CN117683802B - Ralstonia engineering strain for producing isoleucine through methyl malic acid pathway, construction method and production method thereof - Google Patents
Ralstonia engineering strain for producing isoleucine through methyl malic acid pathway, construction method and production method thereof Download PDFInfo
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- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 title claims abstract description 105
- 229960000310 isoleucine Drugs 0.000 title claims abstract description 105
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 title claims abstract description 105
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- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 239000001630 malic acid Substances 0.000 title claims abstract description 49
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- -1 methyl malic acid Chemical compound 0.000 title claims abstract description 49
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 31
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- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 claims abstract description 16
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Abstract
The invention belongs to the technical field of biology, and relates to a Ralstonia engineering strain for producing isoleucine through a methyl malic acid pathway, and a construction and production method thereof. The invention establishes a methyl malic acid pathway to synthesize isoleucine by expressing a methyl malic acid synthase gene; the transport metabolism of the strain to glucose is realized by modifying an endogenous N-acetylglucosamine transport system; accumulation of isoleucine in the fermentation broth is achieved by expression of a branched-chain amino acid-exoprotein gene. The production capacity of the engineering strain for synthesizing the isoleucine through a methyl malic acid way is obviously improved by adjusting coenzyme balance, relieving feedback inhibition, knocking out metabolic bypass and adopting feeding and tank feeding fermentation. The engineering strain takes glucose as a unique carbon source, the yield of isoleucine after 36 hours of fermentation is 857 mg/L, and the yield of isoleucine after 25 days of fermentation is 105 mg/L by taking CO 2 as a unique carbon source.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a Ralstonia engineering strain for producing isoleucine through a methyl malic acid pathway, and a construction and production method thereof.
Background
Isoleucine belongs to branched-chain amino acids and is one of the essential amino acids for human body. Since they cannot be synthesized autonomously and can be obtained only by means of exogenous foods, the market demand for isoleucine is enormous. Isoleucine as a precursor for protein synthesis can directly participate in metabolic processes such as muscle repair and oxidation energy supply, and can supplement isoleucine in food to strengthen nutritional ingredients, rapidly convert into glucose in liver to prevent muscle injury, synergistically promote insulin secretion to regulate blood sugar, and improve food nutritional value. The isoleucine additive is supplemented in the feed, so that the amino acid balance of the feed can be adjusted, the protein level of the feed can be reduced, and the application of low-protein ration can be promoted. Isoleucine can also be used as a signal molecule to participate in blood sugar regulation, and can regulate and control organism metabolism and hormone secretion through an mTOR signal path, promote organism cell proliferation and protein synthesis, and increase immunity; the lipid oxidation products are reduced by regulating intestinal flora, so that the tolerance of oxidative stress of the organism is enhanced. Therefore, the isoleucine has wide application prospect.
Isoleucine synthesis levels are relatively low compared to other amino acids. Due to the two chiral carbon atoms in the isoleucine molecule, it is difficult to obtain high purity isoleucine with single conformation by chemical synthesis. At present, industrial production of high-purity isoleucine is mainly realized by a microbial fermentation method of corynebacterium glutamicum and escherichia coli. In recent years, the metabolic pathway of the production strain is optimized through traditional breeding and genetic engineering means, and methods of enhancing substrate transport, controlling carbon source flow, relieving feedback inhibition and the like are gradually applied to the construction of the high-yield strain, so that the fermentation yield of isoleucine is improved. However, in both Corynebacterium glutamicum and Escherichia coli, the traditional microbial synthesis of isoleucine requires the threonine pathway. Six amino acid synthesis bypasses and five speed-limiting catalytic enzymes with feedback regulation sites exist in the threonine pathway, the complex metabolic pathway and regulation network limit the production efficiency of isoleucine synthesized by the metabolic engineering regulation threonine pathway, and technical breakthrough is difficult to realize by means of the traditional transformation means. Therefore, a brand new chassis organism is searched, a new strategy for efficiently synthesizing the isoleucine is explored, the fermentation yield of the isoleucine can be further improved, and the bottleneck problem of the industrial production of the isoleucine is solved.
Disclosure of Invention
The invention aims to provide a Ralstonia engineering strain for producing isoleucine through a methyl malic acid pathway.
It is a further object of the present invention to provide a method of constructing an engineered strain of ralstonia which produces isoleucine via the methyl malate pathway.
It is another object of the present invention to provide a method for producing isoleucine by fermentation using an engineering strain of Ralstonia.
The method for constructing a rogowski bacterium engineering strain producing isoleucine through a methyl malic acid pathway according to the present invention comprises the steps of:
Modifying an endogenous N-acetylglucosamine transport system of a wild-type Ralstonia strain to realize the utilization of glucose by the engineering strain, wherein the step of modifying the endogenous N-acetylglucosamine transport system of the wild-type Ralstonia strain comprises the following steps: the 793 rd base of N-acetylglucosamine transporter gene nagE based on a phosphotransferase system of a wild-type Ralstonia strain is mutated from G to C, and the nucleotide sequence of the mutated N-acetylglucosamine transporter gene nagE is shown as SEQ ID NO:4, and knocking out a transcriptional regulator gene nagR of the N-acetylglucosamine transporter, wherein the nucleotide sequence of the transcriptional regulator gene nagR is shown as SEQ ID NO:5 is shown in the figure;
Expressing a methyl malate synthase gene cimA and branched-chain amino acid exoprotein genes ygaZ and ygaH, realizing the synthesis and secretion of isoleucine by Ralstonia through a methyl malate pathway, wherein the nucleotide sequence of the methyl malate synthase gene cimA is shown as SEQ ID NO:1, wherein the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaZ is shown in SEQ ID NO:2, the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaH is shown as SEQ ID NO: 3.
The method for constructing a rogowski bacterium engineering strain producing isoleucine through the methyl malic acid pathway according to the present invention, wherein the method further comprises the steps of:
Knocking out endogenous polyhydroxybutyrate synthesis genes phaC1, phaA and phaB1 of the Ralstonia strain, wherein the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaC1 is shown as SEQ ID NO:6, the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaA is shown as SEQ ID NO:7, the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaB1 is shown as SEQ ID NO: shown as 8;
The methyl malate synthase gene cimA is further overexpressed.
The method for constructing a rogowski bacterium engineering strain producing isoleucine through the methyl malic acid pathway according to the present invention, wherein the method further comprises the steps of:
Knocking out lactic acid synthesis genes ldhA1 and ldhA2, and expressing NADPH-dependent acetohydroxyacid isomerase reductase genes ilvC and NADPH coenzyme-dependent branched-chain aminotransferase genes ilvE, wherein the nucleotide sequence of the lactic acid synthesis gene ldhA1 is shown as SEQ ID NO:10, the nucleotide sequence of the lactic acid synthesis gene ldhA2 is shown as SEQ ID NO:11, the nucleotide sequence of the acetohydroxy acid isomerase ilvC gene is shown in SEQ ID NO:12, the nucleotide sequence of the NADPH-dependent branched-chain aminotransferase gene ilvE is shown in SEQ ID NO: 13;
knocking out lactic acid synthesis gene ldh and expressing acetohydroxy acid synthase genes ilvB and ilvN which lose feedback regulation sites, wherein the nucleotide sequence of the lactic acid synthesis gene ldh is shown in SEQ ID NO:9, the nucleotide sequence of the acetohydroxy acid synthase gene ilvB is shown in SEQ ID NO:14, the nucleotide sequence of the acetohydroxy acid synthase gene ilvN is shown in SEQ ID NO: 15.
