CN116732081A - Method for improving bacillus subtilis to synthesize menatetrenone, obtained recombinant strain and application thereof - Google Patents

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

A method for improving the synthesis of heptamenadione by Bacillus subtilis and obtaining recombinant bacteria Strains and their applications

Technical field

The invention relates to the field of biotechnology, and specifically relates to a method for improving the synthesis of menadione by Bacillus subtilis , obtaining a recombinant strain and its application.

Background technique

Menaquinone (MK-7) consists of the main ring of menaquinone and the isoprene side chain of 7 isoprene units connected at the C-3 position of the main ring. MK-7 has a variety of physiological functions on the human body, has good effects in protecting bone health, preventing cardiovascular diseases, etc., and is used in the treatment of diseases such as diabetes, chronic kidney disease, immune disorders, and Alzheimer's disease. plays a key role. As a subtype of vitamin K ₂ (Vitamin K ₂ ), MK-7 has the advantages of high affinity and long half-life in the human body.

As a food safety-grade model organism, Bacillus subtilis has the advantages of non-pathogenicity, clear genetic background, no codon preference, and complete genetic manipulation tools. It has been widely used in biosynthesis and metabolic engineering. research in the field. Bacillus subtilis is the main producer of MK-7. The metabolic process of MK-7 in Bacillus subtilis is mainly divided into four modules: glycerol dissimilation pathway, SA pathway, MEP pathway and MK-7 pathway. After Bacillus subtilis ingests glycerol, it synthesizes glyceraldehyde-3-phosphate (G3P), pyruvate (PYR) and phosphoenolpyruvate (PEP) through the glycerol dissimilation pathway and glycolysis pathway. PYR and G3P are synthesized through the MEP pathway. Heptasoprenyl pyrophosphate (HDP) provides the isoprene side chain for the synthesis of MK-7. PEP and erythrose-4-phosphate (E4P) enter the SA pathway to synthesize chorismate (CHA), and then synthesize 1, 4-dihydroxy-2-naphthoic acid (DHNA) into MK through the seven-step enzymatic reaction of the MK-7 pathway. The synthesis of -7 provides the menadione parent ring, and finally HDP and DHNA are catalyzed by 1,4-dihydroxy-2-naphthoate heptadienyl transferase (menA) and methyltransferase (menG) to finally generate MK- 7. The MEP pathway is the main synthesis pathway of terpenes in bacteria. HDP, the key precursor of MK-7, needs to be synthesized by the cascade amplification reaction of IPP. In the isoprenol synthesis pathway, the nuclear distributed protein encoded by the nudF gene catalyzes the synthesis of two isoprenols, which are isomers of each other, by IPP and DMAPP. This competes with the MK-7 synthesis pathway. Knocking out nudF reduces the consumption of the key intermediate metabolite DMAPP and contributes to the synthesis of HDP, the key precursor of MK-7. MK-7 serves as an electron acceptor in the electron transport chain of Bacillus subtilis and carries the function of transporting electrons, which is of great significance to the life activities of Bacillus subtilis. Strengthening the electron supply of the cell electron transport chain to promote MK-7 synthesis is one of the engineering directions for Bacillus subtilis to synthesize MK-7. The reduced form of flavin coenzyme FMNH ₂ realizes electron transfer by transferring hydrogen atoms to MK-7. transmission on the cell membrane, thereby forming a series of aerobic respiratory activities of Bacillus subtilis. The phenolic acid decarboxylase encoded by the yclB gene catalyzes the amylation process of FMNH2. The consumption of FMNH2 reduces the supply of electrons in the electron transport chain. The knockout of the yclB gene helps enhance the supply of electrons in the electron transport chain of cells.

Although blocking branch pathways in the product synthesis pathway is a common transformation strategy in MK-7 research, the two genes nudF and yclB do not directly appear in the MK-7 synthesis pathway, and by knocking out nudF and yclB There are no relevant research reports on using genes to promote the synthesis of MK-7.

Contents of the invention

Therefore, the present invention conducts a comprehensive analysis of the common intermediate metabolic precursors and electron transfer shared receptors in the strain, selects the genes nudF and yclB as the target points for transformation, and adopts the method of knocking out the branched metabolic pathway genes nudF and yclB respectively or simultaneously. Methods to increase the metabolic flux of the MK-7 synthesis pathway and strengthen the electron supply of the cellular electron transport chain, thereby promoting the synthesis of MK-7 in Bacillus subtilis.

The present invention first provides a method for improving the synthesis of heptamenadione by Bacillus subtilis. The method is achieved by knocking out one or both of the nudF and yclB genes in Bacillus subtilis.

Preferably, the method is achieved by simultaneously knocking out nudF and yclB genes in Bacillus subtilis.

In a specific embodiment, the Genebank ID of the nudF gene is 938719, and the Genebank ID of the yclB gene is 938296.

In a preferred embodiment, the Bacillus subtilis is wild-type Bacillus subtilis. 168 (abbreviated as BS168), and the dhbB, trpE and aroH genes in the branch metabolic pathway of the shikimate pathway are knocked out, and the glycerol dissimilation pathway is knocked out. mgsA gene in branched metabolic pathways.

