentially reducing conversion of phosphoenolpyruvate to pyruvate, also results in limited glucose uptake by the strain, and therefore, the invention enhances glk gene expression, enhances Glk/GalP-mediated glucose uptake, and improves L-cysteine biosynthesis.
(1) The gRNA was subjected to site-directed mutagenesis by PCR amplification (primers pT-glk-F and pT-glk-R) using the pTarget plasmid (ADDGENE PLASMID # 62226) as template. The PCR product was digested with DpnI. Digestion products were transferred to E.coli DH 5. Alpha. And plated on a spectinomycin plate, single colonies were picked for sequencing verification (primers pT-VF and pT-VR), and successfully mutated pTarget-glk plasmids were selected.
(2) The strain BW11 was prepared to a transformation competent, pCas plasmid (ADDGENE PLASMID # 62225) was transformed into BW11 transformation competent by chemical transformation, and applied to a kanamycin resistance plate to give strain BW11/pCas.
(3) The E.coli BW25113 genome is used as a template, and the primers glk-Up-F and glk-Up-R, glk-F and glk-R, glk-Down-F and glk-Down-R are used for PCR amplification to obtain 500bp upstream and downstream of the insertion site and glk genes. The above DNA fragments were fused by fusion PCR to obtain the fragment Donor-glk.
(4) Strain BW11/pCas was made electrotransport competent. The plasmid pTarget-glk and the fragment Donor-glk are electrotransferred to BW11/pCas and coated on a kanamycin and spectinomycin double-resistance plate, single colonies are selected for PCR verification (primers glk-VF and glk-VR), and successfully edited strains are selected to obtain the strain BW11ΔptsG:: glk. The primers are shown in Table 4.
(5) Positive single colonies were picked up and inoculated into LB tubes containing 1mM IPTG and 0.05mg/L kanamycin, incubated overnight at 30℃and streaked on LB plates containing 0.05mg/L kanamycin, incubated for 24h at 30℃and single colonies were picked up and streaked on LB plates containing 0.05mg/L spectinomycin and failed to successfully eliminate the pTarget-glk plasmid on LB plates containing 0.05mg/L spectinomycin. Single colonies successfully eliminated by pTarget-glk plasmid are picked up in LB test tubes, cultured overnight at 37 ℃, streaked with the next day bacterial solution on LB plates, cultured for 12 hours at 37 ℃, single colonies streaked with LB plates containing 0.05mg/L kanamycin are picked up, single colonies on LB plates containing 0.05mg/L kanamycin cannot be successfully eliminated by pCas plasmids, and finally the plasmid BW11ΔptsG: glk is obtained.
(6) The strain BW11ΔptsG:: glk was prepared as a chemocompetent cell, and the pE plasmid constructed in example 2 was transformed into BW11ΔptsG:: glk competent by a chemical transformation method to obtain BW11ΔptsG:: glk/pE strain.
(7) The strain BW11ΔptsG glk/pE was inoculated into 10mL of LB medium and cultured overnight at 30-37℃and 150-200 rpm. 1mL of the preculture is inoculated into a 500mL shaking flask containing 20-50 mL of fermentation medium, and after 4-6 hours of culture, 0.1mM of IPTG is added to ferment for 2-4 days. The fermentation medium is as described in example 2. The fermentation broth was tested according to example 1 and the OD 600 and L-cysteine content of the supernatant of the fermentation broth are shown in FIG. 2.
To ameliorate the problem of limited glucose uptake following inactivation of the phosphotransferase system, the gene glk of the Glk/GalP-mediated glucose uptake system was overexpressed. As can be seen from the results in FIG. 2, the strain BW11ΔptsG:: L-cysteine yield of glk/pE was 1.42g/L. The results indicate that overexpression of the gene glk effectively promotes L-cysteine production. These efforts provide the basis for further enhancing the efficient production of L-cysteine.