An engineered strain of ralstonia for producing isoleucine by the methyl malic acid pathway according to the present invention, which is a wild-type strain of ralstonia having the following engineered characteristics:
has a nucleotide sequence shown as SEQ ID NO:4, a mutated N-acetylglucosamine transporter gene nagE;
the transcriptional regulator gene nagR of the endogenous N-acetylglucosamine transporter is knocked out, and the nucleotide sequence of the transcriptional regulator gene nagR is shown as SEQ ID NO:5 is shown in the figure;
Has exogenous methyl malic acid synthase gene cimA and branched-chain amino acid exoprotein genes ygaZ and ygaH, wherein the nucleotide sequence of the methyl malic acid synthase gene cimA is shown in SEQ ID NO:1, wherein the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaZ is shown in SEQ ID NO:2, the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaH is shown as SEQ ID NO: 3.
The engineering strain of ralstonia for producing isoleucine through the methyl malic acid pathway, according to the present invention, wherein the engineering strain of ralstonia further has the following engineering features:
The endogenous polyhydroxybutyrate synthesis genes phaC1, phaA and phaB1 are knocked out, wherein the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaC1 is shown as SEQ ID NO:6, the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaA is shown as SEQ ID NO:7, the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaB1 is shown as SEQ ID NO: shown as 8;
Has the methyl malate synthase gene cimA which is over-expressed.
The engineering strain of ralstonia for producing isoleucine through the methyl malic acid pathway, according to the present invention, wherein the engineering strain of ralstonia further has the following engineering features:
The endogenous lactic acid synthesis genes ldhA1 and ldhA2 are knocked out and express NADPH-dependent acetohydroxyacid isomerase gene ilvC and NADPH coenzyme-dependent branched-chain aminotransferase gene ilvE, wherein the nucleotide sequences of the lactic acid synthesis genes ldhA1 and ldhA2 are shown as SEQ ID NO:10, the nucleotide sequence of the acetohydroxy acid isomerase ilvC gene is shown in SEQ ID NO:12, the nucleotide sequence of the NADPH-dependent branched-chain aminotransferase gene ilvE is shown in SEQ ID NO: 13;
The endogenous lactic acid synthesis gene ldh is knocked out, and acetohydroxy acid synthase genes ilvB and ilvN losing feedback regulation sites are expressed, and the nucleotide sequence of the lactic acid synthesis gene ldh is shown in SEQ ID NO:9, the nucleotide sequence of the acetohydroxy acid synthase gene ilvB is shown in SEQ ID NO:14, the nucleotide sequence of the acetohydroxy acid synthase gene ilvN is shown in SEQ ID NO: 15;
ralstonia overexpressing the cimA gene.
The Ralstonia has three lactate dehydrogenase genes, ldh, ldhA1, and ldhA2, respectively, wherein ldhA1 and ldhA2 are adjacent on the Ralstonia genome, are separated by only 680 bp, and have identical DNA sequences.
A method for producing isoleucine by fermentation through the methyl malic acid pathway according to the present invention, the method comprising the steps of: the isoleucine is obtained by fermenting the engineering strain of the Ralstonia producing isoleucine through the methyl malic acid pathway in a culture medium.
The method for producing isoleucine by the methyl malic acid way fermentation according to the invention, wherein the formula of the culture medium is 15.14 g/L Na2HPO4·12H2O,3 g/L KH2PO4,5 g/L (NH4)2SO4,300 mg/L NaHCO3,80 mg/L MgSO4·7H2O,10 mg/L ferric citrate ,2 mg/L CaCl2,1.2 mg/L NiSO4·7H2O,1 mg/L CaSO4,1 mg/L ZnSO4·7H2O,0.6 mg/L CoCl2·6H2O,0.6 mg/L H3BO3,0.44 mg/L MnSO4·H2O,0.4 mg/L Na2MoO4·2H2O,0.4 mg/L CuSO4·5H2O.
The method for producing isoleucine by fermentation through the methyl malic acid pathway according to the present invention, wherein the heterotrophic fermentation is performed using glucose as the sole carbon source, the initial concentration of glucose is 30 g/L, and when the initial glucose consumption is completed, the glucose concentration is controlled to be lower than 15 g/L by feeding through a peristaltic pump, wherein the feeding is 500 g/L glucose.
The method for producing isoleucine by means of the methyl malic acid pathway fermentation according to the invention, wherein the autotrophic fermentation is carried out in a closed vessel using CO 2 as the sole carbon source, the fresh mixed gas being displaced every 24 hours in the closed vessel, wherein the components and proportions of the mixed gas are H 2：O2：CO2 =8: 1:1.
The invention applies the methyl malic acid approach to metabolic engineering reconstruction for the first time, and realizes the biosynthesis of isoleucine by taking glucose or CO 2 as the sole carbon source by utilizing the carbon metabolism characteristic of the engineering strain of the Ralstonia. Further, the production capacity of the engineering strain for synthesizing the isoleucine through a methyl malic acid way is obviously improved by utilizing methods of regulating coenzyme balance, relieving feedback inhibition, knocking out metabolic bypass, adopting feeding, tank feeding fermentation and the like, the conversion yield of the glucose heterotrophic fermentation isoleucine reaches 857 mg/L, and the conversion yield of the CO 2 autotrophic fermentation isoleucine reaches 105 mg/L.
Drawings
FIG. 1 shows the results of glucose shake flask fermentation of the engineering strains Cn102, cnIle1 and CnIle of Ralstonia provided in example 1 of the present invention to produce isoleucine;
FIG. 2 shows the results of glucose shake flask fermentation of the engineering strains CnIle, cnIle, and CnIle of Ralstonia provided in example 2 of the present invention to produce isoleucine;
FIG. 3 shows the results of glucose shake flask fermentation of the engineering strains CnIle and CnIle of Ralstonia provided in example 3 of the present invention to produce isoleucine;
FIG. 4 is a graph showing the results of glucose fermentation in the fermentation tanks of the engineering strains CnIle and CnIle of Ralstonia provided in example 4 of the present invention to produce isoleucine;
FIG. 5 is a graph showing the results of the autotrophic fermentation of CO 2 with the engineering strains CnIle and CnIle of Ralstonia provided in example 5 of the present invention to produce isoleucine.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used, unless otherwise specified, are all commercially available.
TABLE 1 list of engineering strains of Ralstonia constructed in examples of the invention
| Strain | Construction process |
| Ralstonia sp | Wild strain |
| Ralstonia-Cn 101 | NagE (G793C) in the genome of a point mutation derived from a wild strain |
| Ralstonia-Cn 102 | Knock-out strain derived from Cn101, nagR gene |
| Ralstonia-CnIle 1 | Gene cimA from Cn102, genomic integration expression |
| Ralstonia-CnIle 2 | Derived from CnIle1, the genome integration expresses ygaZ, ygaH gene |
| Ralstonia-CnIle 3 | Gene knockout strain derived from CnIle2, phaC1AB1 |
| Ralstonia-CnIle 4 | From CnIle, plasmid over-expression cimA gene |
| Ralstonia-CnIle < 3+ > 1 | The replacement genome ldhA1, ldhA2 genes derived from CnIle are ilvE, ilvC genes |
| Ralstonia-CnIle < 3+ > 2 | Derived from CnIle3+1, the replacement genome ldh gene is ilvB and ilvN gene |
| Ralstonia-CnIle 5 | From CnIle3+2, plasmid over-expression cimA gene |
As shown in Table 1 above, the specific examples of the present application increased the yield of isoleucine from Ralstonia by:
First, a methyl malate pathway is constructed in ralstonia by modifying an N-acetylglucosamine transport system in a genome, integrating a methyl malate synthase gene cimA and branched-chain amino acid exoprotein genes ygaZ, ygaH into the genome, and realizing a production process from glucose to isoleucine; secondly, through knocking out polyhydroxybutyrate synthesis genes phaC1, phaA and phaB1 in the genome and over-expressing a methyl malic acid synthase gene cimA by plasmids, the carbon metabolic flux of a methyl malic acid pathway is increased, and the production capacity of synthesizing isoleucine by a roman engineering strain through the methyl malic acid pathway is improved; thirdly, integrating NADPH dependent acetohydroxyacid isomerase reductase genes ilvC, branched-chain aminotransferase genes ilvE and acetohydroxyacid synthase genes ilvB and ilvN which lose feedback regulation sites into a genome by knocking out lactic acid synthesis genes ldh, ldhA1 and ldhA2, and further improving the production capacity of the engineering strain of the Ralstonia through the methyl malic acid pathway under the combined action of reducing byproducts, balancing coenzymes and relieving feedback regulation; fourth, under the fermentation conditions of improving the inoculation amount, adopting a feeding mode and controlling the pH and dissolved oxygen level, the production capacity of the engineering strain of the Ralstonia for synthesizing isoleucine through a methyl malic acid way is improved again by accelerating the growth and metabolic process of the strain; fifth, the engineering strain of ralstonia constructed according to the present invention still has the ability to synthesize isoleucine through the methyl malic acid pathway under autotrophic fermentation conditions using CO 2 as the sole carbon source.
