CN116732081B - Method for improving bacillus subtilis to synthesize menatetrenone, obtained recombinant strain and application thereof - Google Patents
Method for improving bacillus subtilis to synthesize menatetrenone, obtained recombinant strain and application thereof Download PDFInfo
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 43
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 43
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- 229960005481 menatetrenone Drugs 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 23
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- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
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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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
The invention discloses a method for improving bacillus subtilis to synthesize menatetrenone, an obtained recombinant strain and application thereof. According to the invention, through knocking out nudF and yclB genes of bacillus subtilis, DMAPP consumption is reduced, and meanwhile, electron supply of a cell electron transfer chain is enhanced, the synthesis of a precursor of MK-7 is promoted, and the electron supply is enhanced, so that the purpose of improving efficient synthesis of menatetrenone is achieved. Experiments show that the recombinant strain obtained by knocking out nudF and yclB respectively or simultaneously has the yield of the menatetrenone increased to 204%, 183% and 260% of that of the wild BS168 respectively, so that the recombinant strain has a high practical value and is beneficial to reducing the cost of the production of the menatetrenone.
Description
Technical Field
The invention belongs to the technical field of biology, in particular to a method for improving bacillus subtilisBacillus subtilis) A method for synthesizing menatetrenone, a recombinant strain obtained by the method and application thereof.
Background
Menatetrenone (MK-7) consists of the menatetrenone parent ring and the isoprene side chain of 7 isoprene units attached at the C-3 position of the parent ring. MK-7 has various physiological functions on human body, has good effects on protecting bone health, preventing cardiovascular diseases and the like, and plays a key role in treatment schemes of diabetes, chronic kidney diseases, immune disorders, alzheimer's disease and other diseases. As vitamin K 2 （Vitamin K 2 ) MK-7 has the advantages of high affinity, long half-life and the like in human bodies.
Bacillus subtilis @Bacillus subtilis) As a food-safe model organism, the gene expression vector has the advantages of non-pathogenicity, clear genetic background, no codon preference and gene manipulation tool Ji Quandeng, and is widely applied to the research in the fields of biosynthesis and metabolic engineering at present. Bacillus subtilis is the main producer of MK-7, and the metabolic process of MK-7 in Bacillus subtilis is mainly divided into four modules: the glycerol catabolism pathway, the SA pathway, the MEP pathway, and the MK-7 pathway. Glyceraldehyde-3 phosphate (G3P), pyruvic acid (PYR) and phosphoenolpyruvic acid (PEP) are synthesized by bacillus subtilis after taking glycerol through a glycerol catabolism pathway and a glycolysis pathway, and synthetic heptaprenyl pyrophosphoric acid (HDP) of PYR and G3P through a MEP pathway provides an isoprene side chain for MK-7 synthesis. PEP and erythrose-4-phosphate (E4P) enter an SA path to synthesize Chorismate (CHA), then a seven-step enzyme catalytic reaction is performed on the PEP and erythrose-4-phosphate (E4P) to synthesize 1, 4-dihydroxyl-2-naphthoic acid (DHNA) through an MK-7 path to provide a menaquinone parent ring for MK-7 synthesis, and finally HDP and DHNA are catalyzed by 1, 4-dihydroxyl-2-naphthoic acid heptadienyl transferase (menA) and methyltransferase (menG) to finally generate MK-7. The MEP pathway is the main synthesis pathway of terpenes in bacteria, and the key precursor of MK-7, HDP, needs to be synthesized by means of the cascade amplification of IPP. In the prenyl alcohol synthesis pathway, nuclear distribution protein encoded by nudF gene catalyzes IPP and DMAPP to synthesize two isopentenyl alcohols which are isomers respectively, which form a competitive relationship with the MK-7 synthesis pathway, and the consumption of a key intermediate metabolite DMAPP is reduced by knocking out nudF, so that the synthesis of an MK-7 key precursor HDP is facilitated. MK-7 plays a role in transporting electrons as an electron acceptor in an electron transfer chain of bacillus subtilis, and has important significance on life activities of bacillus subtilis. Enhancement of electron supply of cellular electron transfer chain to promote MK-7 synthesis is one of the engineering directions for Bacillus subtilis to synthesize MK-7, reduced form of flavin-coenzyme FMNH 2 The transfer of electrons on cell membranes is achieved by the transfer of a hydrogen atom to MK-7, thus creating a series of aerobic respiratory events in Bacillus subtilis. Whereas phenolic acid decarboxylase encoded by yclB catalyzes the amyl process of FMNH2, consumption of FMNH2 cuts down the electricity in the electron transfer chainThe knockout of the yclB gene helps to enhance the electron supply in the electron transport chain of the cell.
