CN116536341A - Method for constructing recombinant escherichia coli with high yield of gamma-aminobutyric acid and method for producing gamma-aminobutyric acid - Google Patents
Method for constructing recombinant escherichia coli with high yield of gamma-aminobutyric acid and method for producing gamma-aminobutyric acid Download PDFInfo
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- CN116536341A CN116536341A CN202310337105.5A CN202310337105A CN116536341A CN 116536341 A CN116536341 A CN 116536341A CN 202310337105 A CN202310337105 A CN 202310337105A CN 116536341 A CN116536341 A CN 116536341A
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- Prior art keywords
- aminobutyric acid
- gamma
- escherichia coli
- pyridoxal
- phosphate
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- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims abstract description 68
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12Y104/03005—Pyridoxal 5'-phosphate synthase (1.4.3.5), i.e. pyridoxamine 5-phosphate oxidase
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- C12Y401/01—Carboxy-lyases (4.1.1)
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Abstract
The invention relates to the technical field of agricultural biology, in particular to a method for constructing recombinant escherichia coli with high yield of gamma-aminobutyric acid and a method for producing gamma-aminobutyric acid. According to the invention, an engineering strain of Escherichia coli capable of producing gamma-aminobutyric acid is constructed by heterologously expressing a glutamic acid decarboxylase coding gene derived from bacillus Z11 in Escherichia coli. In order to eliminate the dependence of the exogenous expensive cofactor pyridoxal 5 '-phosphate in the process of converting a substrate to produce gamma-aminobutyric acid by the engineering escherichia coli, a synthesis way of endogenous pyridoxal 5' -phosphate is constructed in the engineering escherichia coli strain by screening gene sources, gene combinations and expression regulation modes. The construction of the path not only can completely supply the coenzyme requirement of the glutamate decarboxylase, but also greatly improves the conversion efficiency of the product.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to a method for constructing recombinant escherichia coli with high yield of gamma-aminobutyric acid and a method for producing gamma-aminobutyric acid.
Background
Gamma-aminobutyric acid (gamma-aminobutyric acid, GABA) is a four-carbon non-protein amino acid commonly existing in nature, and has multiple physiological functions in microorganisms, plants and organisms. In microorganisms, the production process of gamma-aminobutyric acid is directly related to the acid-resistant mechanism of the cells. Most eukaryotic microbial cells can promote spore growth by gamma-aminobutyric acid. In plants, gamma-aminobutyric acid can induce synthesis of ethylene in organisms or regulate balance of intracellular pH, and can prevent plants from being influenced by external environments such as oxidative stress or osmotic pressure. In animals, gamma-aminobutyric acid has been receiving more and more attention in the medical field due to its various physiological functions of regulating blood pressure and heart rate, promoting growth hormone secretion, protecting liver and kidney, improving lipid metabolism of the body, slowing down vascular arteriosclerosis, inhibiting cancer cell growth and promoting reproduction. In addition, the gamma-aminobutyric acid also has the functions of reducing activity and promoting animal growth, so that the gamma-aminobutyric acid can be used as a novel feed additive for animal husbandry.
Currently, the industrial preparation of gamma-aminobutyric acid mainly includes chemical synthesis and microbiological synthesis. Although the chemical synthesis method has the advantages of rapid reaction and high yield, the method has the defects of severe reaction conditions, severe reaction, more byproducts and the like. Along with the development of biotechnology, the biosynthesis method with low cost, environment-friendly process, green and safe performance and high production efficiency is applied. At present, strains used in the microbial synthesis method mainly comprise escherichia coli, corynebacterium glutamicum, lactobacillus and the like. Among these, escherichia coli has the advantages of rapid growth, simple culture, low cost, and the like, and can efficiently and rapidly convert L-glutamic acid directly into gamma-aminobutyric acid by only overexpressing glutamate decarboxylase in cells, and has been used in practical production. However, the efficiency of producing gamma-aminobutyric acid by using escherichia coli cells needs to be further improved, and the concentration of pyridoxal 5' -phosphate endogenously synthesized by escherichia coli is low, so that the requirement of high catalytic activity of glutamate decarboxylase to realize efficient synthesis of gamma-aminobutyric acid cannot be met. Therefore, expensive pyridoxal 5' -phosphate needs to be exogenously added in the production process, and the industrial production cost of the method is increased.
Disclosure of Invention
The invention aims to provide a method for constructing recombinant escherichia coli engineering bacteria for efficiently producing gamma-aminobutyric acid.
It is still another object of the present invention to provide a method for producing gamma-aminobutyric acid using a mixture of L-glutamic acid and sodium L-glutamate as a substrate.
The method for constructing recombinant escherichia coli engineering bacteria for efficiently producing gamma-aminobutyric acid comprises the following steps of:
constructing mutant escherichia coli chassis cells with gamma-aminobutyric acid synthesis capacity by utilizing an inducible promoter to heterologously express a coding gene of glutamic acid decarboxylase from bacillus Z11 in escherichia coli strains;
in the obtained mutant escherichia coli chassis cells with the gamma-aminobutyric acid synthesis capability, the encoding genes of pyridoxal 5 '-phosphate synthase and the encoding genes of glutamine aminotransferase derived from bacillus subtilis 168 are respectively subjected to heterologous expression by using an inducible promoter, and an endogenous pyridoxal 5' -phosphate synthesis way is constructed, so that recombinant escherichia coli engineering bacteria for efficiently producing gamma-aminobutyric acid are obtained.
According to the method for constructing the recombinant escherichia coli engineering bacteria for efficiently producing gamma-aminobutyric acid, the inducible promoter is a T7 promoter.
The method for producing gamma-aminobutyric acid through whole cell transformation comprises the following steps:
culturing and inducing the expression of exogenous glutamate decarboxylase and the synthesis of pyridoxal 5' -phosphate in the engineering strain of the escherichia coli by shaking;
and (3) utilizing the obtained escherichia coli engineering strain to perform whole-cell transformation on a mixture of L-glutamic acid and L-sodium glutamate to produce gamma-aminobutyric acid.
The method for producing gamma-aminobutyric acid through whole cell transformation, provided by the invention, comprises the step of shake flask induction of escherichia coli engineering bacteria by utilizing an LB culture medium.
The method for producing gamma-aminobutyric acid through whole cell transformation, disclosed by the invention, comprises the following steps of: tryptone 10 g/L, yeast extract 5 g/L, naCL10 g/L.
According to the method for producing gamma-aminobutyric acid by whole cell transformation, the induction time of a shaking bottle is 37 ℃, the rotating speed is 220 rpm, and the concentration of inducer IPTG is 0.1 mM.
The method for producing gamma-aminobutyric acid by whole cell transformation according to the present invention, wherein ampicillin is added to the LB medium at a final concentration of 100. Mu.g/L.
The method for producing gamma-aminobutyric acid by whole cell transformation according to the present invention, wherein isopropyl-beta-D-thiogalactoside (IPTG) is added to the LB medium to a final concentration of 0.1 mM.
