Bacillus subtilis with high yield of beta-carotene as well as construction method and application of bacillus subtilis

Abstract

The invention relates to bacillus subtilis with high yield of beta-carotene as well as a construction method and application of the bacillus subtilis, and belongs to the technical field of fermentation engineering. According to the invention, extracellular proteases AprE, NprE, Bpr, Mpr, Epr and NprB are knocked out from bacillus subtilis, a repressor protein mutant tetR expression cassette and a beta-carotene synthetic pathway related gene expression cassette are constructed, and a tetracycline inducible promoter is used for expressing beta-carotene synthetic pathway related genes crtE, crtI, crtB and crtY, so that the bacillus subtilis for producing beta-carotene is successfully constructed. The recombinant strain G600-crtEBIY is constructed, the induction time and the inducer concentration are optimized in the fermentation process of the constructed recombinant strain G600-crtEBIY, the yield of the optimized beta-carotene in a shake flask fermentation system reaches up to 18.5 mg / L, stable and efficient production of the beta-carotene is achieved, and industrial production and popularization of the beta-carotene are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, in particular to a Bacillus subtilis with high beta-carotene production and a construction method and application thereof. Background Art

[0002] β-carotene is a natural carotenoid widely found in green and yellow fruits and vegetables. It belongs to the tetraterpenoid hydrocarbons. Its molecular formula is C40H56 and its molecular weight is 536.88. Its molecule contains two β-ionone rings and four isoprene side chains, that is, two β-ionone rings at both ends. The central rupture can produce two vitamin A molecules. There are multiple double bonds between the molecules and the double bonds are conjugated. Studies have shown that β-carotene can be converted into vitamin A in the body and play a role. Vitamin A plays an important role in human life activities. In the case of insufficient supply of vitamin A, it is easy to cause decreased dark vision and even night blindness; dry skin surface, small bumps protruding from pores, etc. Vitamin A can also prevent carcinogens from combining with DNA, improve the immune activity of living organisms, and antagonize the growth of sarcomas. However, if the human body directly ingests excessive vitamin A, it will cause vitamin A poisoning. If β-carotene is absorbed, vitamin A poisoning will not occur and the body's vitamin A needs can be met.

[0003] At present, the preparation methods of β-carotene mainly include extraction and separation of natural raw materials, chemical synthesis and microbial fermentation. The natural raw material extraction method requires the use of a large amount of organic solvents and non-renewable raw materials, which causes serious environmental pollution and is difficult to recycle; the chemical synthesis method has a low yield and low purity; the microbial fermentation method refers to a method of using the biological metabolic pathway of microorganisms to convert cheap renewable raw materials such as glycerol and glucose into β-carotene through the cultivation of microorganisms, and to separate and obtain β-carotene. Although the β-carotene sold on the market is mainly synthesized by chemical methods, with the increasing global market demand for β-carotene, the production of β-carotene by microbial fermentation has broad application prospects.

[0004] Bacillus subtilis has a clear genetic background and mature gene manipulation technology, so it has been used as a model microorganism for studying Gram-positive bacteria. It is also a commonly used production strain in industrial fermentation because of its excellent characteristics such as fast growth rate, strong secretion ability, no obvious codon preference and not susceptible to phage infection. In addition, because it does not produce harmful products such as endotoxins, B. subtilis has also been certified as a GRAS (Generally recognized as safe) strain by the US Food and Drug Administration (FDA), and is a recognized food safety-grade microorganism. However, the current attempts to construct the β-carotene synthesis pathway in B. subtilis have the defect of too low fermentation yield, which affects its industrial application. Summary of the invention

[0005] In order to solve the above technical problems, the present invention knocks out extracellular proteases AprE, NprE, Bpr, Mpr, Epr and NprB in Bacillus subtilis, constructs a repressor protein mutant tetR expression frame and a β-carotene synthesis pathway related gene expression frame, uses a tetracycline-inducible promoter to express the β-carotene synthesis pathway related genes crtE, crtI, crtB and crtY, successfully constructs a β-carotene-producing Bacillus subtilis, and optimizes the induction time and inducer concentration of the constructed recombinant strain G600-crtEBIY during the fermentation process, so that the yield of β-carotene is further improved.

[0006] The first object of the present invention is to provide a Bacillus subtilis with high beta-carotene production, wherein a repressor protein mutant tetR expression frame and a beta-carotene synthesis pathway-related gene expression frame are constructed in the starting strain;

[0007] The repressor protein mutant tetR expression cassette is composed of the Bacillus subtilis constitutive promoter P21, the RBS sequence and the repressor protein mutant encoding gene tetR, wherein the P 21 The nucleotide sequence of is shown in SEQ ID NO.1, the RBS sequence is shown in SEQ ID NO.2, and the nucleotide sequence of tetR is shown in SEQ ID NO.3;

[0008] The β-carotene synthesis pathway related gene expression frame is composed of a tetracycline inducible promoter, an RBS1 sequence and β-carotene synthesis pathway related genes, wherein the β-carotene synthesis pathway related genes include geranylgeranyl pyrophosphate synthase gene crtE, phytoene synthase gene crtI, phytoene dehydrogenase gene crtB and lycopene cyclase gene crtY, wherein the RBS1 sequence is shown in SEQ ID NO.4.