Although blocking the branching pathways in the product synthesis pathway is a common engineering strategy in the study of MK-7, both nudF and yclB genes do not occur directly in the MK-7 synthesis pathway, and there is no study report on the promotion of MK-7 synthesis by knocking out the nudF and yclB genes.
Disclosure of Invention
Therefore, the invention adopts the method of knocking out or knocking out the branch metabolic pathway genes nudF and yclB respectively to increase the metabolic flux of MK-7 synthetic pathway and strengthen the electron supply of cell electron transfer chain by comprehensively analyzing the public intermediate metabolic precursor and electron transfer common receptor in the strain and selecting the genes nudF and yclB as reconstruction targets, thereby promoting the synthesis of MK-7 in bacillus subtilis.
The invention firstly provides a method for improving the synthesis of menatetrenone by bacillus subtilis, which is realized by knocking out one or two of nudF and yclB genes in bacillus subtilis.
Preferably, the method is carried out by simultaneously knocking out nudF and yclB genes in Bacillus subtilis.
In a specific embodiment, the nudF gene has a Genebank ID of 938719 and the yclB gene has a Genebank ID of 938296.
In a preferred embodiment, the Bacillus subtilis starting material is wild-typeBacillus subtilis. 168 (abbreviated as BS 168) in the shikimate pathway, the dhbB, trpE and aroH genes in the branch metabolic pathway, and the mgsA gene in the branch metabolic pathway of the glycerol catabolism pathway.
Specifically, the knockout is achieved by CpfI gene editing or cre/lox recombination systems.
The invention also provides a bacillus subtilis recombinant strain for efficiently synthesizing menatetrenone, which is obtained by the method.
The invention further provides application of the bacillus subtilis recombinant strain for efficiently synthesizing menatetrenone in production of menatetrenone.
The present invention thus provides a method for producing menatetrenone by culturing said recombinant strain of bacillus subtilis which is highly efficient in the synthesis of menatetrenone to produce menatetrenone.
Specifically, the culture conditions are continuous fermentation in a shaker at 38-42℃and 150-250 rpm for 2-6 days. Optionally, further comprising collecting menatetrenone by cell disruption and extraction of the fermentation broth.
According to the invention, through knocking out nudF and yclB genes of bacillus subtilis, DMAPP consumption is reduced, and meanwhile, electron supply of a cell electron transfer chain is enhanced, the synthesis of a precursor of MK-7 is promoted, and the electron supply is enhanced, so that the purpose of improving efficient synthesis of menatetrenone is achieved. Experiments show that the recombinant strain obtained by knocking out nudF and yclB respectively or simultaneously has the yield of the menatetrenone increased to 204%, 183% and 260% of that of the wild BS168 respectively, so that the recombinant strain has a high practical value and is beneficial to reducing the cost of the production of the menatetrenone.
Drawings
FIG. 1 is a graph showing the yield of MK-7 produced by fermentation of recombinant strains BS168, BS1, BS2, BS3, and BS4 after 96h of fermentation.
Detailed Description
The invention is further described in connection with the following embodiments in order to make the technical means, the creation features, the achievement of the purpose and the effect of the invention easy to understand. It should be noted, however, that the examples are not intended to limit the scope of the invention as claimed.
The methods in the following examples are conventional methods unless otherwise specified.
