A Bacillus subtilis strain, a recombinant Bacillus subtilis strain and its application

Abstract

The present invention provides a Bacillus subtilis, a recombinant Bacillus subtilis and an application thereof, and belongs to the technical field of microbial fermentation. The Bacillus subtilis RF1-6 of the present invention is a mutant strain with the highest riboflavin yield obtained by genetic modification and mutagenesis, using the riboflavin high-yield strain RF1 as a starting strain, and the deposit number is CCTCCNO: M2022565. Compared with the riboflavin high-yield strain RF1, the riboflavin yield can be increased by 22.8%.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a Bacillus subtilis, a recombinant Bacillus subtilis and applications thereof. Background Art

[0002] Riboflavin, also known as vitamin B2, is a unique water-soluble vitamin with the property of being easily decomposed by light. It was first isolated from whey in 1879 and named milk pigment. Riboflavin can be crystallized into orange-yellow crystals, but its pure form is difficult to dissolve in water, soluble in sodium chloride solution, easily soluble in dilute sodium hydroxide solution, easily soluble in alkaline solution, and stable in strong acid solution. It is mainly synthesized by plants and microorganisms and is an important animal nutrient. It is an important nutrient for animals and needs to be obtained from the outside. Riboflavin is converted into two active substances in the animal body: flavin adenine dinucleotide (FAD) and flavin mononucleonucleotide (FMN), which act as cofactors of oxidoreductase oxidation pathway enzymes and participate in a series of redox reactions, some of which are essential for the function of aerobic cells. When it is deficient, it affects the body's biological oxidation and causes metabolic disorders. Its lesions are mostly manifested as inflammation in the mouth, eyes and external genitalia.

[0003] Riboflavin is mainly used in industries such as medicine, food additives, feed processing, and is also used for clinical cancer treatment and prevention. At present, microbial fermentation is widely used for industrial production of riboflavin at home and abroad. The microorganisms that can synthesize riboflavin include bacteria, fungi and molds. In industrial production, Bacillus subtilis and Ashu pseudocysts are mainly used as production strains. The highest yield of riboflavin synthesized by Bacillus subtilis reported in the literature is 15.7g / L (see [Wang, Z., Chen, T., Ma, X., Shen, Z. and Zhao, X. Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum [J]. Bioresource Technology, 2011, 102 (4): 3934-3940]), but this yield still cannot meet the demand for industrial production. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a Bacillus subtilis, a recombinant Bacillus subtilis and applications thereof. The Bacillus subtilis and the recombinant Bacillus subtilis of the present invention have high riboflavin production.

[0005] The invention provides a Bacillus subtilis RF1-6 strain, whose deposit number is CCTCCNO: M 2022565.

[0006] The present invention also provides a recombinant Bacillus subtilis, which uses the Bacillus subtilis RF1-6 described in the above scheme as the original strain, comprising a recombinant plasmid; the recombinant plasmid is inserted with the zwf gene, the ywlf gene and the ribBA gene; the original plasmid of the recombinant plasmid is preferably pMA5-sat; the zwf gene, the ywlf gene and the ribBA gene are preferably connected to the original plasmid by homologous recombination.

[0007] Preferably, the zwf gene, ywlf gene and ribBA gene are sequentially inserted between the EcoRI and KpnI restriction sites of the recombinant plasmid.

[0008] Preferably, the recombinant plasmid is also inserted with a strong promoter P 43 .

[0009] Preferably, the strong promoter P 43 Inserted into the EcoRI restriction site upstream of the zwf gene.

[0010] Preferably, the pgi gene in the recombinant Bacillus subtilis is knocked down.

[0011] Preferably, the purR gene in the recombinant Bacillus subtilis is knocked out.

[0012] Preferably, the recombinant Bacillus subtilis includes Bacillus subtilis RF1-6ZYRS, with the deposit number being CCTCCNO: M 2022566.

[0013] The present invention also provides a bacterial agent, comprising the Bacillus subtilis described in the above scheme or the recombinant Bacillus subtilis described in the above scheme.

[0014] The present invention also provides the use of the Bacillus subtilis RF1-6 or the recombinant Bacillus subtilis or the bacterial agent described in the above scheme in synthesizing riboflavin.

[0015] The present invention provides a strain of Bacillus subtilis RF1-6. The strain of Bacillus subtilis RF1-6 is a mutant strain with the highest riboflavin yield obtained by genetic modification and mutagenesis using the riboflavin high-yield strain RF1 as a starting strain, and the deposit number is CCTCCNO: M 2022565. Compared with the riboflavin high-yield strain RF1, the riboflavin yield can be increased by 22.8%.