EXAMPLE 1 construction of an engineering strain of Ralstonia producing isoleucine via the methyl malic acid pathway
1. Construction of Ralstonia-Cn 101 Strain
The pK18mobsacB plasmid with nagE point mutation site and its upstream and downstream homology arms was transformed into E.coli S17-1 intermediate host, transformed into the wild-type strain of Ralstonia by the conjugal transfer process, and single-and double-crossover strain selection was completed by antibiotic selection and sucrose induction sequentially to obtain Ralstonia-Cn 101 strain of nagE (G793C) in the point mutation genome (Table 1). The specific method comprises the following steps:
1. construction of pK18mobsacB plasmid with nagE Gene mutation site and upstream and downstream homology arms thereof
About 500 bp parts of nagE gene mutation positions at the upstream and downstream of each mutation position in the genome of the Ralstonia are selected, amplification primer pairs nagE-up-F (SEQ ID NO: 16)/nagE-up-R (SEQ ID NO: 17) and nagE-down-F (SEQ ID NO: 18)/nagE-down-R (SEQ ID NO: 19) are designed to amplify the genome of the Ralstonia, an upstream and downstream homology arm is obtained, and after each fragment is ligated by overlap PCR, the fragments are integrated into a linearized plasmid pK18mobsacB which is digested with restriction endonucleases EcoRI and SmaI by Gibson assembly. The recombinant system was transformed into E.coli Trans1-T1 competent cells, and screened using LB kanamycin resistance plates. Positive clones were picked and subjected to LB liquid culture. Plasmids were extracted and PCR verified using the M13-47 (SEQ ID NO: 20)/RV-M (SEQ ID NO: 21) primer pair with the fragment of interest 1136 bp. The successfully constructed plasmid was designated pK18-nagE (G793C).
2. Construction of Ralstonia-Cn 101 Strain
The pK18-nagE (G793C) plasmid was electrotransformed into E.coli S17-1 competence and positive transformants were screened by LB kanamycin resistance plates for LB liquid culture. The wild-type strain of Ralstonia was subjected to LB liquid culture. After enrichment of the cells, the cells were isolated according to Ralstonia: escherichia coli = 2:1, dripping the mixed bacterial cells into a non-resistance LB plate at 30 ℃ for culturing 24h for joint transfer, screening positive transformants by a kanamycin and gentamicin dual-resistance LB plate, performing LB liquid culture, diluting and coating the positive transformants on a LB plate containing 5% sucrose for induction, and performing PCR verification and sequencing on the transformants by using primers nagE-up-testF (SEQ ID NO: 22)/nagE-down-testR (SEQ ID NO: 23) to obtain a Ralstonia-Cn 101 strain.
2. Construction of Ralstonia-Cn 102 Strain
The pK18mobsacB plasmid with nagR gene upstream and downstream homology arms is transformed into an intermediate host of escherichia coli S17-1, and is transformed into the Ralstonia-Cn 101 strain through a conjugal transfer process, and single-exchange strain screening and double-exchange strain screening are completed by using antibiotic screening and sucrose induction successively, so that the Ralstonia-Cn 102 strain with nagR gene knocked-out (Table 1) is obtained, and the strain has the glucose metabolism capability. The specific method comprises the following steps:
1. construction of pK18mobsacB plasmid with nagR homology arms upstream and downstream of the Gene
About 500. 500 bp each was selected upstream and downstream of nagR gene in the genome of Ralstonia, and amplification primer pairs nagR-up-F (SEQ ID NO: 24)/nagR-up-R (SEQ ID NO: 25) and nagR-down-F (SEQ ID NO: 26)/nagR-down-R (SEQ ID NO: 27) were designed to amplify the genome of Ralstonia to obtain upstream and downstream homology arms, and after ligation of the fragments by overlap PCR, the fragments were integrated into the linearized plasmid pK18mobsacB digested with the restriction endonucleases EcoRI and SmaI by Gibson assembly. The recombinant system was transformed into E.coli Trans1-T1 competent cells, and screened using LB kanamycin resistance plates. Positive clones were picked and subjected to LB liquid culture. The plasmid was extracted and PCR verified using the M13-47/RV-M primer pair with the fragment of interest 1160 bp. The successfully constructed plasmid was designated pK 18-Delta nagR.
2. Construction of Ralstonia-Cn 102 Strain
The pK18- Δ nagR plasmid was electrotransformed into E.coli S17-1 competence and positive transformants were screened by LB kanamycin resistance plates for LB liquid culture. The Ralstonia-Cn 101 strain was subjected to LB liquid culture. After enrichment of the cells, the cells were isolated according to Ralstonia: escherichia coli = 2:1, dripping the mixed bacterial cells into a non-resistance LB plate at 30 ℃ for culturing 24 h for joint transfer, screening positive transformants by using a kanamycin and gentamicin dual-resistance LB plate, performing LB liquid culture, diluting and coating the positive transformants on a LB plate containing 5% of sucrose for induction, performing PCR verification on the transformants by using primers nagR-uup-F (SEQ ID NO: 28)/nagR-ddown-R (SEQ ID NO: 29), and obtaining a PCR product size of 1283 bp of a successful knockout nagR gene.
3. Construction of Ralstonia-CnIle Strain
The pK18mobsacB plasmid with P phaC1 promoter, cimA gene and upstream and downstream homology arms is transformed into an intermediate host of escherichia coli S17-1, and transformed into a Ralstonia-Cn 102 strain through a conjugal transfer process, and single exchange strain screening and double exchange strain screening are completed by using antibiotic screening and sucrose induction successively, so that the Ralstonia-CnIle 1 strain (Table 1) with genome integration expression cimA gene is obtained, and the strain has the capacity of metabolizing glucose through a methyl malic acid pathway to synthesize isoleucine. The specific method comprises the following steps:
1. Construction of pK18mobsacB plasmid with P phaC1 promoter, cimA Gene and upstream and downstream homology arms
About 500. 500 bp of each of the upstream and downstream genes of phaR in the genome of Ralstonia was selected, and amplification primer pairs phaR-up-F (SEQ ID NO: 30)/phaR-up-R (SEQ ID NO: 31) and phaR-down-F (SEQ ID NO: 32)/phaR-down-R (SEQ ID NO:) were designed to amplify the genome of Ralstonia to obtain upstream and downstream homology arms; designing a primer pair PphaC-phaR-F (SEQ ID NO: 34)/PphaC 1-phaR-R (SEQ ID NO: 35) to amplify the genome of the Ralstonia to obtain a P phaC1 promoter fragment; designing a primer pair cimA-phaR-F (SEQ ID NO: 36)/cimA-phaR-R (SEQ ID NO: 37) to amplify cimA gene (artificially synthesized codon-optimized cimA gene, the nucleotide sequence of which is shown in SEQ ID NO: 1) to obtain cimA gene fragment; after ligation of the fragments by overlap PCR, the fragments were integrated into the restriction endonuclease EcoRI, smaI double digested linearized plasmid pK18mobsacB by Gibson assembly. The recombinant system was transformed into E.coli Trans1-T1 competent cells, and screened using LB kanamycin resistance plates. Positive clones were picked and subjected to LB liquid culture. Plasmids were extracted and PCR verified using the M13-47/RV-M primer pair with a fragment of interest 2706 bp. The successfully constructed plasmid was designated pK18-cimA.