Strain growth conditions: bacillus subtilis was grown in LB liquid medium at 37℃at 200 rpm and in fermentation medium at 40℃at 200 rpm. The LB culture medium comprises 5 g/L yeast extract, 10 g/L peptone and 10 g/L NaCl, and 17.5 g/L agar is added into the solid culture medium; the fermentation medium comprises glycerol 30 g/L, soybean peptone 60 g/L, yeast extract 5 g/L, and K 2 HPO 4 3 g/L，MgSO 4 7H 2 O0.5 g/L. Liquid culture mediumAnd agar plates were supplemented with 50. Mu.g/ml kanamycin or 100. Mu.g/ml spectinomycin, respectively.
Cell disruption and extraction of MK-7: the 5 mL fermentation broth was placed in a 15 mL centrifuge tube, 500. Mu.l of lysozyme buffer was added, and then the lysozyme solution was added to a final concentration of 20 mg/L, and the mixture was placed in a shaker at 37℃and incubated at 200 rpm for 1 hour. Then 5 ml of 15% HCl was added to further break up the cells, boiled in boiling water for 5 min, cooled slightly and then isopropanol and n-hexane extractant (1:2, V/V) were added.
UPLC detection MK-7 yield: the C18 separation column is adopted, a column temperature box is 25 ℃, methanol is used as a mobile phase, the flow rate is 0.5 mL/min, the detection wavelength is 254 nm, and the sample injection amount is 3 mu L.
Example 1: construction of recombinant strains BS2 and BS3 of Bacillus subtilis
nudF gene and yclB gene on the original strain BS1 are respectively knocked out through a cre/lox recombination system, and bacillus subtilis recombination strains BS2 and BS3 are constructed. The specific construction method is as follows:
the starting strain BS1 isBacillus subtilis. 168ΔdhbBΔTrpEΔaroHΔmgsAThe starting strain is a wild type strain deposited in a laboratoryBacillus subtilis. 168, the branch metabolic pathway genes dhbB, trpE and aroH of the shikimate pathway were knocked out respectively by using a CpfI gene editing tool, and the recombinant strain obtained by knocking out the branch metabolic pathway gene mgsA of the glycerol catabolism pathway by using a cre/lox recombination system. Based on the starting strain BS1, the gene manipulation tools cre/lox recombination systems of Bacillus subtilis were used to knock out the branching pathway genes nudF and yclB, respectively, as described in the article (Radeck J, kraft K, bartels J, et al The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis [ J.)]J Biol Eng.2013 Dec 2;7 (1): 29.) the following procedure is followed: respectively amplifying an upstream homology arm F1 and a downstream homology arm F3 of nudF and yclB genes by taking a bacillus subtilis BS168 genome as a template to obtain gene fragments nudF-F1, nudF-F3, yclB-F1 and yclB-F3; the lox71-Spe-lox66 gene knockout cassette was amplified using the p7S6 plasmid as a template to obtain nudF-F2 and yclB-F2. nudF-F1 and nudF-F are respectively2. And carrying out overlap extension PCR on nudF-F3, yclB-F1, yclB-F2 and yclB-F3, and purifying and recovering a connection product to obtain two fusion gene clone fragments nudF-F1-lox71-spe-lox66-nudF-F3 and yclB-F1-lox71-spe-lox66-yclB-F3. The two fusion gene fragments were phosphorylated separately, transferred into BS1 competence and spread onto spectinomycin resistant plates for selection of positive transformants. BS1 competence was prepared by the Spizizen two-step method, see article (Spizizen J Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate [ J)]. Proc Natl Acad Sci USA, 1958, 44: 1072-1075.）。
And (3) selecting a single colony for PCR verification and sequencing verification, and confirming that the fusion gene fragment is successfully integrated into the BS 1. Transfer of p148cre plasmid to eliminate spectinomycin resistance of recombinant Bacillus subtilis which was successfully integrated, transfer of p148cre plasmid to recombinant Bacillus subtilis which was successfully integrated by means of chemical transformation (Radeck J, kraft K, bartels J, et al The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus sublis [ J ] J Biol Eng.2013Dec2; 7 (1): 29), addition of IPTG to induce cre gene expression to recombine two sites lox71 and lox66, and screening of positive clones by means of kanamycin resistant plates. Elimination of p148cre plasmid was successfully eliminated by picking positive transformants and inoculating them into LB liquid medium, adding 0.05% SDS at 51℃and culturing at 200 rpm for 12 h, streaking onto LB plates, and picking single colony replica onto LB plates and K50 plates. Finally, the bacillus subtilis recombinant strain BS2 with nudF gene knocked out on the basis of BS1 and the bacillus subtilis recombinant strain BS3 with yclB gene knocked out on the basis of BS1 are obtained.