TABLE 4 example 4 primers
Primer name Sequence (5 '-3')
glk-Up-F atcggttactggtggaaactgactc
glk-Up-R tccgctcacaattccacacattatacgagccggatgattaattgtcaaaattgagagtgctcctgagt
glk-F aatgtgtggaattgtgagcggataacaatttcacacaggaaacagaccatgacaaagtatgcattagtc
glk-R acgtcttacggattacagaatgtgacctaaggtctg
glk-Down-F cattctgtaatccgtaagacgttggggagactaag
glk-Down-R gtggatgggacagtcagtaaaggg
pT-glk-F ggtaaatcgctgatgctgcgttttagagctagaaatagc
pT-glk-R cagcatcagcgatttaccgactagtattatacctaggactgagc
glk-VF agaaacggcgggtaaattactg
glk-VR atagccgtctgaccaccac
EXAMPLE 5 Effect of overexpression of galP Gene on L-cysteine production
The gene galP is one of the important components of the Glk/GalP-mediated glucose uptake system. To further evaluate the effect of enhancing Glk/GalP-mediated glucose uptake system on L-cysteine production, the gene galP was further over-expressed to enhance L-cysteine production.
(1) The gRNA was subjected to site-directed mutagenesis by PCR amplification (primers pT-galP-F and pT-galP-R) using the pTarget plasmid (ADDGENE PLASMID # 62226) as template. The PCR product was digested with DpnI. The digested products were transferred to E.coli DH 5. Alpha. And plated on a spectinomycin plate, single colonies were picked for sequencing verification (primers pT-VF and pT-VR), and the mutated pTarget-galP plasmid was selected.
(2) The strain BW11ΔptsG:: glk was prepared to a transformation competent, pCas plasmid (ADDGENE PLASMID # 62225) was transformed into BW11ΔptsG:: glk transformation competent by a chemical transformation method, and was coated on a kanamycin resistance plate to obtain the strain BW11ΔptsG:: glk/pCas.
(3) The BW11ΔptsG is taken as a template, and PCR amplification is carried out by using primers galP-Up-F and galP-Up-R, galP-F and galP-R, glk-Down-F and glk-Down-R to obtain 500bp upstream and downstream of the insertion site and galP genes. The DNA fragment was fused by fusion PCR to obtain a fragment Donor-galP.
(4) The strain BW11ΔptsG:: glk/pCas was made electrotransduce competent. The plasmid pTarget-galP and the fragment Donor-galP are electrotransferred to BW11ΔptsG:: glk/pCas, and then coated on a kanamycin and spectinomycin double-resistance plate, single colonies are picked for PCR verification (primers galP-VF and galP-VR), and successfully edited strains are screened to obtain the strain BW11ΔptsG::: glk::: galP. The primers are shown in Table 5.
(5) Positive single colonies were picked up and inoculated into LB tubes containing 1mM IPTG and 0.05mg/L kanamycin, incubated overnight at 30℃and streaked on LB plates containing 0.05mg/L kanamycin, incubated for 24 hours at 30℃and single colonies were picked up and streaked on LB plates containing 0.05mg/L spectinomycin and failed to successfully eliminate the pTarget-galP plasmid on LB plates containing 0.05mg/L spectinomycin. Single colonies successfully eliminated by pTarget-galP plasmids are picked up in LB test tubes, cultured overnight at 37 ℃, streaked with the next day bacterial solution in LB plates, cultured for 12 hours at 37 ℃, single colonies streaked with LB plates containing 0.05mg/L kanamycin are picked up, single colonies not containing 0.05mg/L kanamycin can not be successfully eliminated by pCas plasmids, and finally the plasmid-free BW11ΔptsG:: glk: galP is obtained.
(6) The strain BW11ΔptsG: glk: galP was prepared into a competent cell, and the pE plasmid constructed in example 2 was transformed into BW11ΔptsG: glk: galP competent cells by a chemical transformation method to obtain the BW11ΔptsG:: glk:: galP/pE strain.
(7) The strain BW11ΔptsG:: glk:: galP/pE was inoculated into 10mL of LB medium and cultured overnight at 30-37℃and 150-200 rpm. 1mL of the preculture is inoculated into a 500mL shaking flask containing 20-50 mL of fermentation medium, and after 4-6 hours of culture, 0.1mM of IPTG is added to ferment for 2-4 days. The fermentation medium is as described in example 2. The fermentation broth was tested according to example 1 and the OD 600 and L-cysteine content of the supernatant of the fermentation broth are shown in FIG. 3.
To further enhance glucose uptake, the gene galP of Glk/GalP-mediated glucose uptake system was overexpressed. As can be seen from the results in FIG. 3, the L-cysteine yield of the strain BW11ΔptsG:: glk:: galP/pE was 1.94g/L. This result suggests that further overexpression of the gene galP effectively promotes L-cysteine production.