2. Construction of Ralstonia-CnIle Strain
The pK18-cimA plasmid was electrotransformed into E.coli S17-1 competence and positive transformants were screened by LB kanamycin resistance plates for LB liquid culture. The Ralstonia-Cn 102 strain was subjected to LB liquid culture. After enrichment of the cells, the cells were isolated according to Ralstonia: escherichia coli = 2:1, dripping the mixed bacterial cells into a non-resistance LB plate at 30 ℃ for culturing 24 h for joint transfer, screening positive transformants by a kanamycin and gentamicin dual-resistance LB plate, performing LB liquid culture, diluting and coating the positive transformants on a LB plate containing 5% of sucrose for induction, and performing PCR verification on the transformants by using primers phaR-uup-F (SEQ ID NO: 38)/phaR-ddown-R (SEQ ID NO: 39), wherein the size of a PCR product of a successfully integrated cimA gene is 2751 bp, thereby obtaining a Ralstonia-CnIle 1 strain.
4. Construction of Ralstonia-CnIle Strain
The pK18mobsacB plasmid with P phaC1 promoter, ygaZ and ygaH genes and upstream and downstream homology arms is transformed into an intermediate host of escherichia coli S17-1, and transformed into a Ralstonia-CnIle strain through a conjugal transfer process, and single-crossover strain screening and double-crossover strain screening are completed by utilizing antibiotic screening and sucrose induction successively, so that a Ralstonia-CnIle strain (Table 1) with genome integration expression ygaZ and ygaH genes is obtained, and the strain has the capability of synthesizing and secreting isoleucine through a methyl malic acid pathway. The specific method comprises the following steps:
1. construction of pK18mobsacB plasmid with P phaC1 promoter, ygaZ, ygaH Gene and upstream and downstream homology arms
About 500 bp each of the upstream and downstream of the H16-A2005 gene in the genome of Ralstonia was selected, and amplification primer pairs A2005-up-F (SEQ ID NO: 40)/A2005-up-R (SEQ ID NO: 41) and A2005-down-F (SEQ ID NO: 42)/A2005-down-R (SEQ ID NO: 43) were designed to amplify the genome of Ralstonia to obtain upstream and downstream homology arms; designing a primer pair PphaC-A2005-F (SEQ ID NO: 44)/PphaC-A2005-R (SEQ ID NO: 45) to amplify the genome of the Ralstonia to obtain a P phaC1 promoter fragment; designing a primer pair ygaZH-F (SEQ ID NO: 46)/ygaZH-R (SEQ ID NO: 47) to amplify ygaZ and ygaH genes (artificially synthesized ygaZ and ygaH genes with optimized codons and encoding branched-chain amino acid exoproteins, wherein the amino acid sequence is derived from escherichia coli, and the nucleotide sequences are shown in SEQ ID NO:2 and SEQ ID NO: 3) to obtain ygaZH gene fragments; after ligation of the fragments by overlap PCR, the fragments were integrated into the restriction endonuclease EcoRI, smaI double digested linearized plasmid pK18mobsacB by Gibson assembly. The recombinant system was transformed into E.coli Trans1-T1 competent cells, and screened using LB kanamycin resistance plates. Positive clones were picked and subjected to LB liquid culture. Plasmids were extracted and PCR verified using the M13-47/RV-M primer pair, the fragment of interest was 2639 bp. The successfully constructed plasmid was designated pK18-ygaZH.
2. Construction of Ralstonia-CnIle Strain
The pK18-ygaZH plasmid was electrotransformed into E.coli S17-1 competence and positive transformants were screened by LB kanamycin resistance plates for LB liquid culture. The strain Ralstonia-CnIle was subjected to LB liquid culture. After enrichment of the cells, the cells were isolated according to Ralstonia: escherichia coli = 2:1, dripping the mixed bacterial cells into a non-resistance LB plate at 30 ℃ for culturing 24 h for joint transfer, screening positive transformants by a kanamycin and gentamicin dual-resistance LB plate, performing LB liquid culture, diluting and coating the positive transformants on a LB plate containing 5% of sucrose for induction, and performing PCR verification on the transformants by using primers A2005-uup-F (SEQ ID NO: 48)/A2005-ddown-R (SEQ ID NO: 49), wherein the size of a PCR product of successfully integrating cimA genes is 2782 bp, thus obtaining the Ralstonia-CnIle strain.
5. Shake flask fermentation experiment of engineering strains Cn102, cnIle1 and CnIle2
The Ralstonia-Cn 102, cnIle1 and CnIle were inoculated into 40 mL LB medium, respectively, and cultured overnight at 30℃at 200 r/min as first seed solution. Primary seed liquid OD 600 was measured and inoculated in a corresponding proportion into 50mL medium (100 mL Erlenmeyer flask) with 30 g/L glucose added thereto, the initial OD 600 being about 0.05, wherein the medium was formulated as ：15.14 g/L Na2HPO4·12H2O,3 g/L KH2PO4,5 g/L (NH4)2SO4,300 mg/L NaHCO3,80 mg/L MgSO4·7H2O,10 mg/L ferric citrate ,2 mg/L CaCl2,1.2 mg/L NiSO4·7H2O,1 mg/L CaSO4,1 mg/L ZnSO4·7H2O,0.6 mg/L CoCl2·6H2O,0.6 mg/L H3BO3,0.44 mg/L MnSO4·H2O,0.4 mg/L Na2MoO4·2H2O,0.4 mg/L CuSO4·5H2O. fermentation at 30℃and a total of 120 h in a 200 r/min shaker. Samples were taken every 24 h a, and the concentration of isoleucine in the medium was isolated and measured by a high performance liquid chromatography uv detector using the shimeji's AJS-01 amino acid analysis method. As shown in FIG. 1, the maximum yield of isoleucine in the fermentation broth of Ralstonia-CnIle 2 was 56 mg/L. The results show that the production of isoleucine by the methyl malic acid pathway using glucose as a carbon source by the Ralstonia was successfully achieved by expressing the methyl malate synthase gene cimA and the branched-chain amino acid exoprotein genes ygaZ, ygaH, and engineering the N-acetylglucosamine transport system in the genome of Ralstonia.
Example 2 enhancement of isoleucine-producing ability of Ralstonia engineering Strain by knocking out polyhydroxybutyrate Synthesis Gene and increasing expression intensity of methyl malate synthase Gene
1. Construction of Ralstonia-CnIle Strain
The pK18mobsacB plasmid with homologous arms at the upstream and downstream of the phaC1, phaA and phaB1 genes is transformed into an intermediate host of escherichia coli S17-1, and is transformed into a Ralstonia-CnIle strain through a conjugal transfer process, and single-exchange strain screening and double-exchange strain screening are completed by utilizing antibiotic screening and sucrose induction successively, so that the Ralstonia-CnIle strain with the gene knocked-out phaC1, phaA and phaB1 genes is obtained (Table 1), and the production capacity of isoleucine is increased. The specific method comprises the following steps:
1. construction of pK18mobsacB plasmid with homology arms of upstream and downstream of phaC1, phaA and phaB1 genes
About 500 bp of each of the upper and lower streams of phaC1, phaA and phaB1 genes in the genome of the Ralstonia are selected, and an amplification primer pair PHB-up-F (SEQ ID NO: 50)/PHB-up-R (SEQ ID NO: 51) and PHB-down-F (SEQ ID NO: 52)/PHB-down-R (SEQ ID NO: 53) is designed to amplify the genome of the Ralstonia to obtain upstream and downstream homology arms, and after each fragment is ligated by overlap PCR, the fragments are integrated into a linearized plasmid pK18mobsacB cut by the restriction endonucleases EcoRI and SmaI by Gibson assembly. The recombinant system was transformed into E.coli Trans1-T1 competent cells, and screened using LB kanamycin resistance plates. Positive clones were picked and subjected to LB liquid culture. The plasmid was extracted and PCR verified using the M13-47/RV-M primer pair with a target fragment of 1001 bp. The successfully constructed plasmid was designated pK18- ΔphaC1AB1.
