Biological hybrid based on semi-artificial photosynthesis, synthetic method of biological hybrid and application of biological hybrid in yield increase of breast milk oligosaccharide

Abstract

The invention discloses a biological hybrid based on semi-artificial photosynthesis, a synthetic method of the biological hybrid and application of the biological hybrid to yield increase of breast milk oligosaccharide. The biological hybrid comprises metal sulfide nanoparticles self-synthesized in situ by microorganisms and bacillus subtilis, the metal sulfide nanoparticles are loaded on the surface of the bacillus subtilis and pericytoplasm. According to the invention, H2S is generated by using a sulfate reduction metabolic pathway of bacillus subtilis and reacts with metal ions, and a semiconductor material-biological hybrid loaded with metal sulfide nanoparticles is formed in situ on the surface and periplasmic space of thalli. The in-situ self-synthesis method disclosed by the invention is simple and high in universality, can be used for preparing different hybrids from various metal ions, provides a new way for solar-driven biological manufacturing and waste conversion, and promotes clean production and circular economy. According to the constructed hybrid, the content of NADPH in microorganisms can be increased without genetic engineering operation, the production capacity of 2 '-FL is improved, and the yield is remarkably increased.

Description

Technical Field

[0001] The invention belongs to the technical field of sustainable biomanufacturing and semiconductor nanomaterials, and specifically relates to a biohybrid based on semi-artificial photosynthesis, a synthesis method thereof, and an application in increasing the production of human milk oligosaccharides. Background Art

[0002] As global energy and environmental issues become increasingly severe, the development of renewable clean energy, especially the utilization and conversion of solar energy, has attracted widespread attention. In artificial photosynthetic systems, the photocatalytic system of semiconductor materials can efficiently convert solar energy, but it is difficult to achieve high activity and high selectivity of the product. Therefore, semi-artificial photosynthesis has emerged in recent years. By integrating semiconductor materials that efficiently absorb light energy with living cells that catalyze with high selectivity, an "artificial light cell" system is constructed, and the intracellular catalytic ability of microorganisms is used to convert the light energy absorbed by the semiconductor into chemical energy, which can greatly improve the efficiency of artificial photosynthesis and achieve the specific production of complex compounds, providing a new path for light-driven biomanufacturing technology. In semi-artificial photosynthetic systems, synthetic materials and biological components are indispensable. The former efficiently captures light and generates carriers, while the latter achieves highly specific catalysis with low substrate activation barriers. This system combines the advantages of living cells and semiconductor materials to convert solar energy into chemical energy, opening up new paths for more sustainable production systems. At present, the construction of semi-artificial photosynthetic systems usually adopts biomineralization or artificial assembly strategies. However, biomineralization carries the risk of heavy metal precursor toxicity and environmental pollution, while artificial assembly requires surface modification of materials or microbial cells, which is complex and costly. Summary of the invention

[0003] Purpose of the invention: In view of the problems existing in the above-mentioned prior art, the present invention provides a biohybrid based on semi-artificial photosynthesis. The biohybrid is a semiconductor-biohybrid material, which uses Bacillus subtilis as a carrier and utilizes the aerobic sulfate reduction pathway of the strain itself to enable it to directly utilize heavy metal ions, sulfates and organic matter in the environment, and in situ self-synthesize nanoscale metal sulfide nanoparticles on the cell surface and periplasmic space of the microorganism. Under the catalysis of visible light, the metal sulfide nanoparticles generate photogenerated electrons, and the electrons can promote the formation of reducing power NADPH after entering the cytoplasm, providing sufficient energy for the biosynthesis pathway of 2′-FL, a human milk oligosaccharide, thereby increasing the yield of 2′-FL to 2.1 times. The biohybrid constructed by the present invention realizes the advantageous combination of biological whole-cell catalysts and semiconductor nanomaterials, enabling non-photosynthetic microbial cell factories to use solar energy to produce high-value chemicals, while avoiding the damage caused by current direct biomineralization and artificial assembly, making solar-driven biomanufacturing and waste conversion a step forward, and paving the way for clean production and remediation of heavy metal pollution in the environment.

[0004] The present invention also provides a synthesis method of the biohybrid material based on semi-artificial photosynthesis and an application of increasing the yield of human milk oligosaccharide 2′-FL.

[0005] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses a biohybrid based on semi-artificial photosynthesis, which comprises metal sulfide nanoparticles synthesized in situ by microorganisms and Bacillus subtilis, wherein the metal sulfide nanoparticles are loaded on the surface and periplasm of the Bacillus subtilis.

[0006] The biohybrid utilizes the sulfate reduction metabolic pathway of Bacillus subtilis to generate H 2 S directly combines with metal ions, thereby in situ self-synthesizing and loading nano-scale metal sulfide nanoparticles on the cell surface and its periplasmic space to form a semiconductor material biohybrid. The Bacillus subtilis is a conventional wild-type Bacillus subtilis or a 2′-FL-producing Bacillus subtilis.

[0007] Among them, conventional wild-type Bacillus subtilis such as Bacillus subtilis 168.

[0008] The method for self-synthesis of the semi-artificial photosynthesis biohybrid of the present invention comprises the following steps:

[0009] (1) culturing the activated Bacillus subtilis and inducing expression;

[0010] (2) adding a salt containing heavy metal ions and a sulfur source to the Bacillus subtilis in step (1), and continuing to culture to obtain the metal sulfide-Bacillus subtilis biohybrid, i.e., the semi-artificial photosynthesis biohybrid.