TABLE 5 example 5 primers
Primer name Sequence (5 '-3')
galP-Up-F aaggcggtcacgttgattttg
galP-Up-R gcgtcaggcatggtctgtttcctgtgtgaaattacagaatgtgacctaag
galP-F cattctgtaatttcacacaggaaacagaccatgcctgacgctaaaaaacag
galP-R ccatctggctgttaatcgtgagcgcctatttcg
galP-Down-F tcacgattaacagccagatggctgccttttttac
galP-Down-R ctataaagcggtggatgggacagtcagtaaag
pT-galP-F taagacgttggggagactagttttagagctagaaatagc
pT-galP-R agtctccccaacgtcttacactagtattatacctaggactgagctag
galP-VF tgctgaaaaaagagcatctgattcag
galP-VR atagccgtctgaccaccac
Example 6 accumulation level of L-cysteine precursor L-serine of the strain BW11ΔptsG:: glk:: galP/pE evaluation in E.coli, L-serine is an important precursor for carbon metabolism in L-E.coli. With an increase in L-cysteine carbon metabolic flux, it is necessary to analyze the accumulation level of L-serine as a precondition.
(1) The strain BW11ΔptsG:: glk:: galP/pE was inoculated into 10mL of LB medium and cultured overnight at 30-37℃and 150-200 rpm. 1mL of the preculture is inoculated into a 500mL shaking flask containing 20-50 mL of fermentation medium, and after 4-6 hours of culture, 0.1mM of IPTG is added to ferment for 2-4 days. The fermentation medium is as described in example 2.
(2) The fermentation broth was tested according to example 1 and the OD 600 and L-serine content in the supernatant of the fermentation broth are shown in FIG. 4.
Fermentation experiments showed that the strain BW11ΔptsG:: glk: galP/pE accumulated L-serine at 1.53g/L, although the L-cysteine yield was significantly increased. This result indicates that the conversion of L-serine to L-cysteine is insufficient.
Example 7 construction of the high cysE f expression strain BW11ΔptsG:: glk:: galP/pEX5 in order to achieve efficient conversion of L-serine to L-cysteine, a further increase in the expression level of cysE f, which releases the feedback inhibition, was necessary. To further achieve high expression of cysE f, the Trc promoter driving cysE f expression on the pE plasmid was replaced with a higher level TrcX5 promoter, thereby enhancing the expression level of the target gene.
(1) The plasmid pE is used as a template, the primers X5-F and X5-R are used for PCR, the plasmid pE is transformed into E.coli DH5 alpha, the E.coli DH5 alpha is coated on an ampicillin resistance plate, single colony is picked up and verified by colony PCR (primers 99a-VF and 99 a-VR), and the pEX5 plasmid is obtained by sequencing verification.
(2) The BW11ΔptsG: glk: galP is prepared into chemosensory cells, and the constructed pEX5 plasmid is transformed into BW11ΔptsG:: glk: galP sensory cells by a chemical transformation method to obtain BW11ΔptsG:: glk:: galP/pEX5 strain.
(3) The strain BW11ΔptsG:: glk:: galP/pEX5 was inoculated into 10mL of LB medium and cultured overnight at 30-37℃and 150-200 rpm. 1mL of the preculture is inoculated into a 500mL shaking flask containing 20-50 mL of fermentation medium, and after 4-6 hours of culture, 0.1mM of IPTG is added to ferment for 2-4 days. The fermentation medium is as described in example 2. The fermentation broth was tested according to the method of example 1 and the OD 600 and L-cysteine and L-serine contents in the supernatant of the fermentation broth are shown in FIG. 5.
The fermentation result shows that the yield of L-cysteine of BW11ΔptsG glk galP/pEX5 is obviously improved to 3.04g/L. The result shows that the introduction of TrcX promoter promotes the conversion level of L-serine to L-cysteine to be obviously improved, and the production of L-cysteine is effectively improved.