The method for producing gamma-aminobutyric acid by whole cell transformation according to the present invention, wherein the whole cell transformation condition is a temperature of 37 ℃ and a rotation speed of 120 rpm.
The method for producing gamma-aminobutyric acid according to the present invention, wherein the whole cell transformation substrate is a mixture of 0.6M L-glutamic acid and 0.4M sodium L-glutamate added in a pure water system.
The technical scheme of the application has the advantages that:
1. according to the invention, an engineering strain of Escherichia coli capable of producing gamma-aminobutyric acid is constructed by heterologously expressing a glutamic acid decarboxylase coding gene derived from bacillus Z11 in Escherichia coli. In order to eliminate the dependence of the exogenous expensive cofactor pyridoxal 5 '-phosphate in the process of converting a substrate to produce gamma-aminobutyric acid by the engineering escherichia coli, a synthesis way of endogenous pyridoxal 5' -phosphate is constructed in the engineering escherichia coli strain by screening gene sources, gene combinations and expression regulation modes. The construction of the path not only can completely supply the coenzyme requirement of the glutamate decarboxylase, but also greatly improves the conversion efficiency of the product.
2. The strain of the invention can be cultivated and induced at 37 ℃ for 6 h, and the concentration of the inducer IPTG is 0.1 mM. The obtained E.coli cells (OD 600 Approximately 15) a mixture of 0.6M L-glutamic acid and 0.4M sodium L-glutamate can be completely converted to approximately 1. 1M of gamma-aminobutyric acid in 0.5. 0.5 h. The same batch of bacterial cells can be repeatedly used for 7 times, the conversion rate is above 96%, namely 4.8M L-glutamic acid and 3.2M L-sodium glutamate can be converted into gamma-aminobutyric acid in 4 h, and the production rate can reach205 g/L/h。
Drawings
FIG. 1 shows the effect of induction conditions on the synthesis of gamma-aminobutyric acid by recombinant E.coli;
FIG. 2 shows the effect of gene source and expression regulation on pyridoxal 5' -phosphate synthesis;
FIG. 3 shows the effect of gene combination on the synthesis of gamma-aminobutyric acid from pyridoxal 5' -phosphate-fed strains;
FIG. 4 shows optimization of conditions for efficient synthesis of gamma-aminobutyric acid by recombinant E.coli;
FIG. 5 shows the investigation of a recombinant E.coli cell reuse batch.
Description of the embodiments
The strains used in the examples below were E.coli BL21 (DE 3) host strain, and the plasmid vectors were pET-28a (+), pETDute-1 and pACYADute-1. Restriction enzymes and other biochemical reagents used are all commercially available from Biochemical reagent company. Molecular biology experimental methods, which are not specifically described, are all carried out by referring to the specific methods listed in the "molecular cloning Experimental guidelines" (third edition) J.Sam Brookfield, or according to the kit and the product instructions.
According to the specific embodiment of the invention, a mutant escherichia coli chassis cell with gamma-aminobutyric acid synthesis capability is constructed by utilizing an inducible promoter to heterologously express a gene encoding glutamic acid decarboxylase derived from bacillus Z11 in escherichia coli strains; in the obtained mutant escherichia coli chassis cells with the gamma-aminobutyric acid synthesis capability, the encoding genes of pyridoxal 5 '-phosphate synthase and the encoding genes of glutamine aminotransferase derived from bacillus subtilis 168 are respectively subjected to heterologous expression by using an inducible promoter, and an endogenous pyridoxal 5' -phosphate synthesis way is constructed, so that recombinant escherichia coli engineering bacteria for efficiently producing gamma-aminobutyric acid are obtained.
In the following examples, the gene encoding the Bacillus Z11-derived glutamate decarboxylase is disclosed in genbankgadZ11Genes with accession numbers GenBank ID: MW703456.1; the pyridoxal 5' -phosphate synthase coding gene derived from bacillus subtilis 168 is disclosed in genbankOpen and openpdxSThe nucleotide sequence accession number is Genbank ID:939988; the glutamine aminotransferase encoding gene is disclosed as genbankpdxTThe nucleotide sequence accession number is Genbank ID:939971.
Plasmid vectors used in the present application are shown in Table 1, and E.coli BL21 (DE 3) engineering strains are shown in Table 2:
TABLE 1
| Plasmid name | Traits (3) |
| pET28a-gadZ11 | Expression under the control of T7 promoter using pET-28a (+)gadZ11 |
| pETDute-1-T7-PdxS-T7-PdxT | Expression under the control of two T7 promoters, respectively, using pETDute-1 vectorpdxSAndpdxT |
| pETDute-1-T7-PdxS/PdxT | expression under the control of a T7 promoter in the pETDute-1 vectorpdxSAndpdxT |
| pETDute-1-T7-PdxS | expression under the control of T7 promoter using pETDute-1 vectorpdxS |
| pACYCDute-1-T7-PdxT | Expression under the control of T7 promoter using pACYCDute-1 vectorpdxT |
| pETDute-1-J-PdxS-J-PdxT | Expression Using pETDute-1 vector under the control of two J23100 promoters, respectivelypdxSAndpdxT |
| pETDute-1-J-PdxS/PdxT | expression Using pETDute-1 vector under the control of a J23100 promoterpdxSAndpdxT |
| pETDute-1-J-PdxS | expression Using pETDute-1 vector under the control of J23100 promoterpdxS |
| pACYCDute-1-J-PdxT | Expression under the control of J23100 promoter Using pACYCDute-1 vectorpdxT |
| pETDute-1-T7-SNO1-T7-SNZ1 | Expression under the control of two T7 promoters, respectively, using pETDute-1 vectorSNO1AndSNZ1 |
| pETDute-1-T7-SNO1/SNZ1 | expression under the control of a T7 promoter in the pETDute-1 vectorSNO1AndSNZ1 |
| pETDute-1-T7-SNO1 | expression under the control of T7 promoter using pETDute-1 vectorSNO1 |
| pACYCDute-1-T7-SNZ1 | Expression under the control of T7 promoter using pACYCDute-1 vectorSNZ1 |
| pETDute-1-J-SNO1/SNZ1 | Using pETDute-1 vector at J23100 promoterExpression under control of a promoterSNO1AndSNZ1 |
| pETDute-1-J-SNO1 | expression Using pETDute-1 vector under the control of J23100 promoterSNO1 |
| pACYCDute-1-J-SNZ1 | Expression under the control of J23100 promoter Using pACYCDute-1 vector SNZ1 |
| pCDFDute-1-T7-PdxS-T7-PdxT | Expression under the control of two T7 promoters, respectively, using the pCDFDute-1 vectorpdxSAndpdxT |
| pETDute-1-T7-PdxS-T7-PdxT-T7-GADZ11 | expression under the control of three T7 promoters, respectively, using pETDute-1 vectorspdxS、pdxTAndgadZ11 |
| pETDute-1-T7-PdxS-T7-PdxT-RBS-GADZ11 | expression under the control of two T7 promoters, respectively, using pETDute-1 vectorpdxS、pdxTAndgadZ11 |
TABLE 2
| Strain name | Traits (3) |
| E. coli BL21(DE3) | Large intestine rodBacterial BL21 (DE 3) strain |
| Ec-0 | Coli strain carrying plasmid pETDute-1 |
| Ec-1 | Carrying plasmid pET28a-gadZ11gadZ11Gene-expressed strains |