[0011] Preferably, the method for synthesizing a biohybrid based on semi-artificial photosynthesis according to the present invention comprises the following steps:

[0012] (1) The Bacillus subtilis engineered bacteria activated in LB medium were inoculated into sugar-containing TB liquid medium for aerobic expansion culture, and isopropylthiogalactoside inducer was added to induce expression;

[0013] (2) adding a certain amount of heavy metal ions and a sulfur source to the expanded Bacillus subtilis, and continuing to culture to obtain the metal sulfide-Bacillus subtilis biohybrid;

[0014] The activated Bacillus subtilis in step (1) is cultured by inoculating it in a sugar-containing TB liquid culture medium for aerobic expansion culture, and adding isopropylthiogalactoside inducer for inducing expression.

[0015] Preferably, the sugar-containing TB liquid medium in step (1) comprises 24 g / L yeast extract, 12 g / L peptone, 4 ml / L glycerol, 0.8 g / L K 2 HPO 4 , 0.2g / L KH 2 PO 4 , 60g / L glucose, 20g / L lactose; after the preparation of the sugar-containing TB culture medium is completed, it is placed in a sterilizing pot for sterilization at 115°C for 15min and set aside for later use.

[0016] LB medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, sterilize in an autoclave at 120°C for 20 min and set aside for later use.

[0017] Wherein, the isopropylthiogalactoside inducer has a concentration of 0.2-1.0 mM; the OD of the bacterial solution after fermentation of Bacillus subtilis in TB expansion medium is 600 =8~20.

[0018] Preferably, the OD 600 =10.

[0019] Wherein, the heavy metal ion-containing substance in step (2) includes one or more combinations of anhydrous cadmium chloride, lead acetate trihydrate, and mercuric nitrate, and the heavy metal ion concentration is 0.1-1.0 mM; the sulfur source is L-Cys, and the concentration is 1.0-100 mM. Further, the heavy metal ion solution is prepared by dissolving the corresponding drug in sterile water, vortexing for 15 minutes to fully mix, ultrasonicating for 30 minutes, passing through a 0.45 μm water filter to sterilize, and then storing in a 4°C refrigerator away from light; the L-Cys solution is prepared by dissolving the drug in sterile water, vortexing for 15 minutes to fully mix, ultrasonicating for 30 minutes, passing through a 0.45 μm water filter to sterilize, and then storing in a 4°C refrigerator away from light.

[0020] The induced expression TB induction fermentation culture temperature is 30-37°C, the rotation speed is 200-220rpm, and the fermentation time is 4-5 days.

[0021] Preferably, the induced fermentation conditions are: temperature 37° C., rotation speed 220 rpm, and fermentation time 4 days.

[0022] The semi-artificial photosynthesis biohybrid of the present invention is used in increasing the production of human milk oligosaccharide 2′-FL.

[0023] Among them, the mixed fermentation liquid based on semi-artificial photosynthesis biohybrid is subjected to visible light catalytic reaction to obtain the required solar energy and achieve increased production of the target product 2′-FL.

[0024] Wherein, the light intensity is 0~14mW / cm 2 The total illumination time is 3 to 48 hours; the pH of the environmental conditions is 6 to 9; and the ambient temperature is 30 to 37°C.

[0025] Preferably, the light intensity is 2-6 mW / cm 2 The total lighting duration is 24 to 48 hours.

[0026] More preferably, the light intensity is 4 mW / cm 2 , 12h light-12h dark-alternately twice.

[0027] The semi-artificial photosynthesis-based biohybrid material of the present invention is used to increase the yield in the whole-cell synthesis of 2′-FL. The biohybrid comprises metal sulfide nanoparticles synthesized in situ by microorganisms and Bacillus subtilis. The metal sulfide nanoparticles are loaded on the surface and periplasm of the Bacillus subtilis. The Bacillus subtilis is a Bacillus subtilis that produces 2′-FL.

[0028] Wherein, the preferred and specific preparation process of the application is as follows:

[0029] (1) Inducible 2′-FL production and fermentation optimization

[0030] Under sterile conditions, the logarithmic phase Bacillus subtilis activated by LB medium is inoculated into fresh sugar-containing TB medium at a volume ratio of 6% to 10% for expansion culture, and placed in a constant temperature shaking fermentation shaker for shaking culture; wherein the fermentation culture temperature is 30 to 37°C, the shaking speed is 200 to 220 rpm, and the time is 12 to 16 hours.

[0031] (2) Preparation of precursor solution

[0032] A 0.6M sterile aqueous solution of L-Cys and a 0.1M sterile aqueous solution of cadmium chloride were prepared respectively; the preparation methods of lead acetate and mercuric nitrate were the same as above.

[0033] (3) In situ self-synthesis of metal sulfides mediated by Bacillus subtilis

[0034] Use fresh TB medium to dilute the bacterial solution obtained during the above fermentation time to OD 600 =8~20, preferably, OD 600 = 10, add isopropyl thiogalactoside for induction, and add 0.6M L-Cys and 0.1M heavy metal ion solution (Cd 2+ / Pb 2+ / Hg 2+), the culture conditions were 30-37°C, the rotation speed was 200-220rpm, and the semiconductor-biohybrid material was initially obtained. Samples were taken every 24 hours, and the sampled fermentation liquid was frozen in a -20°C refrigerator. The product 2′-FL was detected by high performance liquid chromatography. Liquid phase conditions: Model: HPX-87H organic acid column; Temperature: 60°C; Detector: differential detector; Mobile phase: 5mM H 2 SO 4 .