TABLE 6 example 7 primers
Primer name Sequence (5 '-3')
X5-F cggataacaaaaagagggcacaatgtcgtgtgaagaactggaaattg
X5-R cacacgacattgtgccctctttttgttatccgctcacaattccacac
99a-VF gtttgacagcttatcatcgactgc
99a-VR agaccgcttctgcgttctg

Claims (10)

1. The genetically engineered bacterium for producing the L-cysteine at high yield is characterized by being constructed and obtained by the following method:
(1) Taking a strain BW08: 125126 glyA:cysM: nrdH/pTrc99a-cysE f as a starting strain, marking the starting strain as a strain BW11/pE, knocking out a gene ptsG in the genome of the starting strain, and inserting a gene glk and a gene galP into the genome of the starting strain to obtain engineering bacteria BW11 DeltatssG:glk:galP/pE;
(2) The Trc promoter of the cysE f gene on the plasmid pTrc99a-cysE f is replaced by a TrcX promoter to obtain a vector plasmid pEX5, and the vector plasmid pEX5 is introduced into engineering bacteria BW11 delta ptsG glk galP to obtain engineering bacteria BW11 delta ptsG glk galP/pEX5, namely the high-yield L-cysteine genetic engineering bacteria.
2. A genetically engineered bacterium capable of producing L-cysteine at high yield as defined in claim 1, wherein the nucleotide sequence of the gene ptsG is shown in SEQ ID NO.1, the nucleotide sequence of the gene glk is shown in SEQ ID NO.2, and the nucleotide sequence of the gene galP is shown in SEQ ID NO. 3.
3. The genetically engineered bacterium for high yield of L-cysteine according to claim 1 or 2, wherein the nucleotide sequence of the TrcX promoter is shown as SEQ ID No. 6.
4. The genetically engineered bacterium for high yield of L-cysteine according to claim 1 or 2, wherein the nucleotide sequence of the gene cysE f is shown as SEQ ID No. 4.
5. A method for constructing the genetically engineered bacterium capable of producing L-cysteine according to any one of claims 1 to 4, wherein the method is characterized in that a strain BW08: 125126 glyA:cysM: nrdH/pTrc99a-cysE f is taken as a starting strain and is recorded as a strain BW11/pE, and the method comprises the following steps:
(1) Knocking out the gene ptsG in the genome to obtain engineering bacteria BW11ΔptsG/pE;
(2) Inserting a gene glk into a genome of engineering bacteria BW11ΔptsG/pE to obtain engineering bacteria BW11ΔptsG: glk/pE;
(3) Inserting a gene galP into a genome of engineering bacteria BW11ΔptsG:: glk/pE to obtain engineering bacteria BW11ΔptsG:: glk:: galP/pE;
(4) The Trc promoter of the cysE f gene on the plasmid pTrc99a-cysE f is replaced by a TrcX promoter to obtain a vector plasmid pEX5, and the vector plasmid pEX5 is introduced into engineering bacteria BW11 delta ptsG glk galP to obtain engineering bacteria BW11 delta ptsG glk galP/pEX5, namely the high-yield L-cysteine genetic engineering bacteria.
6. The use of the genetically engineered bacterium for high-yield of L-cysteine according to any one of claims 1 to 4 or the genetically engineered bacterium for high-yield of L-cysteine constructed by the method according to claim 5 in microbial fermentation preparation of L-cysteine.
7. The use according to claim 6, wherein the genetically engineered strain is inoculated into a fermentation medium, and is subjected to fermentation culture at 28-37 ℃ and 150-200rpm, and after fermentation, the supernatant of the fermentation broth is taken for separation and purification to obtain the L-cysteine.
8. The use according to claim 7, wherein the fermentation medium is inoculated with a medium at a temperature of from 30 to 37℃and at a speed of from 150 to 200rpm for a period of from 12 to 24 hours.
9. The use according to claim 7 or 8, wherein the fermentation medium composition comprises: glucose 20~30g/L、(NH4)2SO45~10g/L、KH2PO42~5g/L、Na2S2O35~10g/L、 g/L, na 2HPO4 g/15 g/L yeast extract, peptone 1 g/L to 5g/L,1ml/L microelement solution, and deionized water as solvent.
10. The use of claim 9, wherein the trace element solution composition :300~500g/LMgSO4·8H2O,2~5g/LMnSO4·8H2O,2~5g/L ZnSO4·7H2O,2~8g/LFe2SO4, solvent is deionized water.
CN202410412745.2A 2024-04-08 2024-04-08 Genetically engineered bacterium for high-yield of L-cysteine, construction method and application Pending CN118147034A (en)

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