Bacillus subtilis strain for producing natural products and construction method thereof

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. Functional genes of a geranyl pyrophosphate synthase gene, a phytoene dehydrogenase gene and a phytoene synthetase gene for expressing and synthesizing lycopene are integrated in a chromosome of a starting bacillus subtilis strain, and one or more of 12 genes in total including 9 genes in an MEP (Methyl Ester Protein) pathway can be further integrated. The method comprises the following steps: constructing a recombinant strain of lycopene, endowing each gene with a to-be-edited RBS composed of eight independent continuous G basic groups to obtain a chassis bacterium, expressing dCas9-AID fusion protein and gRNA of a targeted RBS core region in the chassis bacterium to obtain a mutant library of which the expression levels of 12 genes are regulated at the same time, and performing high-throughput screening to obtain the recombinant strain of high-yield lycopene. According to the invention, a biological engineering strategy is adopted, and the constructed recombinant bacillus subtilis is used for synthesizing lycopene, so that a new idea is provided for high yield of lycopene by a biological method.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to Bacillus subtilis strains for producing natural products and their construction methods, and more specifically to a recombinant Bacillus subtilis mutant library for producing lycopene and its construction methods and applications. Background Technology

[0002] Lycopene possesses excellent pharmacological and physiological activities, including antioxidant, anti-inflammatory, and anti-tumor effects, and is widely used in functional foods, nutritional products, pharmaceuticals, and cosmetics. As a highly promising carotenoid (belonging to the C40 terpenoid class of compounds) found in plant-based foods, lycopene cannot be synthesized by the human body and must be obtained through diet. Large international companies such as Ly-cored Natural Products Industries Ltd. (Israel), Henkel (USA), and Makhtshim (Japan) have developed foods and pharmaceuticals for treating high cholesterol and high blood lipids, and reducing the activity of cancer cells, with significant therapeutic effects. In Japan, lycopene is used as a functional ingredient in functional foods to produce antioxidant health capsules, or combined with other medicinal plants to create medicinal canned foods. Currently, there are few reports of lycopene being used as a food or pharmaceutical ingredient in China.

[0003] Currently, lycopene is available in both natural and synthetic forms, with practical applications primarily through tomato extraction, Blakeslea trispora fermentation, and artificial synthesis. Natural pigments offer advantages such as safety, nutrition, and health benefits; however, tomatoes contain very low levels of lycopene, typically only 20g per ton of tomatoes. Therefore, natural extraction methods suffer from low yields and high prices, failing to meet demand. Blakeslea trispora fermentation is currently an effective method for producing lycopene; however, its yield is low and the fermentation cycle is long, hindering industrialization. Artificial lycopene synthesis is susceptible to contamination by heavy metals and other toxic chemicals, potentially harming human health. Consumers have concerns about synthetic pigments and increasingly favor foods with added natural pigments. Therefore, the use of genetic engineering and enzyme engineering to modify microorganisms for the production of natural lycopene has attracted considerable attention from researchers.

[0004] Lycopene and its derivatives are synthesized in organisms primarily through two pathways: the mevalonic acid pathway (MVA), which begins with acetyl-CoA and is catalyzed by a series of enzymes to produce IPP; and the methylerythritol phosphate pathway (MEP), which begins with glyceraldehyde-3-phosphate and pyruvate and is catalyzed by a series of enzymes to form a mixture of approximately 5:1 IPP and DMAPP. IPP and DMAPP are activated C5 units, upon which various terpenoids can be synthesized. DMAPP then condenses with three molecules of IPP one by one under the action of GGPP synthase to form geranyldiphosphate (GPP), farnesyl diphosphate (FPP), and geranyl-geranyldiphosphate (GGPP). Two molecules of GGPP synthase synthase synthetase synthesize phytoene, which is then converted into lycopene through dehydrogenation and conjugated double bond elongation. Currently, research on the biotransformation method for lycopene synthesis has been reported (Zhao J, Li Q, Sun T, et al. Engineering central metabolic modules of Escherichia coli for improving β-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 Escherichia coli. It is worth noting that Escherichia coli is restricted in the production of food and related substances, while Bacillus subtilis, as a food safety bacterium, has advantages such as biosafety (recognized as GRAS by the US FDA, generally regarded as safe), simple culture conditions, rapid growth, and clear genetic background.Bacillus subtilis itself cannot synthesize lycopene, but it possesses the MEP pathway for terpene precursor biosynthesis. More importantly, the level of isoprene produced by Bacillus subtilis is significantly higher than that of most other eubacteria, including Escherichia coli. Therefore, the synthesis of high-value-added terpene products such as lycopene from Bacillus subtilis has greater application value. However, the reported production levels of lycopene synthesized by Bacillus subtilis as a chassis cell are still relatively low. Existing reports indicate that the lycopene yield of recombinant strains obtained by integrating lycopene biosynthesis genes into the genome is 1.12 mg / L (Liu Y, Cheng H, Li H, Zhang Y, Wang MA Programmable CRISPR / Cas9 Toolkit Improves Lycopene Production in Bacillus subtilis. Appl Environ Microbiol. 2023; 89(6):e0023023.), which is significantly lower than the yields of industrial strains such as Escherichia coli and Saccharomyces cerevisiae. Therefore, it is necessary to develop new engineering strategies to enhance the potential of Bacillus subtilis as a chassis cell to produce high-value-added terpenoids such as lycopene. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for creating a recombinant Bacillus subtilis mutant library and to screen strains that produce high yields of the natural product lycopene from it, so that the recombinant strains can be used in the biosynthesis of lycopene without adding any resistance markers to maintain a high yield. At the same time, the invention also provides a method for constructing and applying the recombinant strain mutant library.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides steps including the following:

[0008] S1 integrates and expresses the functional genes for lycopene synthesis, namely, the gerany pyrophosphate synthase gene, the phytoene dehydrogenase gene, and the phytoene synthase gene, on the chromosome of the starting strain; preferably, the above genes are integrated at the epr gene on the chromosome of the starting strain to obtain the chassis strain.

[0009] More preferably, nine genes from the MEP pathway are integrated to obtain the chassis bacteria, wherein the nine genes from the MEP pathway are dxs, dxr, ispD, ispE, ispF, ispG, ispH, idi, and ispA genes;

[0010] S2 introduced a cytosine base editor into the strain, and after inducing editing, obtained a Bacillus subtilis mutant library that produces lycopene.

[0011] Preferably, the nine genes in the MEP pathway are derived from Bacillus subtilis; the exogenous genes, including the geranyl pyrophosphate synthase gene, the phytoene dehydrogenase gene, and the phytoene synthase gene, are derived from Pantotheca acuminata and are referred to as crtE, crtI, and crtB, respectively; the promoters and terminators controlling each gene are derived from promoters and terminators in Bacillus subtilis.

[0012] The promoter is selected from the modified promoter P. gapDH The terminator is selected from T. M3 Termination of contract.

[0013] More specifically, firstly, the constitutive promoter P is combined gapDH The expression cassettes containing RBS with 8 consecutive G bases were used to integrate expression cassette 1 containing the dxs gene into the amyE gene; then expression cassette 2 containing the dxr and ispD genes was integrated into the amyE gene; expression cassette 3 containing the ispE and ispF genes was further integrated into the thrC gene; then expression cassette 4 containing the ispG and ispH genes was integrated into the thrC gene; then expression cassette 5 containing the ispA and idi genes was integrated into the vpr gene; and expression cassette 6 containing the crtE, crtI, and crtB genes was sequentially integrated into the Bacillus subtilis genome to obtain an editable Bacillus subtilis chassis strain that produces lycopene.

[0014] Preferably, the nucleotides of the gene expression cassette 1 fragment are as shown in SEQ ID NO:1; the nucleotides of the gene expression cassette 2 fragment are as shown in SEQ ID NO:2; the nucleotides of the gene expression cassette 3 fragment are as shown in SEQ ID NO:3; the nucleotides of the gene expression cassette 4 fragment are as shown in SEQ ID NO:4; the nucleotides of the gene expression cassette 5 fragment are as shown in SEQ ID NO:5; and the nucleotides of the gene expression cassette 6 fragment are as shown in SEQ ID NO:6.

[0015] More preferably, a cytosine base editor is introduced into the constructed Bacillus subtilis chassis strain, and after induction editing, a Bacillus subtilis mutant library for producing lycopene is obtained.

[0016] The RBS region of the chassis strain was edited using the CRISPR / dCas9-AID base editing system to generate a mutant library.

[0017] In practice, the gene is integrated into the genome of the originating bacterium using the CRISPR / Cas9 targeted gene integration system.

[0018] In a specific embodiment, the starting bacteria are Bacillus subtilis 168 and Bacillus subtilis SCK6.

[0019] This invention provides a Bacillus subtilis mutant library for producing lycopene obtained by the construction method described above.

[0020] Furthermore, this provides the application of the aforementioned Bacillus subtilis mutant library for producing lycopene in screening for Bacillus subtilis mutant strains that produce high levels of lycopene.

[0021] The present invention also provides a method for screening Bacillus subtilis mutant strains that produce high levels of lycopene, wherein strains from the Bacillus subtilis mutant library that produces lycopene are inoculated into a culture medium, and high-lycopene-producing Bacillus subtilis mutant strains are selected based on the amount of lycopene produced.

[0022] Specifically, the Bacillus subtilis mutant strain that produces lycopene is activated in a seed culture medium and then inoculated into a fermentation culture medium for culture. After culture, the bacterial cells are collected to extract lycopene.

[0023] The seed culture of the Bacillus subtilis mutant strain that produces lycopene was prepared at OD... 600 Inoculate 0.05-0.15g into the fermentation medium and incubate at 35-38℃ and 180-220rpm for 12-72h.