[0016] The present invention also provides an engineered bacterium Bacillus subtilis RF1-6ZYRS constructed based on Bacillus subtilis RF1-6. The maximum riboflavin yield of Bacillus subtilis RF1-6ZYRS reaches 25.2 g / L, which is 69.58% higher than the final yield of Bacillus subtilis RF1-6.

[0017] Biological Deposit Description

[0018] Bacillus subtilis RF1-6 was deposited in the China Center for Type Culture Collection on May 9, 2022, at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number: CCTCC NO: M 2022565.

[0019] Bacillus subtilis RF1-6ZYRS was deposited in the China Center for Type Culture Collection on May 9, 2022, at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number: CCTCC NO: M2022566. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the mutagenesis plasmid map of the present invention;

[0021] Figure 2 To report how the system works;

[0022] Figure 3 The results of shake flask rescreening;

[0023] Figure 4 This is the fermentation result of mutant strain RF1-6 in a fermenter;

[0024] Figure 5 This is the fermentation result of bacterial agent RF1-6ZYPS in a 5L fermenter. DETAILED DESCRIPTION

[0025] The invention provides a strain of Bacillus subtilis RF1-6, whose deposit number is CCTCCNO: M 2022565.

[0026] In the present invention, the Bacillus subtilis RF1-6 uses the riboflavin high-yielding strain RF1 as the starting strain, and after genetic modification and mutagenesis, a mutant with the highest riboflavin yield is screened. Compared with the riboflavin high-yielding strain RF1, the riboflavin yield can be increased by 22.8%.

[0027] The present invention also provides a recombinant Bacillus subtilis, which uses the Bacillus subtilis RF1-6 described in the above scheme as the original strain and comprises a recombinant plasmid; the recombinant plasmid is inserted with a zwf gene, a ywlf gene and a ribBA gene.

[0028] The present invention increases the riboflavin metabolic flow by metabolically modifying the riboflavin high-yield strain RF1-6, thereby increasing the riboflavin yield.

[0029] In the present invention, the zwf gene, ywlf gene and ribBA gene are preferably inserted in sequence between the EcoRI and KpnI restriction sites of the recombinant plasmid; the recombinant plasmid is preferably also inserted with a strong promoter P 43 ; The strong promoter P 43 Preferably, the gene is inserted into the EcoRI restriction site upstream of the zwf gene. The original plasmid of the recombinant plasmid is preferably pMA5-sat; the zwf gene, ywlf gene and ribBA gene are preferably connected to the original plasmid by homologous recombination.

[0030] In the present invention, zwf encodes glucose-6-phosphatase dehydrogenase, ribBA encodes a bifunctional GTP cyclohydrolase II / 3,4-dihydroxy-2-butanone 4-phosphate synthase, and ywlf encodes ribose 5-phosphate isomerase B. These three genes jointly promote the riboflavin synthesis metabolic flow and increase riboflavin production.

[0031] The present invention utilizes the strong promoter P 43 Controls the expression of zwf gene, ywlf gene and ribBA gene.

[0032] In the present invention, the pgi gene in the recombinant Bacillus subtilis is preferably knocked down, and the expression level of the gene pgi is reduced by sRNA knockdown technology, so that the metabolic flow flows to the pentose phosphate pathway. In the present invention, the purR gene in the recombinant Bacillus subtilis is preferably knocked out. By knocking out the purR gene, the feedback inhibition in the cell is released, so that more precursor substance GTP is synthesized in the cell.

[0033] In the present invention, the recombinant Bacillus subtilis preferably includes Bacillus subtilis RF1-6ZYRS, with a deposit number of CCTCC NO: M 2022566.

[0034] In the present invention, the maximum riboflavin yield of the Bacillus subtilis RF1-6 ZYRS reaches 25.2 g / L, which is 69.58% higher than the final yield of the Bacillus subtilis RF1-6.

[0035] The present invention also provides a bacterial agent, comprising the Bacillus subtilis described in the above scheme or the recombinant Bacillus subtilis described in the above scheme.

[0036] The present invention also provides the use of the Bacillus subtilis RF1-6 or the recombinant Bacillus subtilis or the bacterial agent described in the above scheme in synthesizing riboflavin.

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0038] The culture medium involved in the following examples is as follows:

[0039] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 0.2 g / L agar powder.

[0040] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl.