Specifically, the knockout is achieved using CpfI gene editing or cre/lox recombination system.

The present invention also provides a recombinant strain of Bacillus subtilis that can efficiently synthesize heptamenadione obtained by the method.

The present invention further provides the application of the recombinant strain of Bacillus subtilis that can synthesize heptamenaquinone with high efficiency in the production of heptamenaquinone.

The present invention therefore provides a method for producing heptamenaquinone by culturing the Bacillus subtilis recombinant strain that efficiently synthesizes heptamenaquinone to produce heptamenaquinone.

Specifically, the culture conditions are continuous fermentation in a shaker at 38-42°C and 150-250 rpm for 2-6 days. Optionally, further comprising collecting the heptamenadione by cell disruption and extraction of the fermentation broth.

By knocking out the nudF and yclB genes of Bacillus subtilis, the present invention reduces the consumption of DMAPP and at the same time strengthens the electron supply of the cell electron transport chain, promotes the synthesis of the precursor of MK-7 and enhances the electron supply, thereby improving the efficient synthesis of heptamenadione. the goal of. Experiments have shown that the recombinant strains obtained by knocking out nudF and yclB respectively or simultaneously in the present invention can increase the production of heptamenadione to 204%, 183%, and 260% of wild-type BS168, respectively, and therefore have great practical value. , which is beneficial to reducing the cost of producing heptamenadione.

Description of drawings

Figure 1 shows the fermentation production of MK-7 by BS168, BS1, BS2, BS3, and BS4 recombinant strains after 96 hours of fermentation.

Detailed ways

In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments. However, it should be noted that the embodiments do not limit the scope of protection claimed by the present invention.

The methods in the following examples are all conventional methods unless otherwise specified.

Strain growth conditions: Bacillus subtilis was grown in LB liquid medium at 37°C, 200 rpm, and in fermentation medium at 40°C, 200 rpm. The components of the LB medium include 5 g/L yeast extract, 10 g/L peptone, and 10 g/L NaCl. On this basis, the solid medium is added with 17.5 g/L agar; the components of the fermentation medium include 30 g/L glycerol. , soy peptone 60 g/L, yeast extract 5 g/L, K ₂ HPO ₄ 3 g/L, MgSO ₄ 7H ₂ O 0.5 g/L. Liquid medium and agar plates were supplemented with 50 μg/ml kanamycin or 100 μg/ml spectinomycin, respectively.

Cell disruption and MK-7 extraction: Take 5 mL of fermentation broth in a 15 mL centrifuge tube, add 500 μl of lysozyme buffer, and then add the lysozyme aqueous solution to a final concentration of 20 mg/L, and place it on a shaker at 37°C. , incubate at 200 rpm for 1 hour. Then add 5 ml of 15% HCl to further disrupt the cells, boil in boiling water for 5 min, cool slightly and then add isopropanol and n-hexane extractants (1:2, V/V).

UPLC detection of MK-7 production: Use a C18 separation column, column temperature at 25°C, methanol as the mobile phase, flow rate 0.5 mL/min, detection wavelength 254 nm, and injection volume 3 μL.

Example 1: Construction of recombinant strains of Bacillus subtilis BS2 and BS3

The nudF gene and yclB gene on the starting strain BS1 were knocked out respectively through the cre/lox recombination system, and the recombinant strains BS2 and BS3 of Bacillus subtilis were constructed. The specific construction method is as follows:

The starting strain BS1 is Bacillus subtilis. 168ΔdhbBΔTrpEΔaroHΔmgsA . The starting strain is based on the wild-type Bacillus subtilis. 168 preserved in the laboratory and uses the CpfⅠ gene editing tool to knock out the branch metabolic pathway genes dhbB, TrpE and aroH of the shikimate pathway respectively. , a recombinant strain obtained by using the cre/lox recombination system to knock out the branch metabolic pathway gene mgsA of the glycerol dissimilation pathway. On the basis of the starting strain BS1, the gene manipulation tool cre/lox recombination system of Bacillus subtilis was used to knock out the branch pathway genes nudF and yclB respectively. For the method, please refer to the article (Radeck J, Kraft K, Bartels J, et al. The Bacillus BioBrickBox: generation and evaluation of essential genetic building blocks forstandardized work with Bacillus subtilis. [J]J Biol Eng. 2013 Dec 2;7(1):29.), the specific method is as follows: using the Bacillus subtilis BS168 genome as a template The upstream homology arm F1 and the downstream homology arm F3 of the nudF and yclB genes were amplified respectively to obtain gene fragments nudF-F1, nudF-F3 and yclB-F1, yclB-F3; p7S6 plasmid was used as a template to amplify lox71-Spe- lox66 gene knockout cassette, resulting in nudF-F2 and yclB-F2. Overlap extension PCR was performed on nudF-F1, nudF-F2, nudF-F3 and yclB-F1, yclB-F2 and yclB-F3 respectively. After the ligation products were purified and recovered, two fusion gene clone fragments nudF-F1-lox71-spe were obtained. -lox66-nudF-F3 and yclB-F1-lox71-spe-lox66-yclB-F3. The two fusion gene fragments were phosphorylated and self-ligated respectively, then transferred into the competent state of BS1 and spread on spectinomycin-resistant plates to screen for positive transformants. The BS1 competent state was prepared by the Spizizen two-step method. For specific methods, please refer to the article (Spizizen J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate[J]. Proc Natl Acad Sci USA, 1958, 44:1072-1075.).