[0009] Furthermore, the repressor protein mutant tetR is obtained by mutating the phenylalanine at position 119 of the starting sequence of the amino acid sequence such as SEQ ID NO.10 to tyrosine.

[0010] Furthermore, the starting strain knocks out or silences the extracellular proteases AprE, NprE, Bpr, Mpr, Epr and NprB.

[0011] Furthermore, the nucleotide sequence of crtE is shown as SEQ ID NO.5, the nucleotide sequence of crtI is shown as SEQ ID NO.6, the nucleotide sequence of crtB is shown as SEQ ID NO.7, and the nucleotide sequence of crtY is shown as SEQ ID NO.8.

[0012] Furthermore, the tetracycline-inducible promoter is P tet .

[0013] The second object of the present invention is to provide a method for constructing the above-mentioned Bacillus subtilis, comprising the following steps:

[0014] Step S1, knocking out or silencing the extracellular proteases AprE, NprE, Bpr, Mpr, Epr and NprB in Bacillus subtilis 168 to obtain Bacillus subtilis G600;

[0015] Step S2, constructing a recombinant expression vector, wherein the recombinant expression vector uses the pHT01 plasmid as a backbone, includes a repressor protein mutant tetR expression cassette and a β-carotene synthesis pathway related gene expression cassette, wherein the repressor protein mutant tetR expression cassette is driven by the Bacillus subtilis constitutive promoter P 21 , RBS sequence and repressor protein mutant encoding gene tetR, the β-carotene synthesis pathway related gene expression frame is composed of a tetracycline inducible promoter, RBS1 sequence and β-carotene synthesis pathway related genes;

[0016] Step S3, transferring the recombinant expression vector into Bacillus subtilis G600 to obtain the Bacillus subtilis with high β-carotene production.

[0017] The third object of the present invention is to provide a microbial preparation comprising the above-mentioned Bacillus subtilis.

[0018] The fourth object of the present invention is to provide the use of the above-mentioned Bacillus subtilis or the above-mentioned microbial preparation in the production of β-carotene.

[0019] The fifth object of the present invention is to provide a method for producing β-carotene, wherein the above-mentioned Bacillus subtilis or the above-mentioned microbial preparation is added to a fermentation system, and an inducer anhydrotetracycline is added to carry out fermentation production.

[0020] In one embodiment of the present invention, the fermentation system includes 90 g / L glucose, 60 g / L tryptone, 120 g / L yeast powder, 60 g / L urea, 50 g / L glycerol, 30 g / L MgSO4·7H2O, 12.50 g / L K2HPO4·3H2O, and 2.50 g / L KH2PO4.

[0021] Furthermore, the inducer is added 3-6 hours after the start of fermentation.

[0022] Preferably, the inducer is added at 6 hours after the start of fermentation.

[0023] Furthermore, the concentration of the inducer is 4-8 μM.

[0024] Preferably, the concentration of the inducer is 4 μM.

[0025] Beneficial effects of the present invention:

[0026] The invention provides a high-yield beta-carotene Bacillus subtilis and a construction method and application thereof, using a Bacillus subtilis with a short growth cycle, a clear metabolic pathway, and convenient molecular operation as a starting strain, in which six extracellular proteases are inactivated, and by constructing a repressor protein tetR expression frame and a beta-carotene synthesis pathway-related gene expression frame, de novo synthesis and stable production of beta-carotene in Bacillus subtilis are achieved, and the induction time and induction concentration of dehydrotetracycline are optimized, and the optimized recombinant strain has a yield of 18.5 mg / L in a shake flask fermentation system. The dehydrotetracycline inducible recombinant expression vector used in the invention has a simple structure, high activity, strict regulation, no leakage in Bacillus subtilis, and has obvious advantages over other inducible promoters. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0028] Figure 1The recombinant expression vector pHT-P in Example 1 of the present invention tet -Schematic diagram of crtEIBY structure;

[0029] Figure 2 The β-carotene production results of the recombinant strain G600-crtEBIY in Example 2 of the present invention under different induction times of dehydrotetracycline;

[0030] Figure 3 The β-carotene production results of the recombinant strain G600-crtEBIY in Example 2 of the present invention under the induction of different concentrations of dehydrated tetracycline;

[0031] Figure 4 The β-carotene yields expressed using different inducible systems in the comparative examples of the present invention are shown; DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0033] The strains and vectors involved in the examples are as follows:

[0034] The plasmid was constructed in E. coli DH5α, and after the plasmid was constructed, it was transformed into B. subtilis G600 for expression. B. subtilis G600 was constructed by inactivating six extracellular proteases (AprE, NprE, Bpr, Mpr, Epr and NprB) in B. subtilis 168 strain.

[0035] The culture medium involved in the embodiment is as follows:

[0036] E. coli were cultured using LB medium (containing 10 g tryptone, 5 g yeast powder, and 10 g NaCl per liter);

[0037] For plasmid transformation, YN medium (7 g yeast powder and 18 g nutrient broth per liter) was used;

[0038] The seed culture medium was LB medium;

[0039] Shake flask fermentation medium (g / L): glucose 90, tryptone 6, yeast powder 12, urea 6, glycerol 5, MgSO4·7H2O 3, K2HPO4·3H2O 12.5, KH2PO4 2.5. Glucose and MgSO4·7H2O need to be sterilized separately from other ingredients and added before inoculation.

[0040] The detection method involved in the embodiment is as follows:

[0041] Take an appropriate amount of fermentation broth, dilute it to an appropriate multiple, centrifuge it at 12,000 rpm for 5 minutes, pour off the supernatant, resuspend the bacteria with ddH2O, wash twice, resuspend the bacteria with 1 mL of acetone, extract at 55°C in the dark for 15 minutes (mix every 5 minutes), then centrifuge the sample at 12,000 rpm for 10 minutes, and take the supernatant containing β-carotene for liquid phase detection.

[0042] Samples were analyzed using a high performance liquid chromatography system (Agilent Technologies 1260) equipped with a C18 column (4.6 mm × 250 mm; 5 μm; Waters, Ireland) and a variable wavelength scanning UV detector (VWD) set to 474 nm. The mobile phase was 50% (v / v) acetonitrile, 30% (v / v) methanol, and 20% (v / v) isopropanol at a flow rate of 1 mL / min and a temperature of 40 °C.

[0043] Example 1: Construction of recombinant expression vector and recombinant strain

[0044] The crtE, crtI, crtB and crtY gene fragments (nucleotide sequences are shown in SEQ ID NOs. 5-8, respectively) were amplified and integrated using primers pHT-LiF / R and pHT-P tet -sfGFP was used as a template to amplify the linearized vector (pHT-P tet -sfGFP is disclosed in patent CN114645062A, a dehydrated tetracycline-induced Escherichia coli-Bacillus subtilis universal inducible expression system, the nucleotide sequence is shown in SEQ ID NO.9), and the PCR product is recovered using a DNA purification kit. The plasmid vector and the inserted gene fragment are connected using the Biyuntian Seamless Cloning Kit to make pHT-P tet -sfGFP was replaced with crtEIBY to obtain the recombinant expression vector pHT-P tet -crtEIBY, such as Figure 1 The recombinant expression vector was transformed into E. coli DH5α competent cells, and the transformants were verified using primers Ptet-VF, crtE-VR, crtI-VR, crtB-VR and crtY-VR, and the sequencing was correct. The primers involved are shown in Table 1.

[0045] Table 1 Primers involved

[0046]

[0047]

[0048] Take freshly streaked Bacillus subtilis G600, inoculate it in YN medium overnight; take 450 μL, add 2 mL YN medium and 80 μL xylose with a concentration of 500 g / L, culture at 37°C for 2 hours, divide and store in 10% glycerol. Alternatively, take 150 μL after overnight and inoculate it in 2 mL YN medium and then add xylose to induce for 2 hours to make competent cells. Take 1000 ng of the correctly sequenced plasmid and add it to 500 μL of the preserved competent cells, transform at 37°C for 2 hours, and directly plate to obtain the β-carotene producing strain Bacillus subtilis G600-crtEBIY.

[0049] Example 2: Optimization of conditions for producing β-carotene by recombinant strains

[0050] Plate culture: Take Bacillus subtilis G600-crtEBIY stored at -80℃ and streak it on the activation plate, culture it at 37℃ for 12h, and subculture it once;

[0051] Shake flask seed culture: Use an inoculation loop to scrape a ring of slant seeds and inoculate into a 50 mL Erlenmeyer flask containing 10 mL of seed culture medium, seal with nine layers of gauze, and culture at 37°C, 220 rpm for 10 h;

[0052] Shake flask fermentation culture: inoculate 4% of the volume of the seed culture solution into a 250 mL Erlenmeyer flask containing fermentation medium (final volume 40 mL), seal with nine layers of gauze, and culture at 37°C, 200 r / min shaking;

[0053] Optimization of induction time: 1 μM dehydrotetracycline was added at 0, 3, 6, 9, 12, 18 and 24 hours for induction and fermentation. The fermentation results are shown in Figure 2 As shown, it can be seen that the best time to add anhydrotetracycline to induce expression is the 6th hour after the start of shake flask fermentation;

[0054] Optimization of induction concentration: 0, 0.1, 0.5, 1, 2, 4, 6, 8 and 10 μM dehydrotetracycline were added at the optimal time points for fermentation. The fermentation results are shown in Figure 3 As shown, when the concentration of dehydrotetracycline was 4 μM, the strain G600-crtEBIY had the highest yield after 24 hours of fermentation, and the β-carotene content reached 18.5 mg / L.

[0055] Comparative Example: Effects of different inducible expression systems on β-carotene expression

[0056] In order to investigate the effects of different inducible expression systems on the expression of β-carotene, in addition to the anhydrotetracycline inducible promoter, the xylose inducible promoter P shown in SEQ ID NO.11 and SEQ ID NO.12 were also used. xylA and IPTG-inducible promoter P grac100 The corresponding inducible expression system was constructed. The two inducible promoters showed low leakage expression and high induction expression intensity in previous reports.

[0057] The fermentation results are as follows Figure 4 As shown in Figure 2, both the xylose-induced expression system and the IPTG-induced expression system can achieve the expression of β-carotene, but there are obvious differences in the yield of β-carotene. tet The highest β-carotene production was expressed by P xylA and P grac100 15.4 times and 7.4 times.

[0058] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A Bacillus subtilis with high beta-carotene production, characterized in that: Constructing the repressor protein tetR mutant expression cassette and the β-carotene synthesis pathway related gene expression cassette in the starting strain; The repressor protein mutant tetR expression cassette is driven by the constitutive promoter P of Bacillus subtilis. 21 , RBS sequence and repressor protein mutant encoding gene tetR, wherein the P 21 The nucleotide sequence of is shown in SEQ ID NO.1, the RBS sequence is shown in SEQ ID NO.2, and the nucleotide sequence of tetR is shown in SEQ ID NO.3; The β-carotene synthesis pathway related gene expression frame is composed of a tetracycline inducible promoter, an RBS1 sequence and β-carotene synthesis pathway related genes, wherein the β-carotene synthesis pathway related genes include geranylgeranyl pyrophosphate synthase gene crtE, phytoene synthase gene crtI, phytoene dehydrogenase gene crtB and lycopene cyclase gene crtY, wherein the RBS1 sequence is shown in SEQ ID NO.

4.

2. The Bacillus subtilis according to claim 1, characterized in that: The starting strain knocks out or silences the extracellular proteases AprE, NprE, Bpr, Mpr, Epr and NprB.

3. The Bacillus subtilis according to claim 1, characterized in that: The nucleotide sequence of crtE is shown in SEQ ID NO.5, the nucleotide sequence of crtI is shown in SEQ ID NO.6, the nucleotide sequence of crtB is shown in SEQ ID NO.7, and the nucleotide sequence of crtY is shown in SEQ ID NO.

8.

4. The Bacillus subtilis according to claim 1, characterized in that: The tetracycline-inducible promoter is P tet .

5. The method for constructing Bacillus subtilis according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step S1, knocking out or silencing the extracellular proteases AprE, NprE, Bpr, Mpr, Epr and NprB in Bacillus subtilis 168 to obtain Bacillus subtilis G600; Step S2, constructing a recombinant expression vector, wherein the recombinant expression vector uses the pHT01 plasmid as a backbone, includes a repressor protein mutant tetR expression cassette and a β-carotene synthesis pathway related gene expression cassette, wherein the repressor protein mutant tetR expression cassette is driven by the Bacillus subtilis constitutive promoter P 21 , RBS sequence and repressor protein mutant encoding gene tetR, the β-carotene synthesis pathway related gene expression frame is composed of a tetracycline inducible promoter, RBS1 sequence and β-carotene synthesis pathway related genes; Step S3, transferring the recombinant expression vector into Bacillus subtilis G600 to obtain the Bacillus subtilis with high β-carotene production.

6. A microbial preparation comprising the Bacillus subtilis according to any one of claims 1 to 4.

7. Use of the Bacillus subtilis according to any one of claims 1 to 4 or the microbial preparation according to claim 6 in producing β-carotene.

8. A method for producing beta-carotene, characterized in that: The Bacillus subtilis described in any one of claims 1 to 4 or the microbial preparation described in claim 6 is added to the fermentation system, and an inducer, dehydrotetracycline, is added to carry out fermentation production.

9. The method according to claim 8, characterized in that: The time for adding the inducer is 3-6 hours after the start of fermentation.

10. The method according to claim 8, characterized in that: The concentration of the inducer was 4-8 μM.

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