2. Construction of Ralstonia-CnIle Strain
The pK18- ΔphaC1AB1 plasmid was electrotransformed into E.coli S17-1 competence and positive transformants were screened by LB kanamycin resistance plates for LB liquid culture. The Ralstonia-CnIle strain was subjected to LB liquid culture. After enrichment of the cells, the cells were isolated according to Ralstonia: escherichia coli = 2:1, dripping the mixed bacterial cells into a non-resistance LB plate at 30 ℃ for culturing 24 h for joint transfer, screening positive transformants by a kanamycin and gentamicin dual-resistance LB plate, carrying out LB liquid culture, diluting and coating the positive transformants on a LB plate containing 5% of sucrose for induction, carrying out PCR verification on the transformants by using primers PHB-uup-F (SEQ ID NO: 54)/PHB-ddown-R (SEQ ID NO: 55), and successfully knocking out the PCR products of phaC1, phaA and phaB1 genes, wherein the sizes of the PCR products are 1325 bp, thus obtaining the Ralstonia-CnIle strain.
2. Construction of Ralstonia-CnIle strain 4
The pBBR1-MCS2 plasmid with P phaC1 promoter and cimA gene is electrotransformed into Ralstonia-CnIle strain, and the transformation screening is completed by using antibiotics, so as to obtain Ralstonia-CnIle strain (Table 1) with plasmid over-expression cimA gene, and increase the productivity of isoleucine. The specific method comprises the following steps:
1. construction of pBBR1-MCS2 plasmid with P phaC1 promoter and cimA Gene
Designing a primer pair PphaC-cimA-F (SEQ ID NO: 56)/PphaC 1-cimA-R (SEQ ID NO: 57) to amplify the genome of the Ralstonia to obtain a P phaC1 promoter fragment; designing a primer pair cimA-F (SEQ ID NO: 58)/cimA-R (SEQ ID NO: 59) to amplify a cimA gene (an artificially synthesized cimA gene with a nucleotide sequence detailed in SEQ ID NO: 1) to obtain a cimA gene fragment; after ligation of the fragments by overlap PCR, the fragments were integrated into the restriction endonuclease EcoRI, ndeI double digested linearized plasmid pBBR1-MCS2 by Gibson assembly. The recombinant system was transformed into E.coli Trans1-T1 competent cells, and screened using LB kanamycin resistance plates. Positive clones were picked and subjected to LB liquid culture. The plasmid was extracted and PCR verified using the M13F (SEQ ID NO: 60)/M13R (SEQ ID NO: 61) primer pair, the fragment of interest was 1591 bp. The successfully constructed plasmid was designated pBBR1-cimA.
2. Construction of Ralstonia-CnIle strain 4
The Ralstonia-CnIle strain was subjected to LB liquid culture, centrifuged at 4℃and concentrated cells were resuspended 3 times with 10% glycerol, and finally the cells were resuspended to OD 600 of about 60-80 with 10% glycerol to prepare a competent Ralstonia. 600 ng of pBBR1-cimA plasmid was electrotransformed into 100. Mu.L of Ralstonia competent cells using a 2 mm electric beaker, 1 mL non-resistant LB liquid was added, and after incubation at 30℃of 200R/min for 2h, positive transformants were screened by LB kanamycin resistant plates, PCR verification was performed on the transformants using M13F/M13R primers, and the size of the PCR product of the successful plasmid over-expression cimA gene was 1591 bp, obtaining Ralstonia-CnIle 4 strain.
3. Shake flask fermentation experiments with Ralstonia-CnIle 2, ralstonia-CnIle 3, and Ralstonia-CnIle 4
The above-mentioned Ralstonia-CnIle, ralstonia-CnIle and Ralstonia-CnIle were inoculated into 40 mL LB (the medium of CnIle4 contains kanamycin) medium, respectively, and cultured overnight at 30℃at 200 r/min as a first seed solution. Primary seed liquid OD 600 was determined and inoculated in a corresponding proportion into 50: 50 mL medium (100: 100 mL Erlenmeyer flask) with 30: 30 g/L glucose to give an initial OD 600 of about 0.05, wherein the medium had a formulation of ：15.14 g/L Na2HPO4·12H2O,3 g/L KH2PO4,5 g/L (NH4)2SO4,300 mg/L NaHCO3,80 mg/L MgSO4·7H2O,10 mg/L ferric citrate ,2 mg/L CaCl2,1.2 mg/L NiSO4·7H2O,1 mg/L CaSO4,1 mg/L ZnSO4·7H2O,0.6 mg/L CoCl2·6H2O,0.6 mg/L H3BO3,0.44 mg/L MnSO4·H2O,0.4 mg/L Na2MoO4·2H2O,0.4 mg/L CuSO4·5H2O（CnIle4 containing kanamycin. Fermentation was performed in a shaker at 30℃and 200 r/min for 120: 120 h. Samples were taken every 24h a, and the concentration of isoleucine in the medium was isolated and measured by a high performance liquid chromatography uv detector using the shimeji's AJS-01 amino acid analysis method. As shown in FIG. 2, after the phaC1, phaA and phaB1 genes of the Ralstonia-CnIle 2 are knocked out, the highest yield of isoleucine in the fermentation liquor of the Ralstonia-CnIle 3 is obviously improved from 56 mg/L to 114 mg/L; after the cimA gene is over-expressed on the plasmid in the Ralstonia-CnIle, the highest yield of isoleucine in the fermentation liquor of the Ralstonia-CnIle is further obviously improved from 114 mg/L to 248 mg/L. The results show that the isoleucine production capacity of the engineering strain of the Ralstonia is successfully improved by knocking out polyhydroxybutyrate synthesis genes phaC1, phaA and phaB1 and over-expressing methyl malic acid synthase gene cimA.
Example 3 further improvement of isoleucine-producing ability of engineering strain of Ralstonia by knocking out lactic acid Synthesis Gene and expressing NADPH-dependent acetohydroxyacid isomerase Gene, branched-chain aminotransferase Gene, acetohydroxyacid synthase Gene lacking feedback regulatory site
1. Construction of Ralstonia-CnIle 3+1 Strain
The pK18mobsacB plasmid with P phaC1 promoter, ilvC, ilvE gene and upstream and downstream homology arms of ldhA1 and ldhA2 genes is transformed into a middle host of escherichia coli S17-1, and transformed into a strain of Ralstonia-CnIle through a conjugal transfer process, and single-exchange strain screening and double-exchange strain screening are completed by using antibiotic screening and sucrose induction successively, so that the strain of Ralstonia-CnIle 3+1 (Table 1) with replacement genome ldhA1 and ldhA2 genes of ilvC and ilvE genes is obtained, and the production capacity of isoleucine is increased. The specific method comprises the following steps:
1. Construction of a pK18mobsacB plasmid with P phaC1 promoter, ilvC, ilvE Gene and homology arms upstream and downstream of the ldhA1 and ldhA2 Gene
About 600 bp was selected upstream and downstream of the ldhA1 and ldhA2 genes in the genome of Ralstonia, and the amplification primer pairs ldhA1A2-up-F (SEQ ID NO: 62)/ldhA 1A2-up-R (SEQ ID NO: 63) and ldhA1A2-down-F (SEQ ID NO: 64)/ldhA 1A2-down-R (SEQ ID NO: 65) were designed to amplify the genome of Ralstonia to obtain upstream and downstream homology arms; designing a primer pair PphaC-ilvCE-F (SEQ ID NO: 66)/PphaC 1-ilvCE-R (SEQ ID NO: 67) to amplify the genome of the Ralstonia to obtain a P phaC1 promoter fragment; designing a primer pair ilvC-F (SEQ ID NO: 68)/ilvC-R (SEQ ID NO: 69) to amplify an ilvC gene (encoding an acetohydroxy acid isomerase reductase with NADPH coenzyme dependency, the nucleotide sequence of which is shown in SEQ ID NO: 12) in the genome of escherichia coli-MG 1655 to obtain an ilvC gene fragment; designing a primer pair ilvE-F (SEQ ID NO: 70)/ilvE-R (SEQ ID NO: 71) to amplify ilvE gene (encoding branched-chain aminotransferase with NADPH coenzyme dependence, the nucleotide sequence of which is shown in SEQ ID NO: 13) in the genome of escherichia coli-MG 1655 to obtain ilvE gene fragment; after ligation of the fragments by overlap PCR, the fragments were integrated into the restriction endonuclease EcoRI, smaI double digested linearized plasmid pK18mobsacB by Gibson assembly. The recombinant system was transformed into E.coli Trans1-T1 competent cells, and screened using LB kanamycin resistance plates. Positive clones were picked and subjected to LB liquid culture. Plasmids were extracted and PCR verified using the M13-47/RV-M primer pair with a target fragment of 4063 bp. The successfully constructed plasmid was designated pK18-ldhA12-ilvCE.