| Ec-2 | Bacterial strain carrying plasmid pETDute-1-T7-PdxS-T7-PdxT for pyridoxal 5' -phosphate synthesis |
| Ec-3 | Bacterial strain carrying plasmid pETDute-1-T7-PdxS/PdxT for pyridoxal 5' -phosphate synthesis |
| Ec-4 | Bacterial strain carrying plasmids pETDute-1-T7-PdxS and pACYCDute-1-T7-pdxT for pyridoxal 5' -phosphate synthesis |
| Ec-5 | Bacterial strain carrying plasmid pETDute-1-J-PdxS-J-PdxT for pyridoxal 5' -phosphate synthesis |
| Ec-6 | Bacterial strain carrying plasmid pETDute-1-J-PdxS/PdxT for pyridoxal 5' -phosphate synthesis |
| Ec-7 | Bacterial strain carrying plasmids pETDute-1-J-PdxS and pACYCDute-1-J-PdxT for pyridoxal 5' -phosphate synthesis |
| Ec-8 | Pyridoxal 5' -phosphate carrying plasmid pETDute-1-T7-SNO1-T7-SNZ1Synthetic strains |
| Ec-9 | Bacterial strain carrying plasmid pETDute-1-T7-SNO1/SNZ1 for pyridoxal 5' -phosphate synthesis |
| Ec-10 | Bacterial strain carrying plasmids pETDute-1-T7-SNO1 and pACYCDute-1-T7-SNZ1 for pyridoxal 5' -phosphate synthesis |
| Ec-11 | Bacterial strain carrying plasmid pETDute-1-J-SNO1/SNZ1 for pyridoxal 5' -phosphate synthesis |
| Ec-12 | Bacterial strain carrying plasmids pETDute-1-J-SNO1 and/pACYCDute-1-J-SNZ 1 for pyridoxal 5' -phosphate synthesis |
| Ec-13 | Strain carrying plasmids pET28a-gadZ11 and pCDFDute-1-T7-PdxS-T7-PdxT capable of gamma-aminobutyric acid production using endogenously synthesized pyridoxal 5' -phosphate |
| Ec-14 | Strain carrying plasmid pETDute-1-T7-PdxS-T7-PdxT-T7-GADZ11 capable of gamma-aminobutyric acid production using endogenously synthesized pyridoxal 5' -phosphate |
| Ec-15 | Strain carrying plasmid pETDute-1-T7-PdxS-T7-PdxT-RBS-GADZ11 capable of gamma-aminobutyric acid production using endogenously synthesized pyridoxal 5' -phosphate |
。
1. Construction of pET28a-gadZ11 vector
The bacillus Z11 genomic DNA was extracted using a bacterial genomic DNA extraction kit. According to the coding gene of glutamate decarboxylasegadZ11The genome DNA is used as a template to amplify the gadZ11 gene fragment with the size of 1525 bp. The resulting fragment was ligated to plasmid pET-28a (+) using Gibson Assembly ligationEcoRI/NotI site. Chemical transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (kanamycin 50. Mu.g/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pET28a-gadZ11.
2. Construction of recombinant E.coli Ec-1
Transferring the recombinant plasmid pET28a-gadZ11 into escherichia coli BL21 (DE 3) by using a heat shock transformation method to obtain escherichia coli engineering bacteria Ec-1.
3. Culture of recombinant E.coli Ec-1 and optimization of the induction conditions of glutamate decarboxylase GADZ11
Ec-1 was inoculated at an inoculum size of 1% into LB medium (kanamycin 50. Mu.g/mL) of 50 mL, and shake cultured overnight at 37℃and 220 rpm. The bacterial liquid obtained was inoculated into 300 mL of LB medium (kanamycin 50. Mu.g/mL) at an inoculum size of 2%, and cultured at 220 rpm in a shaker at 37℃for about 3 h to OD 600 ≈0.6。
(1) Influence of the Induction temperature on the Synthesis of gamma-aminobutyric acid by recombinant E.coli genetically engineered bacteria
To culture to OD 600 1 mM IPTG was added to the bacterial solution of approximately 0.6 and the mixture was induced at different induction temperatures (16, 20, 25, 30, 37 ℃) in a 200 rpm shaker at 16 h.12000 The cells were collected by centrifugation at rpm for 5 min. Washing the obtained thallus with pure water twice, transferring the thallus to 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with concentration OD 600 About 20 and pyridoxal 5' -phosphate was added at a final concentration of 0.1. 0.1 mM. 2.94 g of L-glutamic acid (final concentration of 1. 1M in the reaction system) was weighed into a 100 mL conical flask, and 20. 20 mL bacterial liquid in the centrifuge tube was added into the conical flask to start the product synthesis reaction. 500. Mu.L of the reaction solution was added to 50 in each of reactions 0.5 and 1. 1 h 0. mu.L of 80% ethanol was used to terminate the reaction, and the supernatant was centrifuged at 12000 rpm for 3 min, and the supernatant was appropriately diluted and subjected to HPLC detection. The detection result is shown in FIG. 1 (a). The result shows that the low-temperature induction is favorable for the expression of glutamate decarboxylase, and when the induction temperature is 16 ℃, the yield of gamma-aminobutyric acid in a system is 1.2M after the conversion reaction is carried out by 1 h, namely, the L-glutamic acid in the system is completely converted. When the induction temperature was raised to 20, 25 and 30 ℃, the substrate conversion rate after reaction 1, h, increased up to 98.6%, slightly below the product conversion rate of the induced cells at 16 ℃. However, when the induction temperature was 37 ℃, the yield of gamma-aminobutyric acid in the system after the conversion reaction was carried out by 1 h was 1.1M, i.e., the L-glutamic acid in the system was theoretically completely converted. This is probably due to the fact that the expression level of glutamate decarboxylase was increased at the optimum growth temperature of E.coli (37 ℃). In practical production, 37 ℃ is easy to realize and more thalli can be obtained for the production of products. Thus, 37 ℃ was finally selected as the induction temperature.
(2) Influence of bacterial strain induction time on synthesis of gamma-aminobutyric acid by recombinant escherichia coli genetically engineered bacteria
To culture to OD 600 1 mM IPTG was added to the bacterial solution of approximately 0.6 and the mixture was induced for various periods of time (2, 4, 6, 10, 16, h) in a 200 rpm shaker at 37 ℃.12000 The cells were collected by centrifugation at rpm for 5 min. Washing the obtained thallus with pure water twice, transferring the thallus to 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with concentration OD 600 About 20 and pyridoxal 5' -phosphate was added at a final concentration of 0.1. 0.1 mM. 2.94 g of L-glutamic acid (final concentration of 1. 1M in the reaction system) was weighed into a 100 mL conical flask, and 20. 20 mL bacterial liquid in the centrifuge tube was added into the conical flask to start the product synthesis reaction. The reaction was terminated by adding 500. Mu.L of 80% ethanol to 500. Mu.L of the reaction mixture at each of reactions 0.5 and 1 and h, and the supernatant was centrifuged at 12000 rpm for 3 min and suitably diluted, followed by detection by HPLC, the detection results being shown in FIG. 1 (b). The results showed that the production of gamma-aminobutyric acid increased with time when the induction time was in the range of 2-6 h. When the induction time is 6 h, the yield of the gamma-aminobutyric acid reaches 1.05M after the conversion reaction is 1 h, i.e. the substrate has reacted completely. Thus, the induction time was chosen to be 6 h.