[0024] More specifically, the seed culture of the Bacillus subtilis mutant library that produces lycopene was inoculated into TSB liquid medium at a temperature of 37°C and a rotation speed of 220 rpm for 24 h of fermentation.

[0025] After collecting the bacterial cells, add lysozyme, incubate at 30°C for 30 min, centrifuge at 14000 r / min for 5 min, discard the supernatant, and collect the bacterial cells; add 1 mL of extraction buffer V. 甲醇 V 丙酮 The bacterial cells were resuspended by vortexing at a ratio of 7:3, and then disrupted by ultrasonication. The cells were extracted at 60°C for 80 min under dark conditions. Finally, the sample was centrifuged at 14000 r / min for 10 min. The supernatant was filtered through a 0.22 μm organic phase microporous membrane, and the lycopene content was determined by high performance liquid chromatography.

[0026] Further, the fermentation medium is TSB liquid medium, and the specific steps are as follows: a single colony is picked from the solid medium containing the recombinant Bacillus subtilis strain and added to the TSB liquid medium, and cultured at 37°C and 200 rpm for 16 hours to obtain the seed culture of the recombinant Bacillus subtilis strain; the percentage of each component in the solid culture relative to the mass of the solid medium is as follows: tryptone 1%; yeast extract 0.5%; NaCl 1%; agar 2%; pH 7.0;

[0027] The percentages of each component in the TSB medium by weight are as follows: tryptone 1.7%; soybean peptone 0.3%; NaCl 0.5%; glucose 0.25%; pH 7.0.

[0028] The seed culture medium was adjusted according to the initial OD. 600 0.1 g was inoculated into TSB liquid medium for fermentation under the following conditions: temperature 37℃, rotation speed 220 rpm, and fermentation culture for 24 h.

[0029] This invention constructs gene elements and modules using specific endogenous and exogenous genes of Bacillus subtilis, and transfers them into the genome of Bacillus subtilis with the amyE, thrC, vpr, and epr genes knocked out, thereby constructing a recombinant lycopene chassis strain. After being introduced into a cytosine base editor and induced to edit, a Bacillus subtilis mutant library producing lycopene is obtained. High-throughput screening yields a high-lycopene-producing Bacillus subtilis strain.

[0030] As a preferred option, the specific gene fragments mentioned above are as follows:

[0031] Bacillus subtilis promoter P gapDH RBS, dxs, and T, which contain eight consecutive G bases M3 Gene fragment 1, formed by sequentially splicing terminators (see schematic diagram) Figure 1 More preferably, the gene fragment 1 is as shown in SEQ ID NO:1.

[0032] Bacillus subtilis promoter P gapDH RBS, dxr, ispD, and T, which contain eight consecutive G bases. M3 Gene fragment 2, formed by sequentially splicing terminators (see schematic diagram) Figure 2 Further preferably, the gene fragment 2 is as shown in SEQ ID NO:2.

[0033] Bacillus subtilis promoter P gapDH RBS and ispE containing eight consecutive G bases, RBS, ispF, and T containing eight consecutive G bases M3 Gene fragment 3, formed by sequentially splicing terminators (see schematic diagram) Figure 3 Further preferably, the gene fragment 3 is as shown in SEQ ID NO:3.

[0034] Bacillus subtilis promoter P gapDH RBS containing 8 consecutive G bases, ispG, RBS containing 8 consecutive G bases, ispH, and T M3 Gene fragment 4, which is formed by sequentially splicing terminators (see schematic diagram) Figure 4Further preferably, the gene fragment 4 is as shown in SEQ ID NO:4.

[0035] Bacillus subtilis promoter P gapDH RBS containing 8 consecutive G bases, ispA, RBS containing 8 consecutive G bases, idi, and T M3 Gene fragment 5, which is formed by sequentially splicing terminators (see schematic diagram). Figure 5 Further preferably, the gene fragment 5 is as shown in SEQ ID NO:5.

[0036] Bacillus subtilis promoter P gapDH RBS containing 8 consecutive G bases, crtE, RBS containing 8 consecutive G bases, crtI, RBS containing 8 consecutive G bases, crtB, and T M3 Gene fragment 6, which is formed by sequentially splicing terminators (see schematic diagram) Figure 6 Further preferably, the gene fragment 6 is as shown in SEQ ID NO:6.

[0037] The Bacillus subtilis mutant library for producing lycopene described in this invention is capable of synthesizing lycopene. Therefore, this invention also provides the application of the recombinant Bacillus subtilis mutant library in the production of lycopene and in the production of products with lycopene as an intermediate.