2. Construction of Ralstonia-CnIle 3+1 Strain
The pK18-ldhA12-ilvCE plasmid was electrotransformed into E.coli S17-1 competence and positive transformants were screened by LB kanamycin resistance plates for LB liquid culture. The strain Ralstonia-CnIle was subjected to LB liquid culture. After enrichment of the cells, the cells were isolated according to Ralstonia: escherichia coli = 2:1, dropwise culturing 24 h in a non-resistant LB plate at 30 ℃ for joint transfer, screening positive transformants by a kanamycin and gentamicin dual-resistant LB plate, performing LB liquid culture, diluting and coating the positive transformants on a LB plate containing 5% of sucrose for induction, and performing PCR verification on the transformants by using primers ldhA1A2-uup-F (SEQ ID NO: 72)/ldhA 1A2-ddown-R (SEQ ID NO: 73), wherein lactic acid synthesis genes ldhA1 and ldhA2 are successfully knocked out, and the PCR products expressing NADPH-dependent acetohydroxyacid isomerase gene ilvC and branched-chain aminotransferase gene ilvE have the size of 4063 bp, thereby obtaining the Ralstonia-CnIle 3+1 strain.
2. Construction of Ralstonia-CnIle 3+2 Strain
The pK18mobsacB plasmid with P phaC1 promoter, ilvB, ilvN gene and the homology arm of the upper and lower stream of the ldh gene is transformed into the intermediate host of the escherichia coli S17-1, and is transformed into the Ralstonia-CnIle 3+1 strain through the joint transfer process, and the single-exchange strain screening and the double-exchange strain screening are completed by utilizing the antibiotic screening and the sucrose induction successively, so that the Ralstonia-CnIle 3+2 strain with the ilvB and ilvN genes as the replacement genome is obtained (Table 1), and the production capacity of isoleucine is increased. The specific method comprises the following steps:
1. construction of pK18mobsacB plasmid with P phaC1 promoter, upstream and downstream homology arms of ilvB, ilvN and ldh Gene
About 600 bp of each of the upstream and downstream of the ldh gene in the genome of Ralstonia, and designing amplification primer pairs ldh-up-F (SEQ ID NO: 74)/ldh-up-R (SEQ ID NO: 75) and ldh-down-F (SEQ ID NO: 76)/ldh-down-R (SEQ ID NO: 77) to amplify the genome of Ralstonia and obtain upstream and downstream homology arms; designing a primer pair PphaC-ilvBN-F (SEQ ID NO: 78)/PphaC-ilvBN-R (SEQ ID NO: 79) to amplify through the genome of Ralstonia to obtain a P phaC1 promoter fragment; designing a primer pair ilvBN-F (SEQ ID NO: 80)/ilvBN-R (SEQ ID NO: 81) to amplify the synthesized ilvB and ilvN genes (encoding acetohydroxy acid synthase genes, corynebacterium glutamicum sources, mutated and lost feedback regulation sites, and the nucleotide sequences of which are shown in SEQ ID NO:14 and SEQ ID NO: 15) to obtain ilvBN gene fragments; after ligation of the fragments by overlap PCR, the fragments were integrated into the restriction endonuclease EcoRI, smaI double digested linearized plasmid pK18mobsacB by Gibson assembly. The recombinant system was transformed into E.coli Trans1-T1 competent cells, and screened using LB kanamycin resistance plates. Positive clones were picked and subjected to LB liquid culture. Plasmids were extracted and PCR verified using the M13-47/RV-M primer pair with a target fragment of 4123 bp. The successfully constructed plasmid was designated pK18-ldh-ilvBN.
2. Construction of Ralstonia-CnIle 3+2 Strain
The pK18-ldh-ilvBN plasmid was electrotransformed into E.coli S17-1 competence and positive transformants were screened by LB kanamycin resistance plates for LB liquid culture. The Ralstonia-CnIle 3+1 strain was subjected to LB liquid culture. After enrichment of the cells, the cells were isolated according to Ralstonia: escherichia coli = 2:1, dripping the mixed bacterial cells into a non-resistance LB plate at 30 ℃ for culturing 24h for joint transfer, screening positive transformants by using a kanamycin and gentamicin dual-resistance LB plate, performing LB liquid culture, diluting and coating the positive transformants on a LB plate containing 5% of sucrose for induction, performing PCR verification on the transformants by using primers ldh-uup-F (SEQ ID NO: 82)/ldh 2-ddown-R (SEQ ID NO: 83), and successfully knocking out a lactic acid synthesis gene ldh and expressing acetohydroxyacid synthase genes ilvB and ilvN which lose feedback regulation sites, wherein the size of a PCR product is 4140 bp, thus obtaining the Ralstonia-CnIle 3+2 strain.
3. Construction of Ralstonia-CnIle Strain
The pBBR1-cimA plasmid was electrotransformed into the Ralstonia-CnIle 3+2 strain, and transformation screening was performed using antibiotics to obtain the Ralstonia-CnIle strain (Table 1) with a plasmid overexpressing cimA gene, further increasing the isoleucine production capacity. The specific method comprises the following steps:
The Ralstonia-CnIle 3+2 strain was subjected to LB liquid culture, centrifuged at 4℃and concentrated cells were resuspended 3 times with 10% glycerol, and finally the cells were resuspended to OD 600 of about 60-80 with 10% glycerol to prepare a competent Ralstonia. The plasmid pBBR1-cimA constructed in example 2 was electrotransformed into 100. Mu.L of competent cells of Ralstonia using a 2 mm electroflask, 1 mL-free LB liquid was added, and after incubation for 2 h at 30℃of 200R/min, positive transformants were screened by LB kanamycin resistance plates, PCR was performed on the transformants using M13F/M13R primers, and the size of PCR product of successful plasmid overexpression cimA gene was 1591 bp, to obtain Ralstonia-CnIle 5 strain.
