Bacillus subtilis for promoting lycopene production by means of extracellular organelle

Abstract

Disclosed in the present invention is Bacillus subtilis for promoting lycopene production by means of an extracellular organelle (membrane nanotube). Specifically, according to the present invention, using a gene recombination technology, the synthesized crtE, crtI, and crtB genes are integrated to construct a plasmid, and the plasmid is transformed into Bacillus subtilis to construct a chassis strain with increased lycopene yield. Then, by means of a gene recombination technology, a dyna gene derived from Bacillus subtilis is integrated to construct a plasmid, and the plasmid is transformed into the chassis strain to construct a recombinant strain with further increased lycopene yield. The results of the present application show that the membrane nanotube has a promoting effect on the production of lycopene from Bacillus subtilis.

Description

Bacillus subtilis, a bacterium that promotes lycopene production through extracellular organelles Technical Field

[0001] This invention relates to a Bacillus subtilis strain that promotes lycopene production through extracellular organelles, belonging to the field of biotechnology. Background Technology

[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. Bacillus subtilis, as a model microorganism, has profound and wide-ranging applications in the biomanufacturing industry, including recombinant protein production and the synthesis of bioactive substances. Functional membrane microdomains (FMMs) are a type of densely structured microdomain on the Bacillus subtilis cell membrane, participating in vital activities such as cell signal transduction, energy metabolism, and protein secretion. FMMs divide the cell membrane into regions, enabling compartmentalized cellular processes and providing a platform for protein and enzyme aggregation. Since the cell membrane surface area is relatively fixed, once the proportion of FMMs in the cell membrane reaches a certain level (nearly 50%), this proportion cannot be further increased through FMM component reconstruction. Therefore, effectively expanding the cell membrane surface area, thereby increasing the FMM scaffold, is of great significance for constructing efficient Bacillus subtilis cell factories.

[0003] Lycopene is an isoprene compound widely found in fruits and vegetables such as tomatoes and red carrots. As an antioxidant, lycopene can effectively enhance the antioxidant capacity of tissues and cells. However, as a hydrophobic isoprene compound, lycopene is mainly stored in the limited space of the cell membrane, which hinders its accumulation. We hypothesize that increasing the cell membrane surface area might effectively solve this problem.

[0004] In 2020, Researchers successfully induced Bacillus subtilis to produce nanotubes by culturing it on coverslips and slides coated with poly-L-lysine and applying a pressure of 80 kPa. Inducing nanotube production in Bacillus subtilis can significantly increase the cell membrane surface area, potentially leading to the efficient synthesis of the target product. However, [further details needed]. In previous work, Bacillus subtilis only produced nanotubes when cultured on coverslips and slides coated with poly-L-lysine and subjected to a pressure of 80 kPa. When poly-L-lysine and pressure were absent, Bacillus subtilis could not produce nanotubes. Considering the current technological limitations of the biomanufacturing industry, this method is not suitable for lycopene production. Therefore, this invention aims to explore how to increase lycopene production by increasing the cell membrane surface area. Summary of the Invention

[0005] To address the aforementioned issues, this invention utilizes molecular biology techniques to construct a Bacillus subtilis strain capable of efficiently expressing and synthesizing lycopene. Furthermore, extracellular organelles (nanotubes) are incorporated into this strain, significantly increasing the lycopene yield of the recombinant Bacillus subtilis strain.

[0006] The first objective of this invention is to provide a recombinant Bacillus subtilis that promotes lycopene production through extracellular organelles, wherein the recombinant Bacillus subtilis expresses the dyna gene encoding the protein shown in SEQ ID NO.7, and at least overexpresses a lycopene synthesis gene, wherein the lycopene synthesis gene includes geranyl-geranyl diphosphate synthase crtE, phytocyanin dehydrogenase gene crtI, and phytocyanin synthase gene crtB.

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

[0008] Further, the sequence of the gerany-gerany 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 will understand that the recombinant strain protected by this invention may also include other modifications, such as increasing the expression level of genes that can further increase lycopene production. These genes include, but are not limited to, the gene ERG10 encoding acetyl-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, and the gene IDI1 encoding isoprene pyrophosphate isomerase, etc.

[0009] Furthermore, the heterologous expression and overexpression can be either genome-integrated expression or free expression.

[0010] Furthermore, free expression via plasmids is preferred. Specifically, vectors expressing the dyna gene include, but are not limited to, the PHT01 plasmid, and vectors expressing the lycopene synthesis gene 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] A second objective of this invention is to provide a method for constructing the recombinant Bacillus subtilis, comprising the following steps:

[0013] S1. Construct recombinant plasmids or integration fragments containing the dyna gene, and construct recombinant plasmids or integration fragments containing the crtE gene, the crtI gene, and the crtB gene, respectively or simultaneously.

[0014] S2. The recombinant plasmid or integrated fragment obtained in S1 is introduced into the Bacillus subtilis host bacteria to obtain the recombinant Bacillus subtilis.

[0015] A third objective of this invention is to provide the application of the recombinant Bacillus subtilis in the preparation of lycopene.

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

[0017] Furthermore, magnesium salts are added to the fermentation system (magnesium salts are added to both the seed culture medium and the fermentation medium).

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

[0019] Furthermore, the recombinant bacteria are inoculated into a seed culture medium to prepare a seed solution, which is then inoculated into a fermentation culture medium and fermented at 30-37°C.

[0020] Furthermore, the fermentation medium without added magnesium salts consists of: 10-15 g / L yeast extract, 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, and 40-80 g / L glucose.

[0021] The beneficial effects of this invention are:

[0022] (1) In order to obtain a recombinant Bacillus subtilis strain capable of efficiently synthesizing lycopene, this invention first integrates the essential gene for lycopene synthesis in Bacillus subtilis into the host strain of Bacillus subtilis to construct strain BS PADK-FQHS, thereby endowing Bacillus subtilis with the ability to synthesize lycopene. Subsequently, based on this strain, the essential gene for the synthesis of extracellular organelles (nanotubes) in Bacillus subtilis is integrated to construct strain BS PADK-FQHS PHT-dyna, thereby endowing Bacillus subtilis with the ability to produce nanotubes.

[0023] (2) This invention successfully constructed a Bacillus subtilis strain capable of producing lycopene, achieving a lycopene yield of 48.5 mg / L. Subsequently, extracellular organelles (nanotubes) were successfully applied to lycopene production. Compared to Bacillus subtilis strains that do not express extracellular organelles (nanotubes), the Bacillus subtilis strain expressing extracellular organelles (nanotubes) achieved a lycopene yield of 81.8 mg / L, representing a 67.1% increase. Attached Figure Description

[0024] Figure 1 shows the colony PCR results of strain BS PADK-FQHS.

[0025] Figure 2 shows the colony PCR results of strain BS PADK-FQHS PHT-dyna.

[0026] Figure 3 shows the lycopene production results of strains BS PADK-FQHS and BS PADK-FQHS PHT-dyna.

[0027] Figure 4 shows the bacterial nanotube production of strain BS PADK-FQHS PHT-dyna, with the left side being the bright field field and the right side being the fluorescence field field. Detailed Implementation

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

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

[0030] Table 1 Primer sequences

[0031] Example 1: Construction of strain BS PADK-FQHS

[0032] (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 as a template and PADK-T and PADK-B as primers, the PADK plasmid fragment (SEQ ID NO.4) was amplified. Subsequently, the fragments were amplified using NEB's Gibson primers. Master Mix fuses the crte, crtI, and crtb gene fragments with the linearized vector PADK (refer to the product instructions for specific steps) to obtain the recombinant plasmid PADK-FQHS. The recombinant plasmid is then sent for sequencing; plasmids with correct sequencing results are stored at -20°C.

[0033] (2) The PADK-FQHS plasmid constructed in step (1) was transformed into Bacillus subtilis 168 via electroporation. The electroporation conditions were 2.5 kV, 5 ms, and recovery at 37°C for 5 h. The plasmid was then plated onto LB agar plates with a final concentration of 50 μg / mL kanamycin resistance and incubated at 37°C for 48 h. Single colonies were picked from the plates, and colony PCR was performed using PADK-JP-0 and PADK-JP-1 as primers. A validation fragment of 5243 bp was amplified (as shown in Figure 1), proving that the plasmid was successfully transformed into Bacillus subtilis 168. This demonstrates the successful construction of strain BS PADK-FQHS.