(3) Influence of inducer concentration on the synthesis of gamma-aminobutyric acid by recombinant E.coli genetically engineered bacteria
To culture to OD 600 0.1, 0.5, 1.0, 1.5, 2.0 and 2.5 mM IPTG were added to the bacterial solutions of approximately 0.6 and 6 h was induced in a 200 rpm shaker at 37 ℃.12000 The cells were collected by centrifugation at rpm for 5 min. Washing the obtained thallus with pure water twice, transferring the thallus to 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with concentration OD 600 About 20 and pyridoxal 5' -phosphate was added at a final concentration of 0.1. 0.1 mM. 2.94 g of L-glutamic acid (final concentration of 1. 1M in the reaction system) was weighed into a 100 mL conical flask, and 20. 20 mL bacterial liquid in the centrifuge tube was added into the conical flask to start the product synthesis reaction. The reaction was terminated by adding 500. Mu.L of 80% ethanol to 500. Mu.L of the reaction mixture at each of reactions 0.5 and 1 and h, and the supernatant was centrifuged at 12000 rpm for 3 min and suitably diluted, followed by detection by HPLC, the detection results being shown in FIG. 1 (c). The results show that different inducer concentrations have no obvious effect on the yield of gamma-aminobutyric acid and that low concentrations of IPTG (0.1 mM) achieve the induction purpose.
4. Recombinant escherichia coli Ec-1 is synthesized into gamma-aminobutyric acid under optimal conditions
Respectively culturing to OD 600 0.1 and 1.0 IPTG were added to the bacterial solutions at a final concentration of approximately 0.6 and induced in shaking tables at 220 rpm at 37 and 16℃respectively, 6 and 16 h.12000 The cells were collected by centrifugation at rpm for 5 min. Washing the obtained thallus with pure water twice, transferring the thallus to 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with concentration OD 600 About 20 and pyridoxal 5' -phosphate was added at a final concentration of 0.1. 0.1 mM. 2.94 g of L-glutamic acid (final concentration of 1. 1M in the reaction system) was weighed into a 100 mL conical flask, and 20. 20 mL bacterial liquid in the centrifuge tube was added into the conical flask to start the product synthesis reaction. The reaction was terminated by adding 500. Mu.L of 80% ethanol to 500. Mu.L of the reaction mixture at each of reactions 0.5 and 1 and h, and the supernatant was centrifuged at 12000 rpm for 3 min and suitably diluted, followed by detection by HPLC, the detection results being shown in FIG. 1 (d). The results show that the cell obtained under the two conditions has no obvious difference in the product yield when transforming L-glutamic acid to produce gamma-aminobutyric acid. After the conversion reaction was carried out at 1 h, the substrates were all completely converted, yielding 1M of gamma-aminobutyric acid. Based on the above study, the induction condition of the E.coli engineering bacteria used in the transformation reaction was set to 37℃and 0.1 mM IPTG was used to induce 6 h.
The whole process of producing gamma-aminobutyric acid can be carried out at normal temperature by the engineering strain Ec-1 of Escherichia coli constructed by the research, but the activity of the enzyme GADZ11 can be exerted by the coenzyme pyridoxal 5' -phosphate when the enzyme GADZ11 catalyzes the conversion of L-glutamic acid to gamma-aminobutyric acid. The pyridoxal 5 '-phosphate synthesized by the escherichia coli is small in quantity and cannot meet the requirement of product synthesis, so that the conversion reaction still needs to add the pyridoxal 5' -phosphate with high price exogenously, the operation steps are increased, and the production cost is increased. The self-supply of this coenzyme is undoubtedly an effective way to solve this problem by establishing the pyridoxal 5' -phosphate synthesis pathway in E.coli. The natural pyridoxal 5' -phosphate synthesis pathway in microorganisms includes two. One is the deoxyxylulose 5-phosphate-dependent pathway, but this pathway is lengthy and requires 7 enzymes to participate in the catalytic reaction. The other is the ribulose 5-phosphate dependent pathway. The method is catalyzed by a complex consisting of only two enzymes, and takes ribose 5-phosphate, glyceraldehyde 3-phosphate and glutamine as substrates to directly synthesize pyridoxal 5' -phosphate. Based on the above, bacillus subtilis [ ] Bacillus subtilis) 168 and Saccharomyces cerevisiaeSaccharomyces cerevisiae) Pyridoxal 5' -phosphate synthase Gene derived from SC288pdxSAndSNO1) And glutamine aminotransferase encoding genepdxTAndSNZ1) Screening of gene sources, promoters and combinations was performed and based thereon, a pyridoxal 5 '-phosphate synthesis pathway was constructed in E.coli BL21 (DE 3), and self-supply of coenzyme pyridoxal 5' -phosphate was achieved. The specific method comprises the following steps:
1. construction of glutaminase Gene and pyridoxal 5' -phosphate synthase Gene expression vector
Genomic DNA of bacillus subtilis 168 and saccharomyces cerevisiae SC288 was extracted using a bacterial genomic DNA extraction kit and a yeast genomic DNA extraction kit, respectively.
(1) Construction of pETDute-1-T7-PdxS-T7-PdxT vector
According topdxSThe gene sequence is designed into a synthetic primer. Amplification with the primers designed as described above using the Bacillus subtilis 168 genome as templatepdxSGene, fragment size 926 bp. The obtained fragment was ligated to plasmid pETDute-1 using Gibson Assembly ligationEcoRI/NotI site. Transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100. Mu.g/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-T7-pdxS.
Then according topdxTThe gene sequence is designed into a synthetic primer. Amplification with the primers designed as described above using the Bacillus subtilis 168 genome as a templatepdxTGene, fragment size 631 bp, the resulting fragment was ligated to plasmid pETDute-1-T7-PdxS using Gibson Assembly ligationEcoRVAnd BglII site. Transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100 [ mu ] g/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was named pETDute-1-T7-PdxS-T7-PdxT.
(2) Construction of pETDute-1-T7-PdxS/PdxT vector
According topdxSAndpdxTthe gene sequence is designed and synthesized, the bacillus subtilis 168 genome is used as a template, and the primers designed are used for amplification and containpdxSAndpdxTis a fragment of the gene of (a) having a fragment size of 1539 and bp. The obtained fragment was ligated to plasmid pETDute-1 using Gibson Assembly ligationEcoRI, and RI systemBglII site. Chemical transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100 [ mu ] g/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-T7-PdxS/T.