4. Shake flask fermentation experiment of engineering strain CnIle4 and CnIle5
The Ralstonia-CnIle and CnIle5 were inoculated into 40 mL LB (containing kanamycin) medium, respectively, and cultured overnight at 30℃at 200 r/min as first seed solution. Primary seed liquid OD 600 was determined and inoculated in a corresponding proportion into 50: 50 mL medium (100: 100 mL Erlenmeyer flask) supplemented with 30: 30 g/L glucose to give an initial OD 600 of about 0.05, wherein the medium had a formulation of ：15.14 g/L Na2HPO4·12H2O,3 g/L KH2PO4,5 g/L (NH4)2SO4,300 mg/L NaHCO3,80 mg/L MgSO4·7H2O,10 mg/L ferric citrate ,2 mg/L CaCl2,1.2 mg/L NiSO4·7H2O,1 mg/L CaSO4,1 mg/L ZnSO4·7H2O,0.6 mg/L CoCl2·6H2O,0.6 mg/L H3BO3,0.44 mg/L MnSO4·H2O,0.4 mg/L Na2MoO4·2H2O,0.4 mg/L CuSO4·5H2O,200 mg/L kanamycin. Fermentation was performed in a shaker at 30℃and 200 r/min for 120: 120 h. Samples were taken every 24 h a, and the concentration of isoleucine in the medium was isolated and measured by a high performance liquid chromatography uv detector using the shimeji's AJS-01 amino acid analysis method. As shown in FIG. 3, the maximum yield of isoleucine in the fermentation broth of Ralstonia-CnIle 5 was significantly increased from 248 mg/L to 483 mg/L after the lactic acid synthesis genes ldh, ldhA1, and ldhA2 of Ralstonia-CnIle 4 were knocked out and the NADPH-dependent acetohydroxy acid isomerase gene ilvC, the branched-chain aminotransferase gene ilvE, and the acetohydroxy acid synthase genes ilvB and ilvN genes at the feedback regulation site were deleted. The results show that the isoleucine-producing ability of the engineering strain of Ralstonia can be further remarkably improved by knocking out the lactic acid synthesis genes ldh, ldhA1, ldhA2 and expressing the NADPH-dependent acetohydroxyacid isomerase gene ilvC, the branched-chain aminotransferase gene ilvE, and the acetohydroxyacid synthase genes ilvB, ilvN which have lost the feedback regulation site.
EXAMPLE 4 production of isoleucine by heterotrophic glucose fermentation by the Methylmalic acid pathway in a fermenter by an engineering strain of Ralstonia
And (3) carrying out heterotrophic fermentation on the constructed Ralstonia-CnIle and Ralstonia-CnIle which are used for producing isoleucine through a methyl malic acid pathway respectively in a 3L fermentation tank, and improving the production capacity of the Ralstonia engineering strain for synthesizing isoleucine through the methyl malic acid pathway by adopting feeding and tank feeding fermentation. The specific method comprises the following steps:
First, the engineering strains of Ralstonia-CnIle and Ralstonia-CnIle were inoculated into 40 mL LB (kanamycin-containing) medium, respectively, and cultured overnight at 30℃at 200 r/min as first seed solution. All the first seed liquid was inoculated into a medium of 4L LB (containing kanamycin) and cultured at 37℃under 200 r/min for 24 h as a second seed liquid. The secondary seed liquid OD 600 was measured, and the cells were concentrated according to the corresponding ratio and inoculated into a medium (3L fermenter) containing 2: 2L and 30: 30g/L glucose to give an initial OD 600 of about 5. Wherein the formula of the culture medium is ：15.14 g/L Na2HPO4·12H2O,3 g/L KH2PO4,5 g/L (NH4)2SO4,300 mg/L NaHCO3,80 mg/L MgSO4·7H2O,10 mg/L ferric citrate ,2 mg/L CaCl2,1.2 mg/L NiSO4·7H2O,1 mg/L CaSO4,1 mg/L ZnSO4·7H2O,0.6 mg/L CoCl2·6H2O,0.6 mg/L H3BO3,0.44 mg/L MnSO4·H2O,0.4 mg/L Na2MoO4·2H2O,0.4 mg/L CuSO4·5H2O,200 mg/L kanamycin. The temperature in the fermentation process is maintained at 30 ℃, the pH is maintained at 6.7 by automatically supplementing ammonia water, and the motor rotation speed is coupled to control the dissolved oxygen level at 10% -30%. When the initial glucose consumption was completed, the glucose concentration was controlled to be lower than 15 g/L by feeding with a peristaltic pump, wherein the feeding was 500 g/L glucose. Samples were taken every 12 h, the fermentation was performed for 84 h, and the concentration of isoleucine in the medium was separated and measured by an ultraviolet detector of high performance liquid chromatography using the Shimadzu AJS-01 amino acid analysis method package. As shown in FIG. 4, in the fermenter in which the inoculum size is increased, the feeding method is used, and the pH and dissolved oxygen level are controlled, the time at which the cumulative isoleucine concentration in the medium is highest is significantly shortened from 120 h in shake flask fermentation to 36 h, the isoleucine yield of Ralstonia-CnIle 4 by the methyl malic acid pathway is significantly increased from 248 mg/L in shake flask fermentation to 627 mg/L, and the isoleucine yield of Ralstonia-CnIle 5 by the methyl malic acid pathway is significantly increased from 483 mg/L in shake flask fermentation to 857 mg/L. The results show that under the fermentation conditions that the inoculation amount is increased in the fermentation tank, the feeding mode is adopted, and the pH and dissolved oxygen level are controlled, the engineering strain of the Ralstonia can heterotrophically ferment glucose through a methyl malic acid way to produce the isoleucine, and the yield of the isoleucine is obviously improved compared with the production efficiency by shaking fermentation.
EXAMPLE 5 production of isoleucine by autotrophic fermentation of CO 2 by the Ralstonia engineering Strain in an anaerobic bottle via the methyl malic acid pathway
According to the nutritional and metabolic characteristics of facultative energy autotrophy, ralstonia can utilize CO 2 as the sole carbon source for growth and metabolism. The constructed Ralstonia-CnIle and Ralstonia-CnIle which produce isoleucine through the methyl malic acid pathway are subjected to autotrophic fermentation in an anaerobic bottle respectively, and the production of isoleucine through the methyl malic acid pathway fermentation CO 2 can be realized in the Ralstonia engineering strain. The specific method comprises the following steps:
First, the engineering strains of Ralstonia-CnIle and Ralstonia-CnIle were inoculated into 40 mL LB (kanamycin-containing) medium, respectively, and cultured overnight at 30℃at 200 r/min as first seed solution. Primary seed liquid OD 600 was measured and inoculated into 50 mL medium (250 mL sealed anaerobic jar) at a corresponding ratio to an initial OD 600 of about 1. Wherein the formula of the culture medium is ：15.14 g/L Na2HPO4·12H2O,3 g/L KH2PO4,5 g/L (NH4)2SO4,300 mg/L NaHCO3,80 mg/L MgSO4·7H2O,10 mg/L ferric citrate ,2 mg/L CaCl2,1.2 mg/L NiSO4·7H2O,1 mg/L CaSO4,1 mg/L ZnSO4·7H2O,0.6 mg/L CoCl2·6H2O,0.6 mg/L H3BO3,0.44 mg/L MnSO4·H2O,0.4 mg/L Na2MoO4·2H2O,0.4 mg/L CuSO4·5H2O,200 mg/L kanamycin. The fermentation is carried out for 35 days in a shaking table with the temperature of 30 ℃ and the speed of 200 r/min, and during the fermentation process, 1 standard atmospheric pressure mixed gas is filled into a sealed anaerobic bottle every day to replace the original gas in the bottle, wherein the components and the proportion of the mixed gas are that H 2：O2：CO2 =8: 1:1. samples were taken every 5 days, and the concentration of isoleucine in the medium was isolated and measured by an ultraviolet detector of high performance liquid chromatography using the Shimadzu AJS-01 amino acid analysis method package. As shown in FIG. 5, in the autotrophic fermentation using CO 2 as the sole carbon source, the time at which the cumulative isoleucine concentration in the medium is highest is 25-30 days, the yield of isoleucine produced by CO 2 conversion of Ralstonia-CnIle 4 by the methyl malate pathway is 105 mg/L, and the yield of isoleucine produced by CO 2 conversion of Ralstonia-CnIle 5 by the methyl malate pathway is 100 mg/L. The results show that under fermentation conditions with CO 2 as the sole carbon source, the engineering strain of Ralstonia can autotrophically ferment CO 2 and produce isoleucine via the methyl malic acid pathway.