[0035] Wherein, the final concentration of L-Cys is 1.0-10 mM, the concentration of heavy metal ion solution is 0-1.0 mM, and the final concentration of isopropyl thiogalactoside is 0.2 mM.

[0036] (4) Photocatalytic increase in the production of Bacillus subtilis:

[0037] The semiconductor-biohybrid mixed fermentation liquid is subjected to photocatalysis, and the light intensity is 0-14 mW / cm 2 The illumination time is 3 to 48 hours. The precipitate and supernatant are collected by centrifugation, and the product 2′-FL is detected by high performance liquid chromatography.

[0038] The present invention proposes a novel strategy: sugar-producing microorganisms synergistically utilize heavy metals in the environment to in situ self-synthesize semiconductor biohybrid materials. The hybrid material can generate electrons through photocatalysis, promote the accumulation of NADPH, thereby enhancing the synthesis of the product 2′-FL and realizing the conversion from solar energy to chemical energy. In Bacillus subtilis, exogenously added glucose synthesizes GDP-4-keto-6-deoxymannose from Glucose-6-P through a series of enzyme-catalyzed reactions, and then generates GDP-L-fucose, which is an important precursor for the generation of 2′-FL. This process consumes reducing power NADPH and releases NADP + Therefore, the supply of NADPH becomes a key factor restricting the sugar production efficiency of Bacillus subtilis. Specifically, the present invention utilizes the aerobic sulfate reduction pathway of Bacillus subtilis itself to generate H 2 S, enabling it to directly utilize heavy metal ions in the environment (such as Cd 2+ , Pb 2+ , Hg 2+ etc.), in situ self-synthesize and load nano-scale metal sulfide nanoparticles on the surface of living Bacillus subtilis and its periplasmic space to form semiconductor material-biohybrid. Semiconductor biohybrid materials generate electron and hole pairs under visible light catalysis, promoting NADP +Reduction generates NADPH, providing sufficient energy for the biosynthesis pathway of human milk oligosaccharide 2′-FL, thereby increasing the yield of high value-added chemical 2′-FL by up to 2.1 times. The present invention can take solar-driven biomanufacturing and waste conversion a step further and pave the way for clean production and circular economy.

[0039] The present invention provides a biohybrid based on semi-artificial photosynthesis and a synthesis method thereof and an application in increasing the production of human milk oligosaccharides, thereby realizing the effective conversion of solar energy into chemical energy. The biohybrid prepared by the present invention generates endogenous H by adding an exogenous sulfur source through the sulfate reduction metabolic pathway of Bacillus subtilis. 2 S, further combined with heavy metal ions in the external environment, thereby in situ self-synthesizing and loading nano-scale metal sulfide nanoparticles on the cell surface and its periplasmic space to form a semiconductor material-biohybrid. This semiconductor biohybrid material has photoelectric properties and can generate electrons under light conditions to promote the cofactor NADP in engineering metabolism. + Reduction generates NADPH, which provides sufficient energy for the biosynthesis pathway of 2′-FL, thereby increasing the yield of 2′-FL. The in-situ self-synthesis method of the present invention is simple and has good universality. It can be used for a variety of heavy metals to prepare semi-artificial photosynthesis biohybrids, making solar-driven biomanufacturing and waste conversion a step forward, and paving the way for clean production and circular economy. This method can achieve the increase of NADPH content in microorganisms without genetic engineering operations, and improve the ability of Bacillus subtilis to synthesize 2′-FL.

[0040] The present invention adopts a specific preparation method to effectively combine the strains transformed by biological metabolic engineering with chemical materials, realizes the biocompatible combination of biological and inorganic materials, and at the same time, the Bacillus subtilis that cannot utilize light energy is assembled into a hybrid that can utilize light energy through this method, which further increases the utilization rate of light energy. It is relatively difficult to assemble inorganic materials on biological surfaces in the prior art, which not only requires further modification of inorganic materials / biological surfaces, but also requires close attention to the damage and lethality of inorganic materials to organisms, and the steps are complicated and the versatility is low. The preparation method of the present invention can be continuously generated along with the biological fermentation process, is simpler and more efficient, and has good biocompatibility, effectively overcoming the problem of inorganic materials on biological surfaces.

[0041] The present invention adopts an in-situ self-synthesis method, and hydrogen sulfide is endogenously produced through the metabolic pathway of the strain itself, and is diffused to the surface to form a semiconductor material with heavy metal ions. This method does not require further chemical modification of the inorganic material, nor does it require surface group modification of the production strain to achieve the purpose of connecting the inorganic material, effectively reducing the damage of the inorganic material to the organism. Since no modification steps are required, the present invention is more concise. The material modification of the prior art has specific requirements for pH, temperature, etc. during the fermentation process of the strain. Some materials can only be synthesized in an organic environment and cannot be formed in a bacterial solution. Some have strict requirements on pH, etc., and some materials are combined with bacteria, but will cause the bacteria to die. Therefore, the metal sulfide-bacteria hybrid in the present invention, in which sulfur is produced by the bacteria itself, is continuously synthesized with exogenous heavy metals during the production process to form a semiconductor material, which can be applied to a variety of metals and has versatility. This process has a certain degree of biological autonomous regulation, which reduces the damage to cells to a certain extent.

