A Bacillus subtilis for promoting lycopene production through extracellular organelles

Abstract

The present invention discloses a Bacillus subtilis that promotes lycopene production through extracellular organelles. Specifically, the synthetic crtE , crtI , crtB genes are integrated into plasmid PADK to construct plasmid PADK-FQHS, and this plasmid is transformed into Bacillus subtilis to construct strain BS PADK-FQHS. Subsequently, the dyna gene derived from Bacillus subtilis is integrated into plasmid PHT01 to construct plasmid PHT-dyna, and this plasmid is transformed into strain BS PADK-FQHS to construct strain BS PADK-FQHS PHT-dyna. By detecting the lycopene yields of strains BS PADK-FQHS and BS PADK-FQHS PHT-dyna, the promoting effect of nanotubes on the lycopene production by Bacillus subtilis is demonstrated, and the yield is increased by 67.1%.

Description

Technical Field

[0001] The present invention relates to a Bacillus subtilis for promoting lycopene production through extracellular organelles, belonging to the field of biotechnology. Background Art

[0002] Microbial metabolic engineering aims to construct stable and efficient cell factories to increase the yield of target products or endow cells with the ability to produce new products. As a model microorganism, Bacillus subtilis has profound and extensive applications in biomanufacturing industries such as recombinant protein production and biosynthesis of bioactive substances. Functional Membrane Microdomains (FMMs) are a class of microdomains with a dense structure on the cell membrane of Bacillus subtilis, which participate in life activities such as cell signal transduction, energy metabolism, and protein secretion. FMMs partition the cell membrane, enabling compartmentalization of cellular processes and providing a platform for the aggregation of proteins and enzymes. Since the cell membrane surface area is relatively fixed, after the proportion of FMMs in the cell membrane reaches a certain level (nearly 50%), it is impossible to further increase this proportion by reconstructing the FMMs components. Therefore, effectively expanding the cell membrane surface area to increase the FMMs scaffold is of great significance for constructing an efficient Bacillus subtilis cell factory.

[0003] Lycopene is an isoprenoid compound widely present in fruits and vegetables such as tomatoes and red carrots. As an antioxidant, lycopene can effectively improve the antioxidant capacity of its own tissues and cells. As a hydrophobic isoprenoid compound, lycopene is mainly stored in limited spaces such as the cell membrane in cells, which will hinder their large accumulation. We speculate that this problem may be effectively solved by increasing the cell membrane surface area.

[0004] In 2020, et al. cultured Bacillus subtilis on cover glasses and slides coated with poly-L-lysine and successfully induced Bacillus subtilis to generate nanotubes by applying a pressure of 80 kPa. Significantly increasing the cell membrane surface area by inducing Bacillus subtilis to produce nanotubes is expected to achieve efficient synthesis of target products. However, in the work of et al., Bacillus subtilis can only produce nanotubes when cultured on cover glasses and slides coated with poly-L-lysine and under a pressure of 80 kPa. When poly-L-lysine and pressure are absent, Bacillus subtilis cannot produce nanotubes. Considering the existing technical conditions in the biomanufacturing industry, this method is not applicable to lycopene production. Therefore, the present invention aims to explore how to increase the yield of lycopene by increasing the cell membrane surface area. Summary of the Invention

[0005] To solve the above problems, based on molecular biology techniques, the present invention constructs a Bacillus subtilis that can efficiently express and synthesize lycopene, and on this basis, applies extracellular organelles (nanotubes) to this strain, greatly improving the lycopene production of the recombinant Bacillus subtilis strain.

[0006] The first object of the present invention is to provide a recombinant Bacillus subtilis that promotes lycopene production through extracellular organelles. The recombinant Bacillus subtilis expresses the coding gene dyna of the protein shown in SEQ ID NO.7 and at least overexpresses lycopene synthesis genes. The lycopene synthesis genes include geranylgeranyl diphosphate synthase crtE, phytoene dehydrogenase gene crtI, and phytoene synthase gene crtB.

[0007] Furthermore, the gene dyna sequence is as shown in SEQ ID NO.5.