(3) Construction of pACYCDute-1-T7-pdxT vector
According topdxTThe gene sequence is designed into a synthetic primer.Amplification with the primers designed as described above using the Bacillus subtilis genome as templatepdxTGene fragment, fragment size 631 and bp, the resulting fragment was ligated into plasmid pACYCDute-1 using Gibson Assembly ligationEcoRI/NotI site. Chemical transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (35 mug/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pACYCDute-1-T7-pdxT.
(4) Construction of pACYCDute-1-J-PdxT vector
The primer containing the J23100 promoter sequence was designed and synthesized according to the pACYCDute-1-T7-PdxT gene sequence. Amplifying target gene by using the vector pACYCDute-1-T7-PdxT as a template and utilizing the designed primer to amplify fragment size 3361 bp, and transforming the obtained fragment into escherichia coliTransIn a 1-T1 host, LB solid plates (35 mug/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pACYCDute-1-J-PdxT.
(5) Construction of pETDute-1-J-PdxS-J-PdxT vector
The primer containing the J23100 promoter sequence is designed and synthesized according to the vector pETDute-1-T7-PdxS gene sequence. The vector pETDute-1-T7-PdxS is used as a template, and the primers designed above are used for amplifying fragments, wherein the sizes of the fragments are 4794 and bp. Transformation of fragments into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100. Mu.g/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-J-PdxS.
The primer containing the J23100 promoter sequence was designed and synthesized according to the pACYCDute-1-J-PdxT gene sequence. The target fragment was amplified using the vector pACYCDute-1-J-PdxT as a template and the fragment size 749 and bp were amplified using the primers designed as described above, and the resulting fragment was ligated to the plasmid pETDute-1-J-pdxS using Gibson Assembly ligationNotI andEcoRV site. Chemical transformation of plasmids into E.coliTransIn a 1-T1 host, an LB solid plate (ampicillin 100 [ mu ] g/mL) was coated at 37 DEG CAfter overnight incubation, the colony PCR was screened for positive clones and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-J-PdxS-J-PdxT.
(6) Construction of pETDute-1-J-PdxS/PdxT vector
A primer containing the J23100 promoter sequence was designed and synthesized based on the vector pETDute-1-T7-PdxS/T sequence. The target fragment was amplified using the above designed primer with the vector pETDute-1-T7-PdxS/T as a template, the fragment size was 5406 bp, and the obtained fragment was ligated into a circular plasmid using Gibson Assembly ligation. Transformation of fragments into E.coli TransIn a 1-T1 host, LB solid plates (ampicillin 100 [ mu ] g/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-J-PdxS/T.
(7) Construction of pETDute-1-T7-SNO1-T7-SNZ1 vector
According toSNO1The gene sequence is designed into a synthetic primer. The Saccharomyces cerevisiae SC288 genome is used as a template, and the primers designed above are used for respective amplificationSNO1Gene fragment, fragment size 717 bp, was ligated into plasmid pETDute-1 using Gibson Assembly ligationEcoRI/NotI site. Transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100 mug/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-T7-SNO1. Then according toSNZ1The gene sequence is designed into a synthetic primer. Amplification with the primers designed as described above using Saccharomyces cerevisiae SC288 genome as templateSNZ1Gene fragment, fragment size 946 bp. The obtained fragment was ligated to plasmid pETDute-1-T7-SNO1 using Gibson Assembly ligationEcoRVAnd BglII site. Chemical transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100 mug/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-T7-SNO1-T7-SNZ1.
(8) Construction of pETDute-1-T7-SNO1/SNZ1 vector
According toSNO1AndSNZ1the gene sequence is designed and synthesized, saccharomyces cerevisiae SC288 genome is used as a template, and the designed primer is used for amplifying target fragments, wherein the fragment size is 1631 and bp. The obtained fragment was ligated to plasmid pETDute-1 using Gibson Assembly ligationEcoRI/NotI site. Transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100 mug/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-T7-SNO1/SNZ1.
(9) Construction of pACYCDute-1-T7-SNZ1 vector
According toSNZ1The gene sequence is designed into a synthetic primer. Amplification with the primers designed as described above using Saccharomyces cerevisiae SC288 genome as templateSNZ1Gene fragment, fragment size 949 bp. The obtained fragment was ligated to plasmid pACYCDute-1 using Gibson Assembly ligationEcoRI/NotI site. Chemical transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (35 mug/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced for verification, and the correctly constructed plasmid was designated pACYCDute-1-T7-SNZ1.
(10) Construction of pETDute-1-J-SNO1/SNZ1 vector
The primer containing the J23100 promoter sequence is designed and synthesized according to the gene sequence of the vector pETDute-1-T7-SNO1/SNZ 1. The target fragment was amplified using the primers designed as described above using the vector pETDute-1-T7-SNO1/SNZ1 as a template, with a fragment size of 5500 and bp. Transformation of fragments into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100 mug/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-J-SNO1/SNZ1.
(11) Construction of pETDute-1-J-SNO1 vector
The primer containing the J23100 promoter sequence is designed and synthesized according to the gene sequence of the vector pETDute-1-T7-SNO 1. The target fragment was amplified using the vector pETDute-1-T7-SNO1 as a template and the primers designed as described above, with a fragment size of 4535 bp. Sheet of materialTransformation of fragments into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100. Mu.g/mL) were coated, cultured overnight at 37℃and positive clones were screened by colony PCR and sequenced for verification. The correctly constructed plasmid was designated pETDute-1-J-SNO1.
(12) Construction of pACYCDute-1-J-SNZ1 vector
The primer containing the J23100 promoter sequence is designed and synthesized according to the pACYCDute-1-T7-SNZ1 gene sequence. The target fragment was amplified using the primers designed as described above using the vector pACYCDute-1-T7-SNZ1 as a template, with fragment size 3622 bp. Chemical transformation of fragments into E.coli TransIn a 1-T1 host, LB solid plates (35 mug/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pACYCDute-1-J-SNZ1.
2. Construction of recombinant E.coli for synthesizing pyridoxal 5' -phosphate and determination of product yield
Recombinant strains Ec-2, ec-3, ec-4, ec-5, ec-6, ec-7, ec-8, ec-9, ec-10, ec-11, ec-12 and Ec-13 were constructed by transforming the above vectors into E.coli BL21 (DE 3), respectively.