[0038] In one specific implementation, the construction method includes:

[0039] Gene fragment 1 was constructed and transformed into wild-type Bacillus subtilis to obtain recombinant Bacillus subtilis strain BSCK6-Dxs;

[0040] Specifically, the Bacillus subtilis endogenous 5-phosphate deoxyxylulose synthase gene dxs is coupled with the promoter P gapDH RBS and T containing 8 consecutive G bases M3 Gene fragment 1, which was sequentially spliced ​​from terminators, was used to construct a CRISPR / Cas9 single plasmid pBAC9987-gRNA1-dxs, which targets the amyE gene insertion site.

[0041] The plasmid pBAC9987-gRNA1-dxs was transformed into the wild-type Bacillus subtilis strain BSCK6-WT. The recombinant Bacillus subtilis strain BSCK6-Dxs was obtained by recombination of the upstream and downstream homologous sequences of the amyE site in gene fragment 1 with the amyE site on the genome of the recombinant Bacillus subtilis strain BSCK6-WT through recombination with the amyE site on the genome of the recombinant Bacillus subtilis strain BSCK6-WT.

[0042] Furthermore, the construction method also includes:

[0043] Gene fragment 2 was constructed, and after gene fragment 2 was transferred into the recombinant Bacillus subtilis strain BSCK6-Dxs, the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspD was obtained.

[0044] Specifically, the endogenous MEP pathway genes dxr and ispD of Bacillus subtilis are combined with the promoter P. gapDH RBS and T containing 8 consecutive G bases dnaA Gene fragment 2, which is composed of the terminator and the upstream and downstream homologous sequences of the Bacillus subtilis amyE site, was used to construct a CRISPR / Cas9 single plasmid pBAC9987-gRNA2-dxr-ispD targeting the amyE site in the gene insertion expression cassette.

[0045] The plasmid pBAC9987-gRNA2-dxr-ispD was transformed into the recombinant Bacillus subtilis strain BSCK6-Dxs via chemical transformation. The recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspD was obtained by recombination of the upstream and downstream homologous sequences of the amyE site in gene fragment 2 with the amyE site on the genome of the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspD.

[0046] Furthermore, the construction method also includes:

[0047] Gene fragment 3 was constructed, and after gene fragments 1 and 2 were introduced, gene fragment 3 was introduced into the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspD to obtain the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEF.

[0048] Specifically, the endogenous MEP pathway genes ispE and ispF of Bacillus subtilis are combined with the promoter P. gapDH RBS and T containing 8 consecutive G bases M3 Gene fragment 3, which was sequentially spliced ​​from terminators, was used to construct a CRISPR / Cas9 single plasmid pBAC9987-gRNA3-ispEF targeting the thrC site in the gene insertion expression cassette.

[0049] The plasmid pBAC9987-gRNA3-ispEF was transformed into the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspD via chemical transformation. The recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspD was integrated into the genome by recombination of the upstream and downstream homologous sequences of the thrC site in gene fragment 3 with the thrC site on the genome of the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspD.

[0050] Most preferably, the construction method further includes:

[0051] Gene fragment 4 was constructed, and after gene fragments 1, 2, and 3 were introduced, gene fragment 4 was introduced into the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEF to obtain the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGH.

[0052] Specifically, the endogenous MEP pathway genes ispG and ispH of Bacillus subtilis are combined with the promoter P. gapDH RBS and T containing 8 consecutive G bases M3 Gene fragment 4, which was sequentially spliced ​​from terminators, was used to construct a CRISPR / Cas9 single plasmid pBAC9987-gRNA4-ispGH targeting the thrC site in the gene insertion expression cassette.

[0053] The plasmid pBAC9987-gRNA4-ispGH was transformed into the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEF via chemical transformation. The recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEF was integrated into the genome through recombination of the upstream and downstream homologous sequences of the thrC site in gene fragment 4 with the thrC site on the genome of the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGH.

[0054] Most preferably, the construction method further includes:

[0055] Gene fragment 5 was constructed, and after gene fragments 1, 2, 3, and 4 were introduced, gene fragment 5 was introduced into the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGH to obtain the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi.

[0056] Specifically, the endogenous MEP pathway genes ispA and idi of Bacillus subtilis are combined with the promoter P. gapDH RBS and T containing 8 consecutive G bases M3 Gene fragment 5, which was sequentially spliced ​​from the terminator, was used to construct a CRISPR / Cas9 single plasmid pBAC9987-gRNA5-ispA-idi targeting the vpr site in the gene insertion expression cassette;

[0057] The plasmid pBAC9987-gRNA5-ispA-idi was transformed into the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGH via chemical transformation. The recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGH was then integrated into the genome through recombination of the upstream and downstream homologous sequences of the vpr site in gene fragment 5 with the vpr site on the genome of the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGH.

[0058] Most preferably, the construction method further includes:

[0059] Gene fragment 6 was constructed, and after gene fragments 1, 2, 3, 4, and 5 were introduced, gene fragment 6 was introduced into the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi to obtain the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi-CrtEIB.

[0060] Specifically, the heterologous lycopene biosynthesis genes crtE, crtI, and crtB are combined with the promoter P gapDH RBS and T containing 8 consecutive G bases M3 Gene fragment 6, which was sequentially spliced ​​from terminators, was used to construct a CRISPR / Cas9 single plasmid pBAC9987-gRNA6-crtEIB targeting the epr site in the gene insertion expression cassette.

[0061] The plasmid pBAC9987-gRNA6-crtEIB was transformed into the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi via chemical transformation. The recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi was integrated into the genome through recombination of the upstream and downstream homologous sequences of the epr site in gene fragment 6 with the epr site on the genome of the recombinant Bacillus subtilis strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi.

[0062] As an example, the gene insertion expression cassette fragment (shown in SEQ ID NO:1): 1-651bp is the promoter P gapDH The RBS sequence contains 8 consecutive G bases; 652-2553bp is the dxs gene sequence; 2554-2602bp is the T gene sequence. M3 Termination subsequence

[0063] Gene fragment 2 (shown as SEQ ID NO: 2): 1-651bp is P gapDHThe sequence consists of an RBS sequence containing eight consecutive G bases; 652-1803 bp is the dxr gene sequence; 1804-1836 is an RBS sequence containing eight consecutive G bases; 1807-2535 bp is the ispD gene sequence; and 2536-2577 bp is a T gene sequence. dnaA Terminate the subsequence.

[0064] Gene fragment 3 (SEQ ID NO: 3): 1-651bp is P gapDH The sequence consists of: 1. An RBS sequence containing 8 consecutive G bases; 652-1521 bp is the ispE gene sequence; 1522-1554 bp is an RBS sequence containing 8 consecutive G bases; 1555-2031 bp is the ispF gene sequence; and 2032-2080 bp is the T gene sequence. M3 Terminate the subsequence.

[0065] Gene fragment 4 (SEQ ID NO: 4): 1-651bp is P gapDH The sequence consists of an RBS sequence containing eight consecutive G bases; 652-1785 bp is the ispG gene sequence; 1786-1818 bp is an RBS sequence containing eight consecutive G bases; 1819-2763 bp is the ispH gene sequence; and 2764-2812 bp is a T gene sequence. M3 Terminate the subsequence.

[0066] Gene fragment 5 (SEQ ID NO: 5): 1-651bp is P gapDH The sequence consists of an RBS sequence containing 8 consecutive G bases; 652-1542bp is the ispA gene sequence; 1543-1575bp is an RBS sequence containing 8 consecutive G bases; 1576-2625bp is the idi gene sequence; and 2626-2674bp is the T gene sequence. M3 Terminate the subsequence.

[0067] Gene fragment 6 (SEQ ID NO: 6): 1-651bp is P gapDH The sequence consists of: 1. RBS sequence containing 8 consecutive G bases; 652-1563bp is the crtE gene sequence; 1564-1596bp is the RBS sequence containing 8 consecutive G bases; 1597-3075bp is the crtI gene sequence; 3076-3108bp is the RBS sequence containing 8 consecutive G bases; 3109-3999bp is the crtB gene sequence; 4000-4048bp is the T gene sequence. M3 Terminate the subsequence.

[0068] According to the technical solution and preferred embodiment of the present invention, the present invention uses Bacillus subtilis SCK6 as the starting strain, and outlines the process of constructing recombinant strains:

[0069] 1) Transform the gene expression cassette 1 fragment into Bacillus subtilis SCK6 and insert the amyE gene locus. The resulting strain was named BSCK6-Dxs.

[0070] 2) Transform the plasmid containing the gene expression cassette 2 fragment into BSCK6-Dxs, and the resulting strain is named BSCK6-Dxs-Dxr-IspD;

[0071] 3) Transform the plasmid containing gene expression cassette 3 into BSSCK6-Dxs-Dxr-IspD, and name the resulting strain BSSCK6-Dxs-Dxr-IspDEF;

[0072] 4) Transform the plasmid containing gene expression cassette 4 into BSCK6-Dxs-Dxr-IspDEF, and name the resulting strain BSCK6-Dxs-Dxr-IspDEFGH;

[0073] 5) Transform the plasmid containing gene expression cassette 5 into BSCK6-Dxs-Dxr-IspDEFGH, and name the resulting strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi;

[0074] 6) Transform the plasmid containing gene expression cassette 6 into BSCK6-Dxs-Dxr-IspDEFGHA-Idi, and name the resulting strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi-CrtEIB.

[0075] The beneficial effects of this invention are:

[0076] Bacillus subtilis is a known food-safe bacterial strain. While it cannot synthesize lycopene itself, it possesses the MEP pathway for terpene precursor biosynthesis. More importantly, Bacillus subtilis produces significantly higher levels of isoprene than most other eubacteria, including Escherichia coli. Therefore, a novel lycopene-producing strain using Bacillus subtilis as a natural product has a potential production capacity far exceeding that of Escherichia coli. This invention constructs a Bacillus subtilis chromosomal gene insertion expression cassette recombinant chassis strain. Through induction editing with a base editor, a large number of mutant libraries can be obtained, resulting in a combined theoretical library capacity of 7.9 × 10⁻⁶. 28 This combination provides a possibility for screening high-yield strains for producing the natural product lycopene.

[0077] In practical examples, by using the pigment deposition condition as a screening criterion of the present invention, from approximately 10 5 Among the colonies, strains with high lycopene production were selected. The strain with the best lycopene yield after 24 hours of fermentation had a yield of 3.9 mg / L, which is 7 times that reported in existing literature. Attached Figure Description

[0078] Figure 1 The diagram shown is a schematic representation of the gene elements in gene expression cassette 1.

[0079] Figure 2 The diagram shown is a schematic representation of the gene elements in gene expression cassette 2.

[0080] Figure 3 The diagram shown is a schematic representation of the gene elements in gene expression cassette 3.

[0081] Figure 4 The diagram shown is a schematic representation of the gene elements in gene expression cassette 4.

[0082] Figure 5 The diagram shown is a schematic representation of the gene elements in gene expression cassette 5.

[0083] Figure 6 The diagram shown is a schematic representation of the gene elements in gene expression cassette 6.

[0084] Figure 7 The diagram shows the integration of the amyE gene into gene expression cassette 1.

[0085] Figure 8 The induction and editing of mutant libraries constructed using Bacillus subtilis SCK6 as the starting strain;

[0086] Figure 9 Preparation of the lycopene standard curve: lycopene concentration standard curve;

[0087] Figure 10 The figure shown is a bar graph of lycopene production of the recombinant Bacillus subtilis strain constructed using Bacillus subtilis SCK6 as the starting strain in this invention. Detailed Implementation

[0088] The preferred embodiments of the present invention are described below, but they are not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

[0089] The gene elements used in constructing the recombinant yeast strain in this invention, such as promoters, terminators, CRISPR / Cas9 target sequences, endogenous genes, and exogenous genes, are all well-known in the art, and their specific sequences are known to those skilled in the art.

[0090] Example 1: Construction of Bacillus subtilis chromosome gene expression cassette 1 recombinant strain BSSCK6-Dxs

[0091] Using the Bacillus subtilis 168 genome as a template, upstream and downstream primers were designed to amplify the 900bp homologous sequences upstream and downstream of the Bacillus subtilis amyE gene using PCR. The following fragments were obtained by PCR using plasmids as templates or by gene synthesis: the Bacillus subtilis endogenous 5-phosphate deoxyxylulose synthase gene dxs and the promoter P. gapDH RBS and T containing 8 consecutive G bases M3 The terminator was used to sequentially assemble the gene insertion expression cassette fragment using OE-PCR. This fragment was then ligated into the vector pBAC8799 (the full genome sequence is shown in SEQ ID NO:7) via homologous recombination to obtain the integration vector pBAC9987-gRNA1-dxs. Using the Bacillus subtilis CRISPR / Cas9 targeted gene integration system, IPTG-induced editing was performed. The upstream and downstream 900bp homologous sequences of the amyE gene in the gene insertion expression cassette fragment recombinated with the amyE gene in the Bacillus subtilis genome, integrating the gene insertion expression cassette fragment into the amyE gene region of the Bacillus subtilis genome. After removing the editing plasmid, the recombinant strain BSCK6-Dxs of the Bacillus subtilis chromosomal gene insertion expression cassette was obtained.

[0092] Example 2: Construction of the recombinant Bacillus subtilis lycopene-editable chassis strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi-CrtEI B

[0093] Following the method in Example 1, based on the Bacillus subtilis mutant strain BSCK6-Dxs, the recombinant strain BSCK6-Dxs-Dxr-IspD was obtained by sequentially transferring the gene expression cassette 2 editing plasmid pBAC9987-gRNA2-dxr-ispD into the culture medium and inducing editing. Using the BSCK6-Dxs-Dxr-IspD recombinant strain as a substrate, the gene expression cassette 3 editing plasmid pBAC9987-gRNA3-ispEF was transferred to obtain the recombinant strain BSCK6-Dxs-Dxr-IspDEF. Using the BSCK6-Dxs-Dxr-IspDEF recombinant strain as a substrate, the gene expression cassette 4 editing plasmid pBAC9987-gRNA4-ispGH was transferred into the culture medium. The recombinant strain BSCK6-Dxs-Dxr-IspDEFGH was obtained; the recombinant strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi was obtained by transfecting the gene expression cassette 5 editing plasmid pBAC9987-gRNA5-ispA-idi with the BSCK6-Dxs-Dxr-IspDEF recombinant strain as the chassis; and the final recombinant Bacillus subtilis lycopene editable chassis strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi-CrtEIB was obtained by transfecting the gene expression cassette 6 editing plasmid pBAC9987-gRNA6-crtEIB with the BSCK6-Dxs-Dxr-IspDEF recombinant strain as the chassis.

[0094] Example 3: Establishment of a library of Bacillus subtilis lycopene mutants

[0095] Using the recombinant strain BSSCK6-Dxs-Dxr-IspDEFGHA-Idi-CrtEIB obtained in Example 2 as the starting strain, a recombinant strain containing a cytosine base editor (CRISPR / dCas9-AID) was introduced. Single colonies were picked from the culture medium and transferred to 5 mL of LB liquid medium, incubated at 30°C and 200 rpm for 12-16 h to obtain the seed culture. The seed culture was then distributed according to OD... 600 0.1 μL was inoculated into 5 mL of fresh LB liquid medium at 30 °C and 220 rpm. Different concentrations of IPTG (0.1 mM, 0.5 mM, 1.0 mM, and 2.0 mM) were added to induce editing of the RBS region in the chassis strain, generating a mutant library. The results are as follows: Figure 8 and Figure 9 As shown, using pigment deposition as the screening criterion, from approximately 10 5 Select strains that produce high levels of lycopene from the colonies.

[0096] Example 4: Extraction and analysis of lycopene from Bacillus subtilis

[0097] After collecting the bacterial cells, add 20 mg / mL lysozyme, incubate at 30°C for 30 min, centrifuge at 14000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Add 1 mL of extraction buffer (V 甲醇 V 丙酮 =7:3), the bacterial cells were first vortexed for 5 min to resuspend them, and then the bacterial cells were broken by ultrasonic disruption. The samples were extracted at 60℃ for 80 min under dark conditions. Finally, the samples were centrifuged at 14000 r / min for 10 min. The supernatant was filtered through a 0.22 μm organic phase microporous membrane and the lycopene content was determined by high performance liquid chromatography.

[0098] Example 5: Preparation of the Lycopene Standard Curve

[0099] A 20 mg / L standard stock solution was prepared by dissolving 2 mg of purified lycopene standard in 100 mL of ethyl acetate. This standard solution was then serially diluted to different concentrations: 0 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 1.5 mg / L, 2 mg / L, and 2.5 mg / L. A standard curve for lycopene in the five different concentrations of standard solutions was determined using high-performance liquid chromatography (HPLC). The results are as follows: Figure 10 As shown, the standard curve R of lycopene standard in standard solution 2 =0.998, good linearity, meets the usage standards.

[0100] Example 6: Production of Lycopene by Shake-Flavor Fermentation of Recombinant Bacillus subtilis Lycopene-Producing Strain

[0101] Single colonies were picked from solid culture media containing wild-type Bacillus subtilis strain SCK6, recombinant Bacillus subtilis lycopene chassis strain BSCK6-Dxs-Dxr-IspDEFGHA-Idi-CrtEIB, and mutant strains 1-3 selected from the mutant library and added to 25 mL of TSB liquid medium. The cultures were incubated at 37°C and 200 rpm for 16 h to obtain the seed culture of the recombinant Bacillus subtilis lycopene-producing strain. The seed culture was then divided according to OD... 600 0.1 mg was inoculated into 50 mL of TSB liquid medium, fermented at 37 °C and 220 rpm for 24 h. Results are as follows: Figure 10 As shown, the lycopene yield of different recombinant strains ranged from 0.6 to 3.9 mg / L. Among them, strain No. 1 had the best lycopene yield after 24 hours of fermentation, reaching 3.9 mg / L, which is 7 times higher than that reported in existing literature.

Claims

1. A method for constructing a recombinant Bacillus subtilis mutant library for producing lycopene, characterized in that, Includes the following steps: S1 integrates and expresses the functional genes for lycopene synthesis—gerany-based pyrophosphate synthase, phytopene dehydrogenase, and phytopene synthase—on the chromosome of the starting strain; preferably, on the chromosome of the starting strain… EPR The above genes were integrated into the gene site to obtain the chassis bacteria; More preferably, nine genes from the MEP pathway are integrated to obtain chassis bacteria, wherein the nine genes from the MEP pathway are... dxs , dxr , ispD , ispE , ispF , ispG , ispH , idi , ispA Gene; S2 introduced a cytosine base editor into the strain, and after inducing editing, obtained a Bacillus subtilis mutant library that produces lycopene.

2. The method for constructing a recombinant Bacillus subtilis lycopene mutant library as described in claim 1, characterized in that, The nine genes in the MEP pathway are derived from Bacillus subtilis; the exogenous genes, including the gerany pyrophosphate synthase gene, phytoene dehydrogenase gene, and phytoene synthase gene, are derived from Pantotheca acuminata and are respectively referred to as... crtE , crtI , crtB The promoters and terminators that control each gene are derived from Bacillus subtilis promoters and terminators. Preferably, the promoter is selected from the modified constitutive promoter P. gapDH The terminator is selected from T. M3 Termination of contract.

3. The method for constructing a recombinant Bacillus subtilis lycopene mutant library as described in claim 2, characterized in that, In S1, the constitutive promoter P is combined. gapDH And a gene expression cassette containing RBS with 8 consecutive G bases, containing dxs Gene expression cassette 1; containing dxr and ispD Gene expression cassette 2; containing ispE and ispF Gene expression cassette 3; containing ispG and ispH Gene expression cassette 4; containing ispA and idi Gene expression cassette 5; containing crtE , crtI , crtB Gene expression cassette 6 was sequentially integrated into the Bacillus subtilis genome to obtain editable Bacillus subtilis chassis bacteria that produce lycopene; Preferably, the nucleotides of expression cassette 1 are as shown in SEQ ID NO:1; the nucleotides of expression cassette 2 are as shown in SEQ ID NO:2; the nucleotides of expression cassette 3 are as shown in SEQ ID NO:3; the nucleotides of expression cassette 4 are as shown in SEQ ID NO:4; the nucleotides of expression cassette 5 are as shown in SEQ ID NO:5; and the nucleotides of expression cassette 6 are as shown in SEQ ID NO:

6. Specifically, the gene is integrated into the genome of the originating bacterium using the CRISPR / Cas9 targeted gene integration system.

4. The method for constructing a Bacillus subtilis mutant library for producing lycopene as described in claim 1, characterized in that, In S2, the RBS region of the basal bacteria was edited using the CRISPR / dCas9-AID base editing system to generate a mutant library.

5. The method for constructing a Bacillus subtilis mutant library for producing lycopene as described in claim 4, characterized in that, The basal bacteria were activated by the culture medium and then inoculated into fresh culture medium for culture. After culture, seed culture was obtained and transferred to culture medium with added inducer to induce bacterial cells and obtain mutant library.

6. The method for constructing a Bacillus subtilis mutant library for producing lycopene as described in claim 5, characterized in that, Seed liquid with OD 600 Inoculate 0.05-0.15 g of the sample into fresh culture medium, add 0.1-2.0 mM of IPTG inducer, and incubate at 25-35℃ and 180-220 rpm for 12-16 h.

7. The method for constructing a Bacillus subtilis mutant library for producing lycopene as described in any one of claims 1 to 6, characterized in that, The originating bacteria were Bacillus subtilis 168 and Bacillus subtilis SCK6.

8. A Bacillus subtilis mutant library for producing lycopene obtained by the construction method according to any one of claims 1 to 7.

9. Application of the Bacillus subtilis mutant library for producing lycopene as described in claim 8 in screening for Bacillus subtilis mutant strains that produce high lycopene.

10. A method for screening Bacillus subtilis mutant strains that produce high levels of lycopene, characterized in that, The strains of the Bacillus subtilis mutant library for producing lycopene as described in claim 8 were inoculated into the culture medium, and high-lycopene-producing Bacillus subtilis mutant strains were selected according to the amount of lycopene produced. Specifically, the Bacillus subtilis mutant strain that produces lycopene is activated in a seed culture medium and then inoculated into a fermentation culture medium for culture. After culture, the bacterial cells are collected to extract lycopene. The seed culture of the Bacillus subtilis mutant strain that produces lycopene was prepared at OD... 600 Inoculate 0.05-0.15g into the fermentation medium and incubate at 35-38℃ and 180-220 rpm for 12-72 h. More specifically, the seed culture of the Bacillus subtilis mutant library that produces lycopene was inoculated into TSB liquid medium at a temperature of 37°C and a rotation speed of 220 rpm for 24 h of fermentation. After collecting the bacterial cells, add lysozyme, incubate at 30°C for 30 min, centrifuge at 14000 r / min for 5 min, discard the supernatant, and collect the bacterial cells; add 1 mL of extraction buffer V. 甲醇 V 丙酮 The bacterial cells were resuspended by vortexing at a ratio of 7:3, and then disrupted by ultrasonic disruption. The cells were extracted at 60°C for 80 min under dark conditions. Finally, the sample was centrifuged at 14000 r / min for 10 min. The supernatant was filtered through a 0.22 μm organic phase microporous membrane, and the lycopene content was determined by high performance liquid chromatography. The fermentation medium is TSB liquid medium, and the specific steps are as follows: a single colony is picked from the solid medium containing the recombinant Bacillus subtilis strain and added to the TSB liquid medium. The culture is carried out at 37°C and 200 rpm for 16 h to obtain the seed culture of the recombinant Bacillus subtilis strain. The percentages of each component in the solid culture medium are as follows: tryptone 1%; yeast extract 0.5%; NaCl 1%; agar 2%; pH 7.

0. The percentages of each component in the TSB medium relative to the total mass of the TSB medium are as follows: tryptone 1.7%; soybean peptone 0.3%; NaCl 0.5%; glucose 0.25%; pH 7.

0. The seed culture medium was adjusted according to the initial OD. 600 0.1 g was inoculated into TSB liquid medium for fermentation under the following conditions: temperature 37°C, rotation speed 220 rpm, and fermentation culture for 24 h.

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