The above embodiments are only for understanding the technical solution of the present application, and do not limit the protection scope of the present application.
Claims (10)
1. A method of constructing an engineered strain of ralstonia that produces isoleucine via the methyl malate pathway, comprising the steps of:
An engineering an endogenous N-acetylglucosamine transport system of a wild-type strain of ralstonia comprising the steps of: the 793 rd base of N-acetylglucosamine transporter gene nagE based on a phosphotransferase system of a wild-type Ralstonia strain is mutated from G to C, and the nucleotide sequence of the mutated N-acetylglucosamine transporter gene nagE is shown as SEQ ID NO:4, and knocking out a transcriptional regulator gene nagR of the N-acetylglucosamine transporter, wherein the nucleotide sequence of the transcriptional regulator gene nagR is shown as SEQ ID NO:5 is shown in the figure;
Expressing a methyl malate synthase gene cimA and branched-chain amino acid exoprotein genes ygaZ and ygaH in a wild-type ralstonia strain, wherein the nucleotide sequence of the methyl malate synthase gene cimA is shown in SEQ ID NO:1, wherein the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaZ is shown in SEQ ID NO:2, the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaH is shown as SEQ ID NO: 3.
2. The method of constructing an engineered strain of ralstonia that produces isoleucine via the methyl malate pathway of claim 1, further comprising the steps of:
Knocking out endogenous polyhydroxybutyrate synthesis genes phaC1, phaA and phaB1 of wild type Ralstonia strains, wherein the nucleotide sequence of the polyhydroxybutyrate synthesis genes phaC1 is shown as SEQ ID NO:6, the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaA is shown as SEQ ID NO:7, the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaB1 is shown as SEQ ID NO: shown as 8;
The methyl malate synthase gene cimA is overexpressed in the wild-type strain of ralstonia.
3. The method of constructing an engineered strain of ralstonia that produces isoleucine via the methyl malate pathway of claim 2, further comprising the steps of:
Knocking out lactic acid synthesis genes ldhA1 and ldhA2 of a wild-type Ralstonia strain, and expressing an NADPH-dependent acetohydroxyacid isomerase gene ilvC and an NADPH-coenzyme-dependent branched-chain aminotransferase gene ilvE, wherein the nucleotide sequence of the lactic acid synthesis gene ldhA1 is shown as SEQ ID NO:10, the nucleotide sequence of the lactic acid synthesis gene ldhA2 is shown as SEQ ID NO:11, the nucleotide sequence of the acetohydroxy acid isomerase ilvC gene is shown in SEQ ID NO:12, the nucleotide sequence of the NADPH-dependent branched-chain aminotransferase gene ilvE is shown in SEQ ID NO: 13;
Knocking out a lactic acid synthesis gene ldh of a wild type Ralstonia strain, and expressing acetohydroxy acid synthase genes ilvB and ilvN which lose feedback regulation sites, wherein the nucleotide sequence of the lactic acid synthesis gene ldh is shown as SEQ ID NO:9, the nucleotide sequence of the acetohydroxy acid synthase gene ilvB is shown in SEQ ID NO:14, the nucleotide sequence of the acetohydroxy acid synthase gene ilvN is shown in SEQ ID NO: 15.
4. An engineered strain of ralstonia that produces isoleucine via the methyl malic acid pathway, wherein the engineered strain of ralstonia is a wild-type strain of ralstonia having the following engineered characteristics:
the nucleotide sequence is shown in SEQ ID NO:4, a mutated N-acetylglucosamine transporter gene nagE;
the transcriptional regulator gene nagR of the endogenous N-acetylglucosamine transporter is knocked out, and the nucleotide sequence of the transcriptional regulator gene nagR is shown as SEQ ID NO:5 is shown in the figure;
expressing exogenous methyl malate synthase gene cimA and branched-chain amino acid exoprotein genes ygaZ and ygaH, wherein the nucleotide sequence of methyl malate synthase gene cimA is shown in SEQ ID NO:1, wherein the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaZ is shown in SEQ ID NO:2, the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaH is shown as SEQ ID NO: 3.
5. The engineered strain of ralstonia that produces isoleucine via the methyl malate pathway of claim 4, further characterized by the engineered strain of ralstonia having the following engineered characteristics:
The endogenous polyhydroxybutyrate synthesis genes phaC1, phaA and phaB1 are knocked out, wherein the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaC1 is shown as SEQ ID NO:6, the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaA is shown as SEQ ID NO:7, the nucleotide sequence of the polyhydroxybutyrate synthesis gene phaB1 is shown as SEQ ID NO: shown as 8;
The methyl malate synthase gene cimA is further overexpressed.
6. The engineered strain of ralstonia that produces isoleucine via the methyl malate pathway of claim 5, further characterized by the engineered strain of ralstonia having the following engineered characteristics:
endogenous lactic acid synthesis genes ldhA1 and ldhA2 are knocked out and express an NADPH dependent acetohydroxyacid isomerase gene ilvC and an NADPH coenzyme dependent branched-chain aminotransferase gene ilvE, wherein the nucleotide sequence of the lactic acid synthesis gene ldhA1 is as set forth in SEQ ID NO:10, the nucleotide sequence of the lactic acid synthesis gene ldhA2 is shown as SEQ ID NO:11, the nucleotide sequence of the acetohydroxy acid isomerase ilvC gene is shown in SEQ ID NO:12, the nucleotide sequence of the NADPH-dependent branched-chain aminotransferase gene ilvE is shown in SEQ ID NO: 13;
The endogenous lactic acid synthesis gene ldh is knocked out, and acetohydroxy acid synthase genes ilvB and ilvN losing feedback regulation sites are expressed, and the nucleotide sequence of the lactic acid synthesis gene ldh is shown in SEQ ID NO:9, the nucleotide sequence of the acetohydroxy acid synthase gene ilvB is shown in SEQ ID NO:14, the nucleotide sequence of the acetohydroxy acid synthase gene ilvN is shown in SEQ ID NO: 15.
7. A method for producing isoleucine by fermentation via the methyl malic acid pathway, said method comprising the steps of: fermenting in a culture medium using the engineering strain of Ralstonia producing isoleucine through the methyl malic acid pathway of any of claims 4 to 6 to obtain isoleucine.
8. The method of producing isoleucine via the methyl malic acid pathway fermentation of claim 7, where the medium has a formulation ：15.14 g/L Na2HPO4·12H2O,3 g/L KH2PO4,5 g/L (NH4)2SO4,300 mg/L NaHCO3,80 mg/L MgSO4·7H2O,10 mg/L ferric citrate ,2 mg/L CaCl2,1.2 mg/L NiSO4·7H2O,1 mg/L CaSO4,1 mg/L ZnSO4·7H2O,0.6 mg/L CoCl2·6H2O,0.6 mg/L H3BO3,0.44 mg/L MnSO4·H2O,0.4 mg/L Na2MoO4·2H2O,0.4 mg/L CuSO4·5H2O,200 mg/L kanamycin.
9. The method for producing isoleucine by the methyl malic acid pathway fermentation of claim 7, where the heterotrophic fermentation is carried out using glucose as the sole carbon source, the initial concentration of glucose is 30 g/L, and when the initial glucose consumption is completed, the feeding is controlled to have a glucose concentration of less than 15 g/L.
10. The method for producing isoleucine by the methyl malic acid pathway fermentation of claim 7, where the autotrophic fermentation is carried out in a closed vessel, using CO 2 as the sole carbon source, where the fresh mixed gas is displaced every 24 hours in the closed vessel, where the mixed gas is 8:1: h 2：O2：CO2 of 1.
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