Ec-2: a strain in which pyridoxal 5' -phosphate is synthesized by T7-PdxS-T7-PdxT;
ec-3: T7-PdxS/PdxT, and a strain for synthesizing pyridoxal 5' -phosphate;
ec-4: strains in which T7-PdxS and T7-pdxT are synthesized from pyridoxal 5' -phosphate;
ec-5: a strain in which pyridoxal 5' -phosphate is synthesized by J-PdxS-J-PdxT;
ec-6: J-PdxS/PdxT, a strain in which pyridoxal 5' -phosphate is synthesized;
ec-7: strains of J-PdxS and-J-PdxT, which undergo pyridoxal 5' -phosphate synthesis;
ec-8: a strain in which pyridoxal 5' -phosphate is synthesized by T7-SNO1-T7-SNZ 1;
ec-9: T7-SNO1/SNZ1, and a strain for synthesizing pyridoxal 5' -phosphate;
ec-10: strains of T7-SNO1 and T7-SNZ1, which undergo pyridoxal 5' -phosphate synthesis;
Ec-11: J-SNO1/SNZ1, a strain for pyridoxal 5' -phosphate synthesis;
ec-12: a strain in which pyridoxal 5' -phosphate synthesis is performed by J-SNO1 and/or J-SNZ 1;
ec-13: gadZ11 and T7-PdxS-T7-PdxT, strains capable of gamma-aminobutyric acid production using endogenously synthesized pyridoxal 5' -phosphate;
ec-14: a strain of T7-PdxS-T7-PdxT-T7-GADZ11 capable of producing gamma-aminobutyric acid using endogenously synthesized pyridoxal 5' -phosphate;
ec-15: T7-PdxS-T7-PdxT-RBS-GADZ11, a strain capable of producing gamma-aminobutyric acid using endogenously synthesized pyridoxal 5' -phosphate.
As a control, E.coli strain Ec-0 transformed with pETDute-1 was used. The strain is inoculated into 50 mL LB culture medium (100 mug/mL of ampicillin and/or 35 mug/mL of chloramphenicol) with corresponding antibiotics in an inoculum size of 1%o, and shake-cultured at 37 ℃ and 220 rpm for overnight. Inoculating the obtained bacterial liquid into 300 mL LB culture medium (ampicillin 100 [ mu ] g/mL and/or chloramphenicol 35 [ mu ] g/mL) with corresponding resistance at an inoculum size of 2%, culturing at 37deg.C and 220 rpm in a shaker until OD 600 ≈0.6。
To culture to OD 600 0.1 mM IPTG was added to the bacterial suspension of approximately 0.6, and 6 h was induced in a 200 rpm shaker at 37 ℃. The absorbance (OD) of the bacterial liquid at a wavelength of 600 nm was measured by a visible light spectrophotometer 600 ). 10 mg (dry cell weight) cells were resuspended in 15 mL phosphate buffer (PBS, pH 7.4) containing 100. Mu.g/mL lysozyme, 10. Mu.g/mL RNaseA and 5. Mu.g/mL DNase I. After incubation on ice for 1 h, the cells were sonicated. Then 20 μg proteinase K was added to the sample and incubated on ice for 30 min, followed by 1.5 mL of 100% trichloroacetic acid (TCA) to precipitate the protein. The samples were vortexed for 1 min and incubated on ice for 15 min. After centrifugation at 12000 rpm for 10 min at 4℃the supernatant was subjected to HPLC detection. The column was an Agilent ZORBAX 300SB-C18 (5 μm, 4.6X1250 mm) column temperature 40 ℃. The sample loading was 10. Mu.L. The mobile phase is composed of 8% acetonitrile and 0.1% C 16 H 37 NO 50 mM (NH) 4 ) 2 HPO 4 Buffer (pH 3.6), detection procedure was: flow rate 0.7 mL/min, detectionFor 20 min, wavelength 292 nm was detected.
The results of the detection are shown in FIG. 2, and the results show that the gene sources and the combination and induction modes are different in pyridoxal 5' -phosphate yield. In general, the product yield of a metabolic pathway constructed using a pyridoxal 5' -phosphate synthesis-related gene derived from Bacillus subtilis is superior to that of a metabolic pathway constructed using a gene derived from Saccharomyces cerevisiae. Meanwhile, the regulation of gene expression by using an inducible promoter is more beneficial to the synthesis of pyridoxal 5' -phosphate, probably because the synthesis of the product has a negative effect on the growth of the strain, while the inducible promoter is in a closed state before the addition of the inducer, and has a smaller effect on the growth of the strain. The strain with highest pyridoxal 5 '-phosphate yield is Ec-2 strain carrying plasmid pETDute-1-T7-PdxS-T7-PdxT, and the pyridoxal 5' -phosphate synthase gene from bacillus subtilis pdxS) And glutaminase Gene [ ]pdxT) Respectively, is controlled by the T7 promoter. Based on this, the gene, combination and induction pattern used to construct the pyridoxal 5' -phosphate synthesis pathway in recombinant E.coli Ec-2 was selected for gamma-aminobutyric acid production.
Based on the selected gene, combination and induction pattern capable of synthesizing the pyridoxal 5' -phosphate in E.coli, the combination pattern of the combination and the glutamic acid decarboxylase GADZ11 was experimentally investigated. The two are overlapped to obtain the high-efficiency synthesis of the product gamma-aminobutyric acid by utilizing endogenous synthesized pyridoxal 5' -phosphate to be matched with glutamate decarboxylase GADZ 11. The specific implementation method is as follows:
1. construction of Gene expression vectors related to glutamate decarboxylase and pyridoxal 5' -phosphate synthase
（1）pCDFDute-1-T7-PdxS-T7-PdxT
According topdxSAndpdxTthe gene sequence is designed into a synthetic primer. The target fragment was amplified using the vector pETDute-1-T7-PdxS-T7-PdxT as a template and the primers designed as described above, with fragment size 1673 bp. The resulting fragment was ligated to plasmid pCDFDute-1 using Gibson Assembly ligationEcoRI/NdeI site. Chemical transformation of plasmids into E.coliTransCoating in a 1-T1 hostLB solid plate (streptomycin 35 [ mu ] g/mL), after overnight culture at 37 ℃, positive clones were screened by colony PCR and sequenced to verify, and the correctly constructed plasmid was named pCDFDute-1-T 7-PdxS-T7-PdxT。
（2）pETDute-1-T7-PdxS-T7-PdxT-T7-GADZ11
According togadZ11The gene sequence is designed and synthesized, the vector pET28a-gadZ11 is used as a template, and the designed primer is used for amplifying the target fragment, wherein the fragment size is 1779bp. The fragment obtained was ligated to plasmid pETDute-1-T7-PdxS-T7-PdxT using Gibson Assembly ligationEcoRV andXhoi site. Chemical transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100. Mu.g/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-T7-PdxS-T7-PdxT 7-GADZ11.
（3）pETDute-1-T7-PdxS-T7-PdxT-RBS-GADZ11
According togadZ11The gene sequence is designed into a synthetic primer. The target fragment was amplified using the primers designed as described above using the vector pET28a-gadZ11 as a template, fragment size 1681 bp. The fragment obtained was ligated to plasmid pETDute-1-T7-PdxS-T7-PdxT using Gibson Assembly ligationEcoRV/XhoI site. Chemical transformation of plasmids into E.coliTransIn a 1-T1 host, LB solid plates (ampicillin 100. Mu.g/mL) were coated, positive clones were screened by colony PCR after overnight incubation at 37℃and sequenced to verify that the correctly constructed plasmid was designated pETDute-1-T7-PdxS-T7-PdxT-RBS-GADZ11.