[0042] The Bacillus subtilis used in the present invention is a metabolically engineered Bacillus subtilis, which is transformed to produce 2′-FL from scratch. Its metabolic network is more complex, and its combination with an inorganic semiconductor can easily lead to bacterial death and no longer produce 2′-FL. The present invention successfully achieves good growth of the strain under the assembly of the two through regulation of sulfur sources, heavy metal ions, light, etc., and further improves the yield of 2′-FL.

[0043] The present invention integrates the best properties of biological whole-cell catalysts and semiconductor nanomaterials, enabling non-photosynthetic industrial microbial cell factories to use solar energy for chemical production, and the output can be further increased to 2.1 times. The method of the present invention can adsorb heavy metal ions, is an environmentally friendly production method, and can provide new ideas for solving heavy metal pollution in the environment and realizing green biomanufacturing.

[0044] The present invention combines the sugar-producing Bacillus subtilis after bioengineering for the first time with chemical materials, and enables it to use solar energy to further achieve a substantial increase in production. The present invention mainly achieves the following purposes: 1. The combination of biological and inorganic materials usually requires modification, the steps are relatively complex and the versatility is low. The combination method of the present invention does not require additional modification and has better biocompatibility. 2. The Bacillus subtilis used in the present invention is a common GRAS (Generally recognized as safe) strain. After metabolic engineering, it produces 2′-FL, and the metabolic network is complex. The present invention proposes for the first time to self-assemble the Bacillus subtilis that produces 2′-FL after transformation with inorganic semiconductors, and increase the yield to 2.1 times. 3. Compared with the common further metabolic transformation, the yield-increasing method of the present invention is simpler and more versatile than the metabolic transformation with strong specificity, and can provide new methods and ideas for further improving the yield of cell factories after metabolic transformation. 4. The present invention can solve the heavy metal pollution in the environment and lay the foundation for green biomanufacturing.

[0045] The present invention is based on the construction of semi-artificial photosynthesis biological hybrids. From the aspects of sulfur source screening, cadmium source screening, light intensity, and illumination time, the successful construction of the semiconductor material-Bacillus subtilis hybrid is determined, and the yield is increased after light catalysis, with the highest increase to 2.1 times the original. This method can be used in heavy metal cadmium, and also has a certain improvement effect in other heavy metals such as lead and mercury.

[0046] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0047] 1. The microorganism used in the present invention is Bacillus subtilis, a model microorganism widely used in the production of nutritional health products and drugs, which facilitates the research and expansion of the method to other organisms for further application potential.

[0048] 2. The present invention provides a material and method for promoting the combination of semiconductor materials and microorganisms to improve the ability of microorganisms to convert solar energy into chemical energy. The obtained semiconductor biohybrid material integrates the best properties of biological whole-cell catalysts and semiconductor nanomaterials, enabling non-photosynthetic microbial cell factories to use solar energy for chemical production.

[0049] 3. The bio-inorganic hybrid system constructed by the present invention reconstructs the positional relationship between microorganisms and inorganic materials. Microorganisms utilize sulfur sources in the environment and continuously convert them into H through their own existing sulfur metabolic pathways. 2S, diffuses to the microbial periplasmic space and cell surface at the same time, and further adsorbs heavy metal ions in the environment, thereby continuously generating semiconductor materials such as cadmium sulfide as it grows. Compared with direct biological adsorption of exogenously added semiconductor materials such as cadmium sulfide that have been prepared in advance, this method avoids the incompatibility of chemically synthesized semiconductor materials with direct biological adsorption, reduces cell damage, and has a higher time-space efficiency of the photocatalyst (Comparative Example 3).

[0050] 4. The present invention utilizes the excellent intracellular catalytic ability of Bacillus subtilis to efficiently convert the photogenerated electrons generated by semiconductors absorbing light energy into bioenergy that can be used by bacteria, potentially greatly improving the efficiency of artificial photosynthesis and the ability to specifically produce complex high-value-added chemicals.

[0051] 5. The method for constructing a biohybrid material based on semi-artificial photosynthesis provided by the present invention has the effect of improving the efficiency and yield of the synthesis of 2′-FL in whole cells. It is simple to prepare, has low modification cost, good enhancement effect, high system stability, high economic, energy and environmental benefits, and has technical potential for large-scale industrial application.

[0052] 6. The preparation method of metal sulfide semiconductors such as cadmium sulfide, lead sulfide, and mercuric sulfide synthesized by Bacillus subtilis provided by the present invention only requires the simple addition of heavy metal ions and cysteine ​​during the bacterial fermentation process, and the use of the sulfur metabolic pathway of Bacillus subtilis itself to self-synthesize photocatalytic semiconductors to enhance the metabolic process of Bacillus subtilis in producing 2′-FL. No high temperature, high pressure, and additional toxic reducing agents and stabilizers are required, and the preparation process is simple, low-cost, and environmentally friendly.