Single colonies were picked for PCR verification and sequencing verification to confirm that the fusion gene fragment was successfully integrated into BS1. The p148cre plasmid was transferred into the successfully integrated recombinant Bacillus subtilis to eliminate spectinomycin resistance, and the p148cre plasmid was transferred into the successfully integrated recombinant Bacillus subtilis through chemical transformation (Radeck J, Kraft K, BartelsJ, et al . The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. [J]JBiol Eng. 2013 Dec 2;7(1):29.), adding IPTG to induce cre gene expression makes both lox71 and lox66 Recombine at each site and screen positive clones using kanamycin-resistant plates. The p148cre plasmid was eliminated by picking positive transformants and inoculating them into LB liquid culture medium, adding 0.05% SDS and culturing at 51°C and 200 rpm for 12 hours, streaking onto the LB plate, picking single colonies and copying them onto the LB plate and K50 Plate to ensure successful elimination of p148cre plasmid. Finally, the recombinant Bacillus subtilis strain BS2 with the nudF gene knocked out on the basis of BS1 and the Bacillus subtilis recombinant strain BS3 with the yclB gene knocked out on the basis of BS1 were finally obtained.

Example 2: Construction of recombinant strain BS4 of Bacillus subtilis

Based on the recombinant strain BS2 obtained in Example 1, a method similar to Example 1 was used to integrate the yclB-F1-lox71-spe-lox66-yclB-F3 gene knockout cassette into the genome of BS2. After spectinomycin resistance screening, colony PCR verification, sequencing verification, transfer of p148cre plasmid, kanamycin resistance screening, IPTG induction, p148cre plasmid elimination, and plate copy verification, we finally obtained simultaneous knockout in the genome of the starting strain BS1. Bacillus subtilis recombinant strain BS4 with nudF gene and yclB gene.

Oligonucleotide sequences used in the examples of Table 1

Example 3: Fermentation production of MK-7 by recombinant strain

Preparation of seed liquid

Streak the starting strains BS168, BS1, and the recombinant strains BS2, BS3, and BS4 constructed in Examples 1 and 2 onto the LB plate, select single colonies and inoculate them into the LB medium for activation for 14 hours to obtain a seed liquid.

fermentation culture

The seed liquid obtained in step (1) was inoculated into 30 ml of fermentation medium at an initial OD of 0.1 for shake flask fermentation. The wild-type strain BS168 and the starting strain BS1 were used as controls, and three parallels were made for each strain. After continuous fermentation in a shaker at 40°C and 200 rpm for 4 days, the production of MK-7 was detected. At the same time, the fermentation broth was diluted 40 times every 24 hours and the biomass of the strain was measured using a UV-spectrophotometer. The 96h fermentation broth was subjected to cell disruption and extraction and then UPLC was used to measure the production of MK-7.

The results showed that compared with the wild-type strain BS168, the production of the starting strain BS1 increased to 125% of that of BS168. In addition, on the basis of BS1, nudF was deleted to obtain the BS2 recombinant strain, yclB was deleted to obtain the BS3 recombinant strain, and nudF and yclB were simultaneously deleted to obtain the BS4 recombinant strain, which increased the MK-7 production of the recombinant strains BS2, BS3, and BS4 to 204%, 183%, and 260% of wild-type strain BS168.

Claims (10)

1. Improve bacillus subtilis @Bacillus subtilis) A method for synthesizing menatetrenone, which is characterized in that one or two of nudF and yclB genes are knocked out in bacillus subtilis.

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 or 2, wherein the bacillus subtilis is wild-typeBacillus subtilis. 168, the dhbB, trpE and aroH genes in the branch metabolic pathway of the shikimate pathway and the mgsA gene in the branch metabolic pathway of the glycerol catabolism pathway were knocked out.

5. The method of claim 4, wherein the knockout is effected using Cpf i gene editing or cre/lox recombination systems.

6. A recombinant strain of bacillus subtilis for the efficient synthesis of menatetrenone obtained by the method according to any one of claims 1 to 5.

7. The use of a recombinant strain of bacillus subtilis for the efficient synthesis of menatetrenone according to claim 6 for the production of menatetrenone.

8. A method of producing menatetrenone, characterized by culturing the recombinant strain of bacillus subtilis for efficient synthesis of menatetrenone of claim 6 to produce menatetrenone.

9. The method of claim 8, wherein the culture conditions are continuous fermentation in a shaker at 38-42℃and 150-250 rpm for 2-6 days.

10. The method of claim 8 or 9, further comprising collecting menatetrenone by cell disruption and extraction of the fermentation broth.