Example 2: construction of recombinant strain BS4 of Bacillus subtilis
The yclB-F1-lox71-spe-lox66-yclB-F3 gene knockout cassette was integrated into the genome of BS2 in a similar manner to example 1 on the basis of the recombinant strain BS2 obtained in example 1. Through spectinomycin resistance screening, colony PCR verification, sequencing verification, p148cre plasmid transfer, kanamycin resistance screening, IPTG induction, p148cre plasmid elimination and plate photocopying verification, the bacillus subtilis recombinant strain BS4 with nudF gene and yclB gene knocked out on the genome of the original strain BS1 is finally obtained.
Table 1 oligonucleotide sequences used in the examples
Example 3: recombinant strain fermentation production of MK-7
Preparation of seed liquid
Starting strains BS168 and BS1 and recombinant strains BS2, BS3 and BS4 constructed in example 1 and example 2 are streaked on LB plates, and single colonies are inoculated into LB culture medium for activation for 14 hours to obtain seed liquid.
Fermentation culture
Inoculating the seed solution obtained in the step (1) into a fermentation medium of 30 ml according to an initial OD of 0.1 for shake flask fermentation, and taking a wild-type strain BS168 and a starting strain BS1 as a control, wherein three strains are parallel to each other. MK-7 production was measured after 4 days of continuous fermentation in a shaker at 40℃and 200 rpm, while the biomass of the strain was measured using an ultraviolet-spectrophotometer after 40-fold dilution of the fermentation broth every 24 hours. After 96h fermentation broth was cell disrupted and extracted, MK-7 production was measured using UPLC.
The results show that: the yield of the starting strain BS1 was increased to 125% of BS168 compared to the wild-type strain BS 168. In addition, on the basis of BS1, nudF is knocked out to obtain a BS2 recombinant strain, yclB is knocked out to obtain a BS3 recombinant strain, and simultaneously nudF and yclB are knocked out to obtain a BS4 recombinant strain, so that MK-7 yield of the recombinant strains BS2, BS3 and BS4 is respectively increased to 204%, 183% and 260% of that of a wild strain BS 168.
Claims (9)
1. A method for improving the synthesis of menatetrenone by bacillus subtilis (Bacillus subtilis), which is characterized in that one or two of nudF and yclB genes are knocked out in the starting bacillus subtilis;
wherein the bacillus subtilis is wild Bacillus subtilis strain, and the dhbB, trpE and aroH genes in the branch metabolic pathway of the shikimate pathway are knocked out, and the mgsA gene in the branch metabolic pathway of the glycerol catabolism pathway is knocked out.
2. The method of claim 1, wherein said method is carried out by simultaneously knocking out nudF and yclB genes in bacillus subtilis.
3. The method of claim 1, wherein the nudF gene has a Genebank ID of 938719 and the yclB gene has a Genebank ID of 938296.
4. The method of claim 1, wherein the knockout is effected using Cpf i gene editing or cre/lox recombination systems.
5. A recombinant strain of Bacillus subtilis for the efficient synthesis of menatetrenone obtained by the process according to any one of claims 1 to 4.
6. The use of a recombinant strain of bacillus subtilis for the efficient synthesis of menatetrenone according to claim 5 for the production of menatetrenone.
7. A method of producing menatetrenone, characterized by culturing the recombinant strain of bacillus subtilis for efficient synthesis of menatetrenone of claim 5 to produce menatetrenone.
8. The method of claim 7, wherein the culturing conditions are continuous fermentation in a shaker at 38-42℃and 150-250 rpm for 2-6 days.
9. The method of claim 7 or 8, further comprising collecting menatetrenone by cell disruption and extraction of the fermentation broth.
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