[0053] 7. The method for self-synthesizing semiconductor-bacteria biohybrid materials based on semi-artificial photosynthesis provided by the present invention utilizes microorganisms as carriers and synthetic substrates, is applicable to a variety of heavy metal ion sources, and has multiple advantages such as high photoelectron excitation ability and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 In order to adopt the method provided in Example 1, only Cd is added 2+ Optical density measurement graph.

[0055] Figure 2 For the Cd provided in Example 1 2+ -Cys (1 mM) optical density measurement graph.

[0056] Figure 3 The Cd prepared in Example 1 2+ -Cys (10 mM) optical density measurement graph.

[0057] Figure 4This is the SEM-EDS image of the cadmium sulfide-bacteria hybrid prepared in Example 2.

[0058] Figure 5 The XRD pattern of the cadmium sulfide-bacteria hybrid prepared in Example 2 is shown in FIG.

[0059] Figure 6 This is the photocurrent density spectrum of cadmium sulfide prepared in Example 2.

[0060] Figure 7 This is the Mott-Schottky curve of the cadmium sulfide prepared in Example 2.

[0061] Figure 8 This is a comparison chart of different simulated sunlight intensities prepared in Example 3.

[0062] Fig. 9 This is a comparison chart of different simulated light exposure times prepared in Example 4.

[0063] Fig.10 This is a diagram of the dark-light-dark natural lighting simulation prepared using Example 5.

[0064] Fig.11 The graph shows the yield of the pure bacterial system and the hybrid prepared in Example 6.

[0065] Fig.12 The growth curves under the addition of different cadmium sources prepared in Comparative Examples 1-2 are shown.

[0066] Fig.13 The Pb-only prepared in Example 7 2+ growth curve.

[0067] Fig.14 The Pb-only prepared in Example 7 2+ -Growth curve of Cys (1 mM).

[0068] Fig.15 The Pb-only prepared in Example 7 2+ -Growth curve of Cys (10 mM).

[0069] Fig.16 The Hg-only 2+ growth curve.

[0070] Fig.17 The Hg-only 2+ -Growth curve of Cys (1 mM).

[0071] Fig.18 The Hg-only2+ -Growth curve of Cys (10 mM).

[0072] Fig.19 The yields of 2′-FL prepared in Example 8 under different heavy metal ions are shown in FIG.

[0073] Fig. 20 The yield graph of 2′-FL prepared by exogenous addition and in situ self-synthesis in Comparative Example 3.

[0074] Fig.21 This is the XRD pattern of the exogenous Cds prepared in Comparative Example 3.

[0075] Fig. 22 Schematic diagram of the principle of light-induced yield increase based on semi-artificial photosynthesis biohybrids. DETAILED DESCRIPTION

[0076] The present invention can be better understood according to the following examples. It is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0077] The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Among them, cadmium chloride, cadmium sulfate, cadmium nitrate, lead acetate, and mercuric sulfate were purchased from McLean Reagent Co., Ltd. Glucose and lactose were purchased from Sinopharm Chemical Reagent Co., Ltd. 2′-FL was purchased from Shanghai Baosen Reagent Co., Ltd.

[0078] Mannose-6-phosphate isomerase gene (manA, Bacillus subtilis, GenBank: QJR45642.1), phosphomannose mutase gene (manB, Escherichia coli, GenBank: WQE50532.1), mannose-1-phosphate guanosyltransferase gene (manC, Escherichia coli, GenBank: QZI63055.1), GDP-mannose dehydratase gene (gmd, Escherichia coli, GenBank: QJZ12613.1), GDP-fucose synthase gene (wcaG, Escherichia coli, GenBank: QJZ12612.1), α-1,2-fucosyltransferase gene (futc, Thermosynechococcus vestitus, GeneBank: WP_011056838.1), Cm rThe resistance gene (Escherichia coli, GenBank: CP095137.1) was artificially synthesized according to the sequence on NCBI.

[0079] The 2′-FL-producing Bacillus subtilis in the present invention can be any 2′-FL-producing Bacillus subtilis constructed in the prior art.

[0080] The 2′-FL-producing Bacillus subtilis used in the embodiments of the present invention was obtained by the applicant based on genome information engineering and molecular biology technology on NCBI and provided by Nanjing Normal University. The main operation steps include: S1 using the cre / loxp system to knock out two β-galactosidase genes (yesZ, Bacillus subtilis, GenBank: WOA99808.1 and ganA, Bacillus subtilis, GenBank: QJR47995.1) in Bacillus subtilis 168 (Bacillus subtilis 168, NC-000964.3), blocking the lactose consumption pathway in B.subtilis, improving the lactose conversion rate, and screening to obtain the B.SubWT1 strain; S2 in the B.SubWT1 strain, overexpressing manA from Bacillus subtilis, introducing manB, manC, gmd, wcaG genes from Escherichia coli and futC from Thermosynechococcusvestitus, thereby introducing a de novo synthesis pathway of 2'-FL, and obtaining the B.SubWT2 strain as the Bacillus subtilis used in various embodiments of the present invention.