[0041] Shake flask fermentation medium: 20g / L glucose, 20g / L yeast powder, 4g / L ammonium citrate, 1g / L K 2 HPO 4 , 1g / LKH 2 PO 4 , 2g / LMgSO 4 7H 2 O, 0.04 g / LMnCl 2 , 0.06 g / L CaCl 2 , 2g / L CuSO 4 , pH 6.8.

[0042] Seed culture medium: 40 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl and 10 μg / mL chloramphenicol.

[0043] Fed-batch fermentation medium: 20 g / L glucose, 20 g / L yeast powder, 6 g / L (NH 4 ) 2 HPO 4 , 5g / LK 2 HPO 4 , 1.5g / LMgSO 4 7H 2 O, 0.03g / LZnSO4 7H 2 O, 0.05 g / LMnCl 2 , 0.02g / LFeSO 4 7H 2 O.

[0044] Feed medium: 600 g / L glucose, 10 g / L yeast powder, 6 g / L (NH 4 ) 2 HPO 4 , 5g / LK 2 HPO 4 , 0.5g / LMgSO 4 7H 2 O.

[0045] The above culture media all use water as solvent.

[0046] The detection methods involved in the following embodiments are as follows:

[0047] The cell growth was monitored at OD600 nm using a spectrophotometer.

[0048] The prepared fermentation broth was diluted with 0.01M NaOH, then centrifuged at 12000rpm for 2min, and the supernatant was taken to determine the riboflavin concentration. The supernatant was transferred to a new EP tube and diluted to an appropriate concentration range (0.3-0.8), and the absorbance value was measured at OD444 nm using a spectrophotometer. The riboflavin concentration was calculated according to the riboflavin concentration standard curve. The calculation formula according to the riboflavin standard curve is: OD444*dilution factor*30 / 1000.

[0049] Glucose concentration was measured using Glucose analysis (Model-SBA40, Shandong, China).

[0050] Example 1 Construction of mutagenic plasmid

[0051] 1. Construction of mutagenic plasmid: Amplify dam and seq genes from plasmid MP6, and the ugi gene is derived from the Escherichia coli genome. Separate the PCR products by agarose gel electrophoresis, and cut the gel to recover the target PCR product. The three fragments were fused by overlapping PCR. First, the upstream and downstream fragments were mixed in a volume ratio of 1:1, and an equal volume of PCR enzyme was added for fusion PCR reaction. The conditions were 98°C, 3min, 98°C, 8s, 61°C, 5s, 72°C, 2min, and 13 cycles of amplification. The product after this step of reaction was used as a template, and the fusion fragment was amplified using primers. The reaction conditions were: 98°C, 3min, 98°C, 10s, 58°C, 15s, 72°C, 1min, and 34 cycles of amplification. Recover the PCR product, and use the Gibson assembly kit to connect the fusion fragment to the HindIII and BamHI sites of the pBT2 plasmid, which is driven by the strong promoter P 43 Control gene expression and construct pBT2-M3 plasmid. pBT2 plasmid is a temperature-sensitive plasmid. It will be lost at 42°C and will not affect the genetic stability of the mutant strain. The gene mutL is derived from Escherichia coli. It is amplified and connected to the plasmid pET28a according to the above method to construct the plasmid pET28a-mutL. ​​The plasmid pET28a-mutL is amplified using reverse amplification primers. The primers contain mutation sites. Mutations are introduced into the gene mutL to construct the plasmid pET28a-mutL (E32K). Using the constructed mutant gene mutL as a template, the PCR fragment of the gene mutL is amplified, and the mutant mutl gene is connected to the pBT2-M3 plasmid according to the above-mentioned Gibson assembly method to construct the mutagenic plasmid pBT2-M4.

[0052] 2. Construction of reporter plasmid: Clone the FMNswitch sequence at the 5' end of the upstream of the rib operon from the genome of Bacillus subtilis 168, amplify the reporter gene gfp, and fuse the FMN riboswitch with gfp according to the overlapping PCR method to form a fused FMNswitch-gfp fragment. Amplify the upstream homology arm (1000bp), downstream homology arm (1000bp) and Marker fragment (containing the bleomycin resistance gene and lox66-lox71 recombination site) of the gene amyE, separate the PCR products by agarose gel electrophoresis, and cut the gel to recover the target PCR product. According to the overlapping PCR method, integrate the fused PCR fragment into the genome of the riboflavin high-producing strain RF1.