2. Recombinant E.coli construction for gamma-aminobutyric acid synthesis using endogenous pyridoxal 5' -phosphate
Simultaneously transferring the recombinant plasmids pET28a-gadZ11 and pCDFDute-1-T7-PdxS-T7-PdxT into escherichia coli BL21 (DE 3), and transferring the recombinant plasmids pETDute-1-T7-PdxS-T7-GADZ 11 and pETDute-1-T7-PdxS-T7-PdxT-RBS-GADZ11 into escherichia coli BL21 (DE 3) to obtain escherichia coli engineering bacteria Ec-13, ec-14 and Ec-15. Carrying plasmid pET28a-gadZ11gadZ11Gene-expressed strain Ec-1 was used as a control, and was added to the transformation reaction systemPyridoxal 5' -phosphate at a final concentration of 0.1. 0.1 mM.
Ec-14, ec-15 and Ec-16 were inoculated at an inoculum size of 1% in LB medium (kanamycin 50. Mu.g/mL, streptomycin 50. Mu.g/mL or ampicillin 100. Mu.g/mL) containing the corresponding antibiotics at 50 mL and shake-cultured overnight at 37℃and 220 rpm. The bacterial liquid obtained was inoculated into 300 mL of LB medium (kanamycin 50. Mu.g/mL) at an inoculum size of 2%, and cultured in a shaker at 37℃and 220 rpm to OD 600 And approximately 0.6. To the bacterial liquid was added IPTG at a final concentration of 0.1 mM, and 6 h was induced in a shaker at 37℃and 2200 rpm. 12000 The cells were collected by centrifugation at rpm for 5 min. Washing the obtained thallus with pure water twice, transferring the thallus to 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with concentration OD 600 And approximately 20. 2.94 g of L-glutamic acid (final concentration of 1. 1M in the reaction system) was weighed into a 100 mL conical flask, and 20. 20 mL bacterial liquid in the centrifuge tube was added into the conical flask to start the product synthesis reaction. The reaction was terminated by adding 500. Mu.L of 80% ethanol to 500. Mu.L of the reaction mixture at 0.5 and 1. 1 h, respectively, and the supernatant was centrifuged at 12000 rpm for 3 min to appropriately dilute the supernatant, followed by HPLC detection. The detection results are shown in FIG. 3. The results show that the strain constructed with the endogenous pyridoxal 5 '-phosphate synthesis pathway is capable of efficiently converting L-glutamic acid to gamma-aminobutyric acid even without exogenously adding pyridoxal 5' -phosphate. At the same time, the reaction rate is significantly higher than that of the control strain. When the reaction time was 0.5. 0.5 h, the product concentration in the control strain system was 0.7. 0.7M, and the product concentrations in the reaction systems of the strains Ec-13, ec-14 and Ec-15 all reached about 1. 1M. This is probably due to the fact that exogenously added pyridoxal 5' -phosphate, although in higher concentrations, does not enter the cell completely to participate in the catalytic reaction. Furthermore, it has been reported that intracellular pyridoxal 5' -phosphate contributes to the expression of glutamate decarboxylase, which increases the amount of enzyme participating in the catalytic reaction in the cell. Based on this, the strain Ec-14 having the highest production of gamma-aminobutyric acid after reaction 1 h was selected as the subsequent production strain.
Coli strain Ec-15 was inoculated at an inoculum size of 1% into 50 mL LB medium (kanamycin 50. Mu.g/mL) and shake cultured overnight at 37℃and 220 rpm. Inoculating the obtained bacterial liquid with 2% of inoculation amountSeed into 300 mL LB medium (kanamycin 50 [ mu ] g/mL), culture in shaker at 37℃at 220 rpm to OD 600 And approximately 0.6. To the bacterial liquid was added IPTG at a final concentration of 0.1 mM, and 6 h was induced in a shaker at 37℃and 2200 rpm. 12000 The cells were collected by centrifugation at rpm for 5 min.
1. Condition optimization for producing gamma-aminobutyric acid by recombinant escherichia coli strain Ec-15 whole cell transformation
(1) Influence of L-glutamic acid with different concentrations on product synthesis is added into a reaction system
Washing the obtained thallus with pure water twice, transferring the thallus to 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with concentration OD 600 And approximately 20. Different amounts of L-glutamic acid (final concentrations of 0.5, 1, 2, 3 and 4M in the reaction system) were weighed into a 100 mL conical flask, and 20 mL bacterial liquid in the centrifuge tube was added into the conical flask to initiate the product synthesis reaction. After reaction 1 h, 500. Mu.L of the reaction mixture was quenched by adding 500. Mu.L of 80% ethanol, and the supernatant was centrifuged at 12000 rpm for 3 min, and the supernatant was diluted appropriately and subjected to HPLC detection. The detection result is shown in fig. 4 (a). The results showed that the yield of gamma-aminobutyric acid gradually increased with increasing substrate L-glutamic acid concentration, whereas the product conversion gradually decreased. When the substrate L-glutamic acid was 1M and below, the conversion was 100%. When the concentration is higher than 1. 1M, the product concentration slightly increases to about 1.2. 1.2M, but the conversion rate drastically decreases to 60% or less. This is probably due to the inhibition of glutamate decarboxylase by the product gamma-aminobutyric acid, i.e. at too high a product concentration, the catalytic activity of glutamate decarboxylase GADZ11 is inhibited and no substrate catalysis is performed.
(2) Influence of the concentration of thallus in the reaction System on the Synthesis of the product
Washing the obtained thallus with pure water twice, transferring the thallus to 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with concentration OD 600 2, 10, 15, 20 and 30, respectively. L-glutamic acid with the final concentration of 6M in the reaction system is weighed and added into a 100 mL conical flask, and 20 mL bacterial liquid in the centrifuge tube is added into the conical flask to start the product synthesis reaction. The reaction was carried out at 37℃and 120 rpm for 2 h, and 500. Mu.L of the reaction mixture was added with an equal volume of 80% ethanol to terminate the reactionAfter the supernatant was collected by centrifugation and diluted appropriately, the concentration of gamma-aminobutyric acid was measured by HPLC. The detection result is shown in FIG. 4 (b). As a result, when the cell amount was OD 600 About 2-15, the yield of product increases in sequence and at OD 600 At 15, a maximum of 1.5. 1.5M is reached. When the amount of the cells is further increased, the yield of the product is decreased.
(3) Influence of the conversion reaction time on the product Synthesis
Washing the obtained thallus with pure water twice, transferring the thallus to 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with concentration OD 600 15. L-glutamic acid with final concentrations of 1, 2, 3 and 4M in the reaction system is weighed and added into a 100 mL conical flask, and 20 mL bacterial liquid in the centrifuge tube is added into the conical flask to start the product synthesis reaction. The reaction was carried out at 37℃and 120 rpm, and after 10, 20, 30, 60 and 90 min, 500. Mu.L of the reaction solution was added with an equal volume of 80% ethanol to terminate the reaction, and after the supernatant was collected by centrifugation and moderately diluted, the concentration of gamma-aminobutyric acid was measured by HPLC. The detection result is shown in FIG. 4 (c). The results show that the yield of product increases with the reaction time at different substrate concentrations. Among them, the conversion rate was the fastest with a substrate of 1M. After 20 min of reaction, the concentration of the gamma-aminobutyric acid can reach 1.0M, namely 100 percent conversion is realized. Whereas for substrates 2, 3 and 4M, 1 h was required to achieve a product concentration of 1M. The optimal duration of the reaction was thus determined to be 30 min. This is a 50% reduction in the reaction time compared to when pyridoxal 5' -phosphate is exogenously added. Indicating that synthesis of endogenous pyridoxal 5' -phosphate is beneficial for the enhancement of the reaction rate.
(4) Influence of the ratio of L-glutamic acid to L-glutamic acid on the Synthesis of the product in the reaction System
The glutamic acid decarboxylase can fully exert the catalytic activity of converting L-glutamic acid into gamma-aminobutyric acid under acidic conditions. But the substrate of L-glutamic acid is expensive, which increases the production cost. However, when low-cost L-sodium glutamate is used as a substrate, a lower conversion rate is caused in a water reaction system, a large amount of acid is required to be added to keep the reaction system acidic, and the operation increases the operation steps and increases the production cost. Therefore, the substrate L-glutamic acid can be replaced by a mixture of L-glutamic acid and sodium L-glutamate, so that the solution can be kept in an acidic condition, the conversion reaction can be carried out, and the production cost can be reduced.
Washing the obtained thallus with pure water twice, transferring the thallus to 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with concentration OD 600 And approximately 20. L-glutamic acid and sodium L-glutamate (1:0, 4:1, 3:2, 1:1, 2:3, 1:4 and 0:1) with different proportions of total final concentration of 1M are weighed into a 100 mL conical flask, and 20 mL bacterial liquid in the centrifuge tube is added into the conical flask to start a product synthesis reaction. After the reaction was carried out at 37℃and 120 rpm for 0.5. 0.5 h, 500. Mu.L of the reaction mixture was added with an equal volume of 80% ethanol to terminate the reaction, and the supernatant was collected by centrifugation and diluted appropriately, and then the concentration of gamma-aminobutyric acid was measured by HPLC. The detection result is shown in FIG. 4 (d). The result shows that when 20% -40% of L-glutamic acid in the reaction system is replaced by sodium L-glutamate, no obvious influence is caused on the production of gamma-aminobutyric acid, and the conversion rate is over 97.7%. When the proportion is increased to more than 50%, the concentration of the gamma-aminobutyric acid is reduced to less than 0.8 and M. Thus, a mixture of 0.6M L-glutamic acid and 0.4M sodium L-glutamate was selected for gamma-aminobutyric acid production by adding to the transformation system.
2. Efficient production of gamma-aminobutyric acid by recombinant E.coli strain Ec-15
Washing the thallus obtained after culture induction with pure water twice, transferring the thallus into 50 mL centrifuge tube, adding water 20 mL for resuspension to obtain bacterial liquid with OD concentration 600 And approximately 15. L-glutamic acid and sodium L-glutamate with final concentrations of 0.6M and 0.4M in the reaction system are weighed and added into a 100 mL conical flask, and 20 mL bacterial liquid in the centrifuge tube is added into the conical flask to start the product synthesis reaction. After the reaction was carried out at 37℃and 120 rpm for 0.5. 0.5 h, the reaction mixture was centrifuged, and the cells were collected, washed twice with clean water, and then added again to the same reaction system to carry out the synthesis reaction of gamma-aminobutyric acid. The cells of the engineering strain in the same batch are utilized for 8 times. The product concentration and conversion per reaction were determined and are shown in FIG. 5. The results show that the yield and conversion rate of the gamma-aminobutyric acid product in each batch are both above 0.9 and M and 96 percent. Indicating bacteriaThe body can be reused, which is beneficial to saving the production cost. The comprehensive experimental result shows that the strain can produce gamma-aminobutyric acid with the concentration of more than 820 g/L in 4 h, the production rate reaches 205 g/L/h, and the strain has extremely high industrialized application prospect.
The above embodiments are only used for explaining the technical solution of the present application, and do not limit the protection scope of the present application.
Claims (7)
1. A method for constructing recombinant escherichia coli engineering bacteria for efficiently producing gamma-aminobutyric acid comprises the following steps:
constructing mutant escherichia coli chassis cells with gamma-aminobutyric acid synthesis capacity by utilizing an inducible promoter to heterologously express a coding gene of glutamic acid decarboxylase from bacillus Z11 in escherichia coli strains;
in the obtained mutant escherichia coli chassis cells with the gamma-aminobutyric acid synthesis capability, the encoding genes of pyridoxal 5 '-phosphate synthase and the encoding genes of glutamine aminotransferase derived from bacillus subtilis 168 are respectively subjected to heterologous expression by using an inducible promoter, and an endogenous pyridoxal 5' -phosphate synthesis way is constructed, so that recombinant escherichia coli engineering bacteria for efficiently producing gamma-aminobutyric acid are obtained.
2. The method for constructing a recombinant escherichia coli engineering bacterium for efficiently producing gamma-aminobutyric acid according to claim 1, wherein the inducible promoter is a T7 promoter.
3. The recombinant escherichia coli engineering bacteria for efficiently producing the gamma-aminobutyric acid are characterized by being constructed by the following steps:
constructing mutant escherichia coli chassis cells with gamma-aminobutyric acid synthesis capacity by utilizing an inducible promoter to heterologously express a coding gene of glutamic acid decarboxylase from bacillus Z11 in escherichia coli strains;
In the obtained mutant escherichia coli chassis cells with the gamma-aminobutyric acid synthesis capability, the encoding genes of pyridoxal 5 '-phosphate synthase and the encoding genes of glutamine aminotransferase derived from bacillus subtilis 168 are respectively subjected to heterologous expression by using an inducible promoter, and an endogenous pyridoxal 5' -phosphate synthesis way is constructed, so that recombinant escherichia coli engineering bacteria for efficiently producing gamma-aminobutyric acid are obtained.
4. A method for producing gamma-aminobutyric acid by whole cell transformation, the method comprising the steps of:
inducing recombinant escherichia coli engineering bacteria for efficiently producing gamma-aminobutyric acid according to claim 3 through shaking;
and adding a whole cell transformation substrate, and carrying out whole cell transformation to produce gamma-aminobutyric acid, wherein the whole cell transformation substrate is a mixture of L-glutamic acid and sodium L-glutamate.
5. The method for producing gamma-aminobutyric acid by whole cell transformation according to claim 4, wherein the induction time of shaking bottle is 37 ℃, the rotation speed is 220 rpm, and the concentration of inducer IPTG is 0.1 mM.
6. The method for producing gamma-aminobutyric acid by whole cell transformation according to claim 4, wherein the condition of whole cell transformation is a temperature of 37 ℃ and a rotation speed of 120 rpm.
7. The method for producing gamma-aminobutyric acid by whole cell transformation according to claim 4, wherein the whole cell transformation substrate is a mixture of L-glutamic acid and sodium L-glutamate having a total concentration of 1M, comprising 0.6M L-glutamic acid and 0.4M sodium L-glutamate.
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