[0081] S1 includes the following steps:

[0082] Preparation of competent cells of Bacillus subtilis. The Bacillus subtilis 168 stored in the laboratory was streaked on solid LB medium and cultured in a constant temperature incubator at 37°C for 12 hours. A single colony was picked and inoculated into 1 mL PAB medium (20 g / L tryptone, 6 g / L yeast extract, 6 g / L beef extract, 14 g / L sodium chloride, 14.8 g / L KH 2 PO 4 , 5.2g / LK 2 HPO 4After preparation, adjust the pH to 7.0 with saturated NaOH, sterilize at 115°C for 20 min), and culture at 37°C, 220 rpm for 12 h to obtain an activated bacterial solution. Subsequently, transfer the bacterial solution to 3 mL PAB medium containing 3% glucose and culture at 37°C, 220 rpm for 2 h. Finally, add glycerol with a final concentration of 10% to the bacterial solution, mix well, and dispense into sterile 1.5 mL EP tubes at 500 μL / piece to obtain B. subtilis competent state, which is stored in a -80°C refrigerator for later use.

[0083] The cre / loxp knockout system was used to determine the upstream and downstream sequences of yesZ (Bacillus subtilis, GenBank: WOA99808.1) and ganA (Bacillus subtilis, GenBank: QJR47995.1) according to the genome information published on NCBI. Primers P8-F / P8-R and P9-F / P9-R were used to amplify the upstream and downstream 1000 bp fragments of yesZ and ganA as homology arms, and primers P10-F / P10-R were used to amplify the resistance gene Cm r , the three DNA fragments were fused by overlap extension PCR technology to obtain the knockout frames of yesZ and ganA, and transferred into the above B.subtilis competent state. The chloramphenicol-resistant LB plate was used for screening, and after PCR verification, the plasmid PDG148 (GenBank: CP157215.1) expressing Cre was transferred; the transformants carrying the PDG148 plasmid were inoculated in liquid LB containing 0.2mM isopropylthiogalactoside, cultured for 24h, 1μL of the culture solution was inoculated in 2mL LB liquid culture medium, cultured at 50℃ and 220rpm for 10h, and then streaked on a non-resistance LB solid plate; the colonies obtained by screening were spotted on ampicillin and chloramphenicol and non-resistance LB solid plates, and only those that can grow on non-resistance solid plates are engineering strains that knock out the target gene and eliminate the plasmid. The successfully transformed transformants were inoculated in 3mL LB liquid culture medium to obtain B.SubWT1 engineering strains with knockout yesZ and ganA genes.

[0084] S2 introduces the 2'-FL synthesis pathway

[0085] Heterologous expression of synthetic manA, manB, manC, gmd, wcaG and futc genes. Plasmid pHT01 (NZ CP148130.1) in the NCBI database was selected as the backbone, and P1-F / P1-R was used as primers to obtain a linearized vector by PCR amplification; P2-F / P2-R, P3-F / P3-R, P4-F / P4-R, P5-F / P5-R, P6-F / P6-R, P7-F / P7-R were used as primers to PCR amplify the synthetic genes manA, manB, manC, gmd, wcaG and futc, respectively, using NEB's Gibson Assembly Cloning kit, assemble the above target fragment and linearized vector to obtain plasmid pHT-ABCGWF (where A represents manA, B represents manB, C represents manC, G represents gmd, W represents wcaG and F represents futc). Pipette 500ng of plasmid pHT-ABCGWF and add it to the competent cells of B.SubWT1 above, culture at 37℃ for 2h, centrifuge at 5000rpm for 2min, retain about 100μL of culture medium, blow and mix the centrifuged bacteria, culture at 37℃ for 2h, and spread on LB agar plate containing ampicillin for overnight culture. Screen the single colony that has been successfully transformed and inoculate it into 3mL of liquid LB culture medium to obtain B.SubWT2 strain.

[0086] The obtained B.SubWT2 strain was activated by LB, inoculated in a sugar-containing TB medium at a volume ratio of 6% for expansion culture for 12, and then isopropylthiogalactoside was added for induction, and placed in a constant temperature shaking fermentation shaker at 37°C and 220rpm for shaking culture for 4 days. Samples were taken every 24 hours, and the supernatant was collected by centrifugation at 12000rpm for 10 minutes. The samples were filtered through a 0.22μm water filter membrane for high performance liquid chromatography detection of 2′-FL. The strain was used for the subsequent construction of biological hybrids. The primer sequences used above are shown in Table 1.

[0087] Table 1 Primer sequences

[0088]

[0089]

[0090] The composition of sugar-containing TB liquid medium: 24g / L yeast extract, 12g / L peptone, 4ml / L glycerol, 0.8g / L K 2 HPO 4 , 0.2g / L KH 2 PO 4, 60g / L glucose, 20g / L lactose; after the sugar-containing TB medium is prepared, place it in a sterilizer for sterilization at 115℃ for 15min and set it aside. LB medium: 10g peptone, 5g yeast extract, 10g NaCl, place it in a sterilizer for sterilization at 120℃ for 20min and set it aside.

[0091] As used herein, the term "L-Cys" refers to L-cysteine.

[0092] As used herein, the term "2'-FL" refers to 2'-fucosyllactose.

[0093] As used herein, the term "NADP + ” refers to nicotinamide adenine dinucleoside phosphate.

[0094] As used herein, the term "NADPH" refers to reduced nicotinamide adenine dinucleotide phosphate.

[0095] As used herein, the term "Glucose-6-P" refers to glucose-6-phosphate.

[0096] As used herein, the term "GDP-4-keto-6-deoxymannose" refers to guanosine diphosphate-4-keto-6-deoxymannose.

[0097] As used herein, the term "GDP-L-fucose" refers to guanosine diphosphate-L-fucose.