[0053] 3. Construction of mutation library: The mutagenic plasmid pBT2-M4 was introduced into the riboflavin high-producing strain RF1 containing the reporter plasmid, and the cells were cultured to the logarithmic phase (OD 600=0.6), and then the cells in the logarithmic phase were subjected to room temperature plasma mutagenesis (ARTP) mutagenesis treatment. The treated mutant library was cultured in an inorganic salt medium for 12 h, and the bacteria were sampled and collected, washed three times with PBS buffer, and then the washed cells were re-selected with PBS, and then diluted to an appropriate bacterial concentration and used for flow cytometry sorting, and the bacterial colonies with fluorescence intensity lower than that of the control strain (ARTP untreated) were selected and post-cultured.

[0054] 4. Spread the isolated mutants on the resistance plate, culture at 37°C for 24 hours, and then use a high-throughput colony picker to pick the colonies with weaker fluorescence intensity on the plate into a 96-well plate containing inorganic salt culture medium. Shake and culture for 24 hours to measure the OD 444 The absorbance value at , riboflavin has the maximum absorption peak at this wavelength, which can indirectly reflect the riboflavin concentration. 1000 cells were selected in each round for repeated screening and identification. Calculate the riboflavin synthesis capacity (OD 444 / OD 600 ) and select the mutant with the highest yield.

[0055] 5. Shake flask rescreening: inoculate the screened mutants into a 250 ml shake flask containing 50 ml fermentation medium, culture at 200 r / min and 41°C for 48 h, measure the riboflavin concentration, and screen the mutant with the highest yield again.

[0056] 6. The mutant strains screened were purified by drawing lines on the plate, and the purified colonies were inoculated into LBG medium and cultured at 42°C, the mutagenic plasmid was discarded, and the final yield was determined by shake flask fermentation. After repeated screening, 10 mutant strains with the highest yield were selected for shake flask fermentation. The results showed that the mutant strain with the highest yield increased riboflavin production by 22.8%, named RF1-6, and the deposit number was CCTCC NO: M 2022565.

[0057] The riboflavin yield of the mutant strain was identified in a 5L fermenter. The riboflavin yield of the high-yielding mutant strain RF1-6 obtained by the shake flask experiment was determined in a 5L fermenter. The specific steps are as follows:

[0058] (1) Bacillus subtilis strain RF1-6 cultured in 10 mL LB medium for 24 h was inoculated into 100 mL seed medium at a volume ratio of 3% at a temperature of 41° C. and a rotation speed of 180 rpm. After culturing for 16 h, a seed solution (OD600=21.2) was prepared;

[0059] (2) The prepared 100 mL seed solution was inoculated into a 5 L fermentation tank containing 1900 mL fermentation medium for batch fed fermentation.

[0060] By controlling the feed medium flow rate, the residual glucose concentration in the fermentation broth was kept at no less than 5 g / L. During the fermentation process, the pH of the fermentation broth was 6.8, and 1 M H 2 SO 4 and 50% ammonia water. Before starting batch feeding, the speed was kept at 400 rpm, then increased to 800 rpm until the end of fermentation, and the temperature was always kept at 41°C. After 60 hours of fed-batch fermentation, the riboflavin yield was measured and the riboflavin concentration reached 14.86 g / L. Compared with the riboflavin concentration of 9.8 g / l of the starting strain RF1, the yield increased by 48.9%. Therefore, the mutagenesis system and high-throughput screening system can effectively improve the performance of industrial strains and improve metabolite synthesis.

[0061] 7. In order to further increase riboflavin production, we used traditional genetic manipulation to metabolically modify the high-yield strains to increase riboflavin metabolic flux, thereby increasing riboflavin production. First, we constructed an overexpression plasmid and connected the genes zwf, ywlf, and ribBA to the pMA5-sat plasmid by homologous recombination. 43 Control gene expression, construct the bacterial agent RF1-6ZYP, and then use sRNA knockdown technology to reduce the expression level of the gene pgi, so that the metabolic flow will flow to the pentose phosphate pathway. In order to increase the synthesis of the precursor substance GTP, the purR gene was knocked out to release the feedback inhibition in the cell and synthesize more GTP in the cell. The obtained bacterial agent was named RF1-6ZYRS, and the preservation number was CCTCC NO: M 2022566. After a series of metabolic modifications, the bacterial agent obtained had a significant increase in riboflavin production at the 5L fermentation tank level, with the highest yield reaching 25.2g / L, and the final yield increased by 69.58%.

[0062] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis )RF1-6ZYRS, the deposit number is CCTCC NO: M2022566.

2. A bacterial agent comprising the Bacillus subtilis RF1-6ZYRS according to claim 1.

3. Use of the Bacillus subtilis RF1-6ZYRS according to claim 1 or the bacterial agent according to claim 2 in synthesizing riboflavin.

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