[0034] Example 2: Construction of strain BS PADK-FQHS PHT-dyna

[0035] (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 PHT01-M plasmid (Miaoling) as a template and PHT01-M-0 and PHT01-M-1 as primers, the PHT01-GJ linearized vector (SEQ ID NO.6) was amplified. Subsequently, the vector was processed using NEB's Gibson... Master Mix fuses the dyna gene fragment with the linearized vector PHT01-GJ (refer to the product instructions for specific steps) to obtain the recombinant plasmid PHT01-dyna. The recombinant plasmid is then sent for sequencing, and plasmids with correct sequencing results are stored at -20°C.

[0036] (2) The PHT01-dyna plasmid constructed in step (1) was transformed into BS PADK-FQHS via electroporation. The electroporation conditions were 2.5 kV, 5 ms, and recovery at 37°C for 5 h. The plating was then spread onto LB plates containing 5 μg / mL chloramphenicol and 50 μg / mL kanamycin, and incubated at 37°C for 48 h. Single colonies were picked from the plates, and colony PCR was performed using PHT-JP-0 and PHT-JP-1 as primers. A validation fragment of 4238 bp was amplified (as shown in Figure 2), proving that the plasmid was successfully transformed into BS PADK-FQHS. This demonstrates the successful construction of the strain BS PADK-FQHS PHT-dyna.

[0037] Example 3: Validation of lycopene production by strains BS PADK-FQHS and BS PADK-FQHS PHT-dyna

[0038] (1) Select single colonies of the constructed strains BS PADK-FQHS and BS PADK-FQHS PHT-dyna and incubate them in liquid LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, with additional 5 mM MgSO4 and 1 mM IPTG) with a final concentration of 5 mM MgSO4 and 1 mM IPTG for 12 h. Take 1.5 mL of the above-cultured seed culture and inoculate it into a 250 mL baffle shaker 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 ferment for 72 h.

[0039] (2) Add 0.6 mL of fermentation broth to 0.6 mL of ethyl acetate. Add 0.1 mL of cell disruption glass beads (0.1 mm in diameter). Use a cell disruptor at a linear velocity of 4 m / s. Each disruption cycle is 60 s, for a total of 10 cycles. Centrifuge at 10,000 rpm for 10 min. After collecting the upper organic phase, filter it through an organic filter membrane and analyze it using liquid chromatography (C18 column, mobile phase V). 乙腈 V 甲醇 V 异丙醇 = 5:3:2, flow rate 1 mL / min, UV absorption wavelength 450 nm). The results are shown in Figure 3. As can be seen from the figure, the lycopene yield of strain BS PADK-FQHS PHT-dyna is 167.1% of that of strain BS PADK-FQHS.

[0040] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant Bacillus subtilis strain that promotes lycopene production via extracellular organelles, characterized in that, The recombinant Bacillus subtilis expressed the dyna gene encoding the protein shown in SEQ ID NO.7, and at least overexpressed lycopene synthesis genes, which include the geraniol-geraniol diphosphate synthase gene crtE, the phytoene dehydrogenase gene crtI, and the phytoene synthase gene crtB.

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

3.

3. The recombinant Bacillus subtilis according to claim 1, characterized in that, The heterologous expression and overexpression are either genome-integrated expression or free expression.

4. The recombinant Bacillus subtilis according to claim 3, characterized in that, Free expression is achieved through plasmids, with the vector for expressing the dyna gene including the PHT01 plasmid and the vector for expressing the lycopene synthesis gene including the PADK plasmid.

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

6. The 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, Includes the following steps: The recombinant Bacillus subtilis as described in any one of claims 1-4 is used for fermentation production.

8. The method according to claim 7, characterized in that, The recombinant bacteria were inoculated into a seed culture medium to prepare a seed solution, which was then inoculated into a fermentation culture medium and fermented at 30-37℃.

9. The method according to claim 7, characterized in that, Add magnesium salt to the fermentation system.

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

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