[0098] Example 1

[0099] This Example 1 investigates the ability of L-Cys to promote the formation of cadmium sulfide-Bacillus subtilis hybrids, and the semi-artificial photosynthesis biohybrids are prepared by the following method:

[0100] (1) Bacillus subtilis in the logarithmic phase after LB activation was inoculated into fresh sugar-containing TB medium at a volume ratio of 6%, mixed evenly, and placed in a constant temperature shaking fermentation shaker at 37° C. and 220 rpm for shaking culture for 12 h.

[0101] (2) Prepare a 0.6M L-Cys sterile aqueous solution and a 0.1M cadmium chloride sterile aqueous solution. Expand the above TB culture of Bacillus subtilis to OD 600=10, add 0.2M isopropyl thiogalactoside for induction, add prepared 0.6M L-Cys and 0.1M cadmium chloride solutions respectively at the beginning of induction expression, and culture in a constant temperature shaking fermentation shaker at 37°C and 220rpm. Ferment for 4 days to preliminarily obtain the semiconductor-biohybrid material, take samples every 12h, and freeze the sampled fermentation liquid in a -20°C refrigerator.

[0102] Among them, set three groups respectively, set to add only Cd 2+ , the concentration gradient is 0.0, 0.1, 0.3, 0.4, 0.5, 1.0mM; the amount of L-Cys added is set to 1mM, Cd 2+ The concentration gradient was 0.0, 0.1, 0.3, 0.4, 0.5, and 1.0 mM; the amount of L-Cys added was set to 10 mM, and the amount of Cd 2+ The concentration gradient was 0.0, 0.1, 0.3, 0.4, 0.5, and 1.0 mM. Three groups were set up in parallel, and samples were taken every 12 hours to measure the OD 600 , draw a growth curve.

[0103] (3) The fermentation liquid was centrifuged at 12000 rpm for 10 min to obtain bacterial precipitate and supernatant. The bacterial precipitate obtained was freeze-dried to obtain solid powder, which was retained for later use.

[0104] The optical density of the fermentation broth at different fermentation times and concentrations was measured at λ = 600 nm using a UV-spectrophotometer. Figure 1-3 As shown, in the absence of L-Cys, the growth of the engineered Bacillus subtilis was inhibited by Cd 2+ In the presence of L-Cys, the engineered Bacillus subtilis was severely inhibited by Cd 2+ The tolerance concentration of Bacillus subtilis to Cd was as high as 0.5 mM, which had almost no adverse effect on growth. The L-Cys concentration was fixed at 1 mM. In addition, when the L-Cys concentration was increased from 1 mM to 10 mM, Bacillus subtilis 2+ The tolerance concentration also increases accordingly. High concentration of L-Cys oxidation will produce too much reactive oxygen species leading to oxidative stress, while reducing the pH value of the culture medium, and L-Cys and its oxidation products interfere with enzyme activity and protein folding. When the final concentration of L-Cys added to the culture medium exceeds 10mM, high concentration of L-Cys leads to toxic effects, resulting in the death of a large number of Bacillus subtilis in the culture medium, which is not conducive to the construction of later biohybrids.

[0105] Depend on Figures 1 to 3It can be concluded that cysteine ​​can promote biomineralization and form material-biohybrids. The optimal solution ratio was determined to be: L-Cys final concentration of 10 mM, heavy metal ion solution final concentration of 0.1 mM, and isopropylthiogalactoside final concentration of 0.2 mM.

[0106] In the present invention Figure 1-3 For Cd alone at different concentrations 2+ , only add L-Cys, add Cd 2+ +Cys, the OD values ​​of the three groups were tested and it was found that 1mM Cd 2+ Under these conditions, a sharp drop in OD value can indirectly indicate the death of bacteria, and the death of a large number of bacteria can also be directly observed under a microscope.

[0107] Example 2

[0108] In this Example 2, the material properties and photoelectric properties of the cadmium sulfide-Bacillus subtilis hybrid under the optimal ratio were investigated.

[0109] (1) The LB-activated Bacillus subtilis was inoculated into a fresh sugar-containing TB medium at a volume ratio of 6%, mixed evenly, and placed in a constant temperature shaking fermentation shaker at 37° C. and 220 rpm for shaking culture for 12 h.

[0110] (2) Prepare a 0.6M L-Cys sterile aqueous solution and a 0.1M cadmium chloride sterile aqueous solution. Expand the above TB culture of Bacillus subtilis to OD 600 =10, add 0.2M isopropyl thiogalactoside for induction, add 0.6M L-Cys and 0.1M cadmium chloride solution respectively when induction expression begins, shake culture in a constant temperature shaking fermentation shaker at 37°C and 220rpm, ferment for 4 days, and preliminarily obtain the semiconductor-biohybrid material. Samples are taken every 24h, and the sampled fermentation liquid is frozen in a -20°C refrigerator.

[0111] Wherein, the final concentration of L-Cys is 10 mM, the final concentration of the heavy metal ion solution is 0.1 mM, and the final concentration of isopropyl thiogalactoside is 0.2 mM.

[0112] (3) The fermentation broth was centrifuged at 12,000 rpm for 10 min to obtain a bacterial precipitate and a supernatant; the bacterial precipitate was freeze-dried and stored for later use.

[0113] The morphology of the semiconductor-biohybrid materials is as follows Figure 4As shown, under a scanning electron microscope, it can be observed that the in situ self-synthesized cadmium sulfide nanoparticles are distributed on the surface of the bacteria, and the elements Cd and S are evenly distributed on the cells; by testing the above-mentioned bacterial precipitate freeze-dried powder, the XRD spectrum confirms that these nanoparticles are CdS, such as Figure 5 In addition, the photoinduced current of CdSNPs is 0.26 μA / cm 2 ( Figure 6 ), the flat conduction band is ~ -0.72ev( Figure 7 ), which is close to the range reported in the literature, further proving that the nanoparticles loaded on the bacteria are CdS and have good photoelectric properties.

[0114] Example 3

[0115] In this Example 3, the ability of the CdS-Bacillus subtilis hybrid to photosynthetically produce 2′-FL under different light intensities was investigated.

[0116] (1) The LB-activated Bacillus subtilis was inoculated into a fresh sugar-containing TB medium at a volume ratio of 6%, mixed evenly, and placed in a constant temperature shaking fermentation shaker at 37° C. and 220 rpm for shaking culture for 12 h.

[0117] (2) Prepare a 0.6M L-Cys sterile aqueous solution and a 0.1M cadmium chloride sterile aqueous solution. Expand the above TB culture of Bacillus subtilis to OD 600 =10, add 0.2M isopropyl thiogalactoside for induction, add 0.6M L-Cys and 0.1M cadmium chloride solutions at the same time of induction expression, and culture in a constant temperature shaking fermentation shaker at 37°C and 220rpm for 4 days.

[0118] Wherein, the final concentration of L-Cys is 10 mM, the concentration of heavy metal ion solution is 0.1 mM, and the final concentration of isopropyl thiogalactoside is 0.2 mM.

[0119] (3) When the mixed solution is cultured for 12 hours, turn on the shaker light source with a light intensity of 0 to 14 mW / cm 2 After 24 hours of illumination, turn off the light source and continue fermentation. Take samples every 12 hours, collect the precipitate and supernatant by centrifugation, and detect the product 2′-FL in the supernatant by high performance liquid chromatography. Liquid phase conditions: Model: Rezex TM ROA-Organic Acid H + (8%) chromatographic column; temperature: 60°C; detector: differential detector; mobile phase: 5 mM diluted H 2 SO 4 ; Flow rate: 0.6mL / min.

[0120] By detecting the production of 2′-FL under different light intensities for 24 hours, it was found that the production of 2′-FL increased with the increase of the light intensity of simulated natural light. However, excessive light intensity may cause photooxidative damage to the cell membrane and excessive concentration of reactive oxygen species in the cell, resulting in decreased cell viability. Therefore, when the light intensity is 4 mW / cm 2 The 2′-FL yield of 2.01±0.2 g / L was obtained at the optimal light power density. Figure 8 shown.

[0121] Example 4

[0122] In this Example 4, the ability of the CdS-Bacillus subtilis hybrid to photosynthetically produce 2′-FL under different illumination times was investigated.

[0123] The difference between this embodiment and embodiment 3 is that the illumination duration in step (3) is changed to 3, 12, 24, 36, and 48 hours; the illumination intensity is 4 mW / cm 2 .

[0124] like Fig. 9 As shown, at 4mW / cm 2 Under the light intensity of , the yield gradually increased with the increase of illumination time. At 24 h, the photocatalytic yield was the highest. After 24 h, the yield gradually decreased. This may be due to the photocorrosion of CdS NPs under long-term illumination, which leads to reduced activity or even decomposition failure, as well as electron-hole recombination, which reduces the redox capacity and leads to reduced catalytic efficiency.

[0125] Example 5

[0126] In this Example 5, the bacterial growth and 2′-FL production ability of the cadmium sulfide-Bacillus subtilis hybrid under the light-dark culture mode simulating natural light were investigated.

[0127] The difference between this embodiment and embodiment 4 is that the illumination condition in step (3) is changed to 4 mW / cm 2 During the culture process of the constant temperature shaking incubator with a light intensity of 100 nm, the light switch of the shaking incubator was switched every 12 h (light-dark cycle).

[0128] By observing the production of 2′-FL by the CdS-Bacillus subtilis hybrid under light-dark natural light simulation, it was found that Bacillus subtilis was more obvious under dark conditions, which is similar to the nocturnal respiration process of natural photosynthesis. The alternation of light and darkness can affect the metabolic pathways of bacteria, such as changing the activity of enzymes, and thus affecting the yield of products, such as Fig.10 shown.

[0129] Example 6

[0130] In this Example 6, the 2′-FL yield of the pure bacterial system and the cadmium sulfide-Bacillus subtilis hybrid under the optimal conditions of the above Examples 1-5 was investigated. Fig. 22 shown.

[0131] (1) Bacillus subtilis in the logarithmic phase after LB activation was inoculated into fresh sugar-containing TB medium at a volume ratio of 6% for expansion culture, and placed in a constant temperature shaking fermentation shaker for culture; wherein the fermentation culture conditions are: 37° C., 220 rpm, and culture for 12 h.

[0132] (2) Use a UV-spectrophotometer to measure the OD value of the fermentation liquid at λ = 600 nm. 600 =10, add isopropyl thiogalactoside inducer, L-Cys and cadmium chloride solