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CN-122012628-A - Bacillus subtilis strain for producing natural products and construction method thereof

CN122012628ACN 122012628 ACN122012628 ACN 122012628ACN-122012628-A

Abstract

The invention relates to the technical field of genetic engineering, and discloses a bacillus subtilis strain for producing natural products and a construction method thereof. The functional genes of geranyl pyrophosphate synthase gene, phytoene dehydrogenase gene and phytoene synthase gene for synthesizing lycopene are integrated and expressed in the chromosome of the bacillus subtilis strain, one or more of 12 genes of 9 genes of MEP path can be integrated, and each gene is endowed with 8 independent continuous G bases to form RBS to be edited to obtain chassis bacteria, the dmas 9-AID fusion protein and gRNA of targeted RBS core region are expressed in the chassis bacteria, a mutant library with 12 simultaneous regulation and control of gene expression levels is obtained, and the recombinant strain with high lycopene yield is obtained through high-throughput screening. The invention adopts a bioengineering strategy, and the constructed recombinant bacillus subtilis is used for synthesizing lycopene, thereby providing a new idea for high-yield lycopene by a biological method.

Inventors

  • WANG MENG
  • LIU YANG
  • Cao Xianhai
  • ZHAO YUE

Assignees

  • 中国科学院天津工业生物技术研究所
  • 天工生物科技（天津）有限公司

Dates

Publication Date
20260512
Application Date
20241108

Claims (10)

  1. 1. The construction method for producing lycopene mutant library by recombinant bacillus subtilis is characterized by comprising the following steps: s1, integrating and expressing functional genes geranyl pyrophosphate synthase gene, phytoene dehydrogenase gene and phytoene synthase gene for synthesizing lycopene on chromosome of an original strain, preferably integrating the genes at epr gene of chromosome of the original strain to obtain chassis bacteria; Further preferably, the chassis fungus is obtained by integrating 9 genes of the MEP pathway, wherein the 9 genes of the MEP pathway are dxs, dxr, ispD, ispE, ispF, ispG, ispH, idi, ispA genes; s2, introducing a cytosine base editor into the strain, and obtaining a bacillus subtilis mutant library for producing lycopene after induction editing.
  2. 2. The method for constructing a library of recombinant bacillus subtilis-derived lycopene mutants according to claim 1, wherein the 9 genes of the MEP pathway are derived from bacillus subtilis, the exogenous genes comprise geranyl pyrophosphate synthase genes, phytoene dehydrogenase genes and phytoene synthase genes derived from pantoea agglomerans, respectively designated crtE, crtI, crtB; Preferably, the promoter is selected from the group consisting of the modified constitutive promoter P gapDH and the terminator is selected from the group consisting of the T M3 terminator.
  3. 3. The method for constructing a library of recombinant bacillus subtilis production lycopene mutants according to claim 2, wherein in S1, a gene expression cassette comprising a combination of a constitutive promoter P gapDH and an RBS comprising 8 consecutive G bases, an expression cassette 1 comprising dxs gene, an expression cassette 2 comprising dxr and ispD genes, an expression cassette 3 comprising ispE and ispF genes, an expression cassette 4 comprising ispG and ispH genes, an expression cassette 5 comprising ispA and idi genes, and an expression cassette 6 comprising crtE, crtI, crtB genes are sequentially integrated into the bacillus subtilis genome to obtain an editable bacillus subtilis chassis strain producing lycopene; Preferably, the nucleotide of the expression cassette 1 is shown as SEQ ID NO. 1, the nucleotide of the expression cassette 2 is shown as SEQ ID NO. 2, the nucleotide of the expression cassette 3 is shown as SEQ ID NO. 3, the nucleotide of the expression cassette 4 is shown as SEQ ID NO. 4, the nucleotide of the expression cassette 5 is shown as SEQ ID NO. 5, and the nucleotide of the expression cassette 6 is shown as SEQ ID NO. 6; Specifically, the gene is integrated onto the genome of the starting bacterium by a CRISPR/Cas9 targeted gene integration system.
  4. 4. A method of constructing a library of bacillus subtilis mutants producing lycopene according to claim 1, wherein in S2, the RBS region of the chaetobacter is edited by CRISPR/dCas9-AID base editing system to generate the library of mutants.
  5. 5. A method for constructing a library of Bacillus subtilis mutant for producing lycopene according to claim 4, wherein the chassis bacteria are inoculated into fresh culture medium for culture after being activated by the culture medium, seed liquid is obtained after the culture, and bacterial cells are induced by transferring the culture medium added with an inducer to obtain the mutant library.
  6. 6. A method of constructing a library of mutants of Bacillus subtilis for producing lycopene according to claim 5, wherein the seed solution is inoculated into fresh medium at OD 600 of 0.05-0.15, and cultured for 12-16h with the addition of inducer IPTG of 0.1-2.0 mM,25-35℃and 180-220 rpm.
  7. 7. A method of constructing a library of mutants of bacillus subtilis for the production of lycopene according to any of claims 1 to 6, wherein the starting bacteria are bacillus subtilis 168, bacillus subtilis SCK6.
  8. 8. A library of lycopene-producing bacillus subtilis mutants obtainable by the construction method of any of claims 1-7.
  9. 9. Use of a library of lycopene-producing bacillus subtilis mutants according to claim 8 for screening bacillus subtilis mutant strains for high lycopene production.
  10. 10. A method for screening a bacillus subtilis mutant strain for high lycopene production, characterized in that a bacillus subtilis mutant strain for high lycopene production according to claim 8 is inoculated to a culture medium, and the bacillus subtilis mutant strain for high lycopene production is selected according to the lycopene production amount; specifically, the bacillus subtilis mutant strain for producing lycopene is inoculated into a fermentation medium for culture after being activated by a seed culture medium, and cells of thalli are collected after the culture for extracting lycopene; inoculating seed solution of the bacillus subtilis mutant strain for producing lycopene into a fermentation medium at an OD 600 of 0.05-0.15, and culturing at 35-38 ℃ and 180-220 rpm for 12-72 h; more specifically, seed culture solution of bacillus subtilis mutant library for producing lycopene is inoculated into TSB liquid culture medium, the temperature is 37 ℃, the rotating speed is 220 rpm, and the fermentation culture is 24 h; Adding lysozyme, incubating at 30deg.C for 30min, centrifuging at 14000 r/min for 5min, discarding supernatant, collecting thallus, adding 1 mL extract V Methanol ：V Acetone (acetone) =7:3, shaking to resuspend thallus, crushing thallus by ultrasonic and ultrasonic crushing method, extracting at 60deg.C under dark condition for 80 min, centrifuging at 14000 r/min for 10 min, filtering supernatant with microporous membrane of 0.22 μm organic phase, and measuring lycopene content by high performance liquid chromatograph; The fermentation medium is a TSB liquid medium, and comprises the specific steps of selecting single colony from a solid medium stored with recombinant bacillus subtilis strain into the TSB liquid medium, and culturing at 37 ℃ and 200 rpm for 16 h to obtain seed culture solution of the recombinant bacillus subtilis strain, wherein the solid culture comprises 1% of tryptone, 0.5% of yeast extract, 1% of NaCl, 2% of agar and 7.0% of pH; The weight percentage of each component in the TSB culture medium is 1.7 percent of tryptone, 0.3 percent of soytone, 0.5 percent of NaCl, 0.25 percent of glucose and 7.0 percent of pH value; seed culture solution is inoculated into TSB liquid culture medium according to the initial OD 600 of 0.1 for fermentation under the conditions of 37 ℃ of temperature, 220 of rotating speed and rpm of rotating speed, and fermentation culture is 24 and h.

Description

Bacillus subtilis strain for producing natural products and construction method thereof Technical Field The invention relates to the technical field of biology, in particular to a bacillus subtilis strain for producing natural products and a construction method thereof, and more particularly relates to a bacillus subtilis mutant library for producing lycopene recombination, and a construction method and application thereof. Background Lycopene has good pharmacological and physiological activities such as oxidation resistance, inflammation resistance, tumor resistance and the like, and is widely applied to functional foods, nutritional products, medicines and cosmetics. Lycopene, which is a carotenoid (belonging to C40 terpenoid compounds) present in a very potent vegetable food, cannot be synthesized by itself in humans and must be obtained by dietary supplements and the like. Some large-scale companies such as Ly-cored Natural Products Industries ltd in israel, henkel company in united states, japan MAKHTSHIM company and the like develop foods and medicines for treating hypercholesterolemia, hyperlipidemia, reducing activities of cancer cells and the like, and have remarkable curative effects. The lycopene is used as functional factor of functional food to make into antioxidant health capsule, or can be mixed with other medicinal plants to make into medicated diet. At present, lycopene is rarely reported as a food or drug raw material in China. At present, lycopene has natural and artificial synthesis, and the actual application mainly comprises tomato extraction, blakeslea trispora (Blakesleatrispora) fermentation and artificial synthesis. The natural pigment has the advantages of safety, nutrition, health care and the like, but the lycopene content in tomatoes is very low, and only 20g of lycopene is usually contained in each ton of tomatoes, so that the natural extraction method has low yield and high price and cannot meet the requirements, the Blakeslea trispora fermentation is an effective method for producing lycopene at present, however, the yield is low, the fermentation period is long, the industrialization is not realized, and the synthetic lycopene is easily polluted by toxic chemical substances such as heavy metals and the like in the synthetic process and can also cause harm to human bodies. Consumers have a problem with artificial pigments, and are increasingly favoured by foods incorporating natural pigments. Therefore, engineering microorganisms to produce natural lycopene by genetic engineering, enzymatic engineering, and the like have received attention from many researchers. Lycopene and its derivatives have two major synthetic pathways in organisms, one of which is the mevalonate pathway (Mevalonic ACID PATHWAY, MVA pathway), the starting substrate of which is acetyl CoA, which is catalyzed by a range of enzymes to produce IPP. The other is the 2-C-methyl-D-erythritol 4-phosphate pathway (Methylerythritol Phosphate Pathway, MEP pathway), whose starting substrates are glyceraldehyde-3-phosphate and pyruvate, catalyzed by a range of enzymes to form an approximately 5:1 mixture of IPP and DMAPP, which are activated C5 units, upon which various terpenes can be synthesized. DMAPP is condensed with three molecules of IPP one by one under the action of GGPP synthetase to form yak-base pyrophosphate (geranyl diphosphate, GPP), farnesyl pyrophosphate (farnesyl diphosphate, FPP) and yak-base pyrophosphate (geranyl-geranyldiphosphate, GGPP). Under the action of phytoene synthase, two molecules of GGPP synthesize phytoene, and then through dehydrogenation reaction and extension of common double bond, lycopene is formed. At present, a study on lycopene synthesis by bioconversion has been reported (Zhao J,Li Q,Sun T,et al.Engineering central metabolic modules of Escherichia coli for improvingβ-carotene production.Metab Eng.2013;17:42-50;Li Q,Fan F,Gao X,et al.Balanced activation of IspG and IspH to eliminate MEP intermediate accumulation and improve isoprenoids production in Escherichia coli.Metab Eng.2017;44:13-21.), and relatively high yields can be achieved in E.coli. It is noted that E.coli is limited in the production of food and the like, and Bacillus subtilis (Bacillus subtilis) is used as a food-safe bacterium, and has the advantages of biosafety (recognized as GRAS, GENERALLY REGARDED AS SAFE by FDA in USA), simple culture conditions, rapid growth, clear genetic background and the like. Bacillus subtilis itself cannot synthesize lycopene but has a MEP pathway for the biosynthesis of terpenoid precursors and, more importantly, it produces isoprene at significantly higher levels than most other eubacteria, including e. Therefore, the high added value terpenoid products such as lycopene and the like synthesized in the bacillus subtilis have higher application value. However, the reported production levels of lycopene by bacillus subtilis as a chassis cell are still low. It has been reported tha