[0008] Furthermore, the sequence of the geranylgeranyl diphosphate synthase gene crtE is preferably the sequence shown in SEQ ID NO.1, the sequence of the phytoene dehydrogenase gene crtI is preferably the sequence shown in SEQ ID NO.2, and the sequence of the phytoene synthase gene crtB is preferably the sequence shown in SEQ ID NO.3. Of course, those skilled in the art can understand that the recombinant strains protected by the present invention may also include other modifications, such as enhancing the expression level of genes that can further increase lycopene production. The genes include but are not limited to the gene ERG10 encoding acetoacetyl-CoA thiolase, the gene ERG13 encoding HMG-CoA synthase, the gene tHMG1 encoding HMG-CoA reductase, the gene ERG12 encoding mevalonate kinase, the gene ERG8 encoding mevalonate-5-phosphate kinase, the gene MVD1 encoding mevalonate pyrophosphate decarboxylase, the gene IDI1 encoding isopentenyl diphosphate isomerase, etc.

[0009] Furthermore, the heterologous expression and overexpression can be genomic integration expression or free expression.

[0010] Furthermore, free expression is preferably carried out through plasmids. Specifically, the vectors expressing the gene dyna include but are not limited to the PHT01 plasmid, and the vectors expressing the lycopene synthesis genes include but are not limited to the PADK plasmid.

[0011] Furthermore, the host cell of the recombinant Bacillus subtilis can be any strain of Bacillus subtilis, such as Bacillus subtilis WB600, Bacillus subtilis 168, etc.

[0012] The second object of the present invention is to provide a method for constructing the recombinant Bacillus subtilis, including the following steps:

[0013] S1. Construct a recombinant plasmid or an integration fragment containing the gene dyna, and construct recombinant plasmids or integration fragments containing the genes crtE, crtI, and crtB separately or simultaneously.

[0014] S2. Introduce the recombinant plasmid or integration fragment obtained in S1 into the Bacillus subtilis host bacterium to obtain the recombinant Bacillus subtilis.

[0015] The third object of the present invention is to provide the use of the recombinant Bacillus subtilis in the preparation of lycopene.

[0016] The fourth object of the present invention is to provide a method for producing lycopene, comprising the following steps: performing fermentation production using the recombinant Bacillus subtilis.

[0017] Further, a magnesium salt is added to the fermentation system (the magnesium salt is added to the seed medium and the fermentation medium).

[0018] Further, in the seed medium, the final concentration of magnesium ions is 3 - 7 mM, and in the fermentation medium, the final concentration of magnesium ions is 3 - 7 mM.

[0019] Further, the recombinant bacteria are inoculated into the seed medium to prepare a seed solution, and the seed solution is inoculated into the fermentation medium, and fermentation production is carried out at 30 - 37 °C.

[0020] Further, the composition of the fermentation medium without added magnesium salt includes: 10 - 15 g / L yeast powder, 4 - 8 g / L tryptone, 4 - 8 g / L (NH4)2SO4, 10 - 15 g / L K2HPO4·3H2O, 1 - 5 g / L KH2PO4, 1 - 5 g / L MgSO4, 40 - 80 g / L glucose.

[0021] The beneficial effects of the present invention:

[0022] (1) In order to obtain a genetically recombinant Bacillus subtilis capable of efficiently synthesizing lycopene, the present invention first integrates the genes necessary for Bacillus subtilis to synthesize lycopene into the Bacillus subtilis host bacterium to construct the strain BS PADK - FQHS, endowing Bacillus subtilis with the ability to synthesize lycopene. Then, based on this strain, the genes necessary for Bacillus subtilis to synthesize extracellular organelles (nanotubes) are integrated to construct the strain BS PADK - FQHS PHT - dyna, endowing Bacillus subtilis with the ability to produce nanotubes.

[0023] (2) The present invention successfully constructed Bacillus subtilis that can produce lycopene, with the lycopene yield reaching 48.5 mg / L. Subsequently, extracellular organelles (nanotubes) were successfully applied to lycopene production. Compared with Bacillus subtilis that does not express extracellular organelles (nanotubes), the Bacillus subtilis expressing extracellular organelles (nanotubes) had a lycopene yield of 81.8 mg / L, an increase of 67.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the colony PCR result of strain BS PADK-FQHS.

[0025] Figure 2 It is the colony PCR result of strain BS PADK-FQHS PHT-dyna.

[0026] Figure 3 It is the lycopene yield results of strain BS PADK-FQHS and strain BS PADK-FQHS PHT-dyna.

[0027] Figure 4 It is the production of bacterial nanotubes of strain BS PADK-FQHS PHT-dyna. Among them, the left side is the bright field view, and the right side is the fluorescence field view. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0029] The primer sequences involved in the present invention are as follows:

[0030] Table 1 Primer Sequences

[0031]

[0032] Example 1 Construction of Strain BS PADK-FQHS

[0033] (1) Using the synthesized gene crte as a template, and crte-T and crte-B as primers, the crte gene fragment (SEQ ID NO.1) was amplified; using the synthesized gene crtI as a template, and crtI-T and crtI-B as primers, the crtI gene fragment (SEQ ID NO.2) was amplified; using the synthesized gene crtb as a template, and crtb-T and crtb-B as primers, the crtb gene fragment (SEQ ID NO.3) was amplified; using the plasmid PADK plasmid as a template, and PADK-T and PADK-B as primers, the PADK plasmid fragment (SEQ ID NO.4) was amplified. Subsequently, the crte, crtI, and crtb gene fragments were fused with the linearized vector PADK using the Gibson Master Mix from NEB (the specific steps refer to the product manual), to obtain the recombinant plasmid PADK-FQHS. The recombinant plasmid was sent for sequencing, and the plasmids with correct sequencing were stored at -20°C.

[0034] (2) The PADK-FQHS plasmid constructed in step (1) was transformed into Bacillus subtilis 168 by electroporation. The electroporation conditions were a voltage of 2.5 Kv, an electroporation time of 5 ms, and a recovery at 37°C for 5 h. Then it was spread on an LB plate with a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C for 48 h. Single colonies on the plate were picked, and colony PCR was performed using PADK-JP-0 and PADK-JP-1 as primers to amplify a verification fragment of 5243 bp (as Figure 1 shown), proving that the plasmid was successfully transferred into Bacillus subtilis 168. It was proved that the strain BS PADK-FQHS was successfully constructed.

[0035] Example 2 Construction of Strain BS PADK-FQHS PHT-dyna

[0036] (1) Using Bacillus subtilis 168 as a template, and dyna-0 and dyna-1 as primers, the dyna gene fragment (SEQ ID NO.5) was amplified; using the PHT01-M plasmid (Miaoling) as a template, and PHT01-M-0 and PHT01-M-1 as primers, the linearized vector PHT01-GJ (SEQ ID NO.6) was amplified. Subsequently, the dyna gene fragment was fused with the linearized vector PHT01-GJ using the Gibson MasterMix from NEB (the specific steps refer to the product manual), to obtain the recombinant plasmid PHT01-dyna. The recombinant plasmid was sent for sequencing, and the plasmids with correct sequencing were stored at -20°C.

[0037] (2) The constructed PHT01-dyna plasmid in step (1) was transformed into BS PADK-FQHS by electroporation. The electroporation conditions were a voltage of 2.5 Kv, an electroporation time of 5 ms, and a 5-hour recovery at 37°C. Then it was spread on an LB plate containing chloramphenicol resistance at a final concentration of 5 μg / mL and kanamycin at 50 μg / mL, and cultured at 37°C for 48 h. Single colonies on the plate were picked, and using PHT-JP-0 and PHT-JP-1 as primers, colony PCR was performed to amplify a verification fragment of 4238 bp in size (as shown in Figure 2 ), proving that the plasmid was successfully transferred into BSPADK-FQHS. It was proved that the strain BS PADK-FQHS PHT-dyna was successfully constructed.

[0038] Example 3 Verification of Lycopene Production by Strain BS PADK-FQHS and Strain BS PADK-FQHS PHT-dyna

[0039] (1) Single colonies of the constructed strain BS PADK-FQHS and strain BS PADK-FQHS PHT-dyna were picked into a liquid LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and additionally supplemented with 5 mM MgSO4 and 1 mM IPTG) containing a final concentration of 5 mM MgSO4 and a final concentration of 1 mM IPTG, and cultured for 12 h. 1.5 mL of the above-prepared seed liquid was aspirated and inoculated into a 250 mL baffled flask containing 30 mL of lycopene medium (12 g / L yeast extract, 6 g / L tryptone, 6 g / L (NH4)2SO4, 12.5 g / L K2HPO4·3H2O, 2.5 g / L KH2PO4, 3 g / L MgSO4, 60 g / L glucose, 5 mM MgSO4, 1 mM IPTG), and fermented for 72 h.

[0040] (2) 0.6 mL of the fermentation broth was aspirated and added to 0.6 mL of ethyl acetate. Glass beads for cell disruption with a diameter of 0.1 mm and a total volume of 0.1 mL were added. A cell disruptor was used with a linear velocity of 4 m / s. Each disruption cycle was 60 s, and a total of 10 cycles were performed. Centrifugation was carried out at 10000 rpm for 10 min. After aspirating the upper organic phase, it was filtered through an organic filter membrane and then detected by liquid chromatography (C18 column, mobile phase V 乙腈 : V 甲醇 : V 异丙醇 = 5:3:2, flow rate of 1 mL / min, ultraviolet absorption wavelength of 450 nm). The results are as shown in Figure 3 . It can be seen from the figure that the lycopene production of strain BS PADK-FQHS PHT-dyna is 167.1% of that of strain BS PADK-FQHS.

[0041] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

Claims

1. A recombinant Bacillus subtilis for promoting lycopene production through extracellular organelles, characterized in that, The recombinant Bacillus subtilis overexpresses the coding gene of the protein shown in SEQ ID NO. 7 dyna , and at least overexpresses lycopene synthesis genes, and the lycopene synthesis genes include geranylgeranyl diphosphate synthase gene crtE , phytoene desaturase gene crtI and phytoene synthase gene crtB .

2. The recombinant Bacillus subtilis according to claim 1, characterized in that, Gene dyna The sequence is as shown in SEQ ID NO.5, and the gene crtE has a sequence as shown in SEQ ID NO.1, and the gene crtI has a sequence as shown in SEQ ID NO.2, and the gene crtB has a sequence as shown in SEQ ID NO.

3.

3. The recombinant Bacillus subtilis according to claim 1, characterized in that, The overexpression is genomic integration expression or episomal expression.

4. The recombinant Bacillus subtilis according to claim 3, characterized in that, Free expression is carried out through plasmids, where the genes to be expressed dyna The vector includes the PHT01 plasmid, and the vector for expressing the lycopene synthesis gene includes the PADK plasmid.

5. The method for constructing the recombinant Bacillus subtilis according to any one of claims 1 to 4, characterized in that, Comprising the following steps: S1. Construct a recombinant plasmid or an integration fragment containing gene dyna , and construct recombinant plasmids or integration fragments containing gene crtE , gene crtI and gene crtB respectively or simultaneously; S2. Introduce the recombinant plasmid or integration fragment obtained in S1 into the Bacillus subtilis host bacterium to obtain the recombinant Bacillus subtilis.

6. Use of the recombinant Bacillus subtilis according to any one of claims 1-4 in the preparation of lycopene.

7. A method for producing lycopene, characterized in that, Comprising the following steps: Perform fermentation production using the recombinant Bacillus subtilis according to any one of claims 1-4.

8. The method according to claim 7, characterized in that, Inoculate the recombinant bacterium into a seed medium to prepare a seed solution, inoculate the seed solution into a fermentation medium, and perform fermentation production at 30-37 °C.

9. The method according to claim 7, wherein Add a magnesium salt to the fermentation system.

10. The method according to claim 9, characterized in that, In the seed medium, the final concentration of magnesium ions is 3-7 mM; in the fermentation medium, the final concentration of magnesium ions is 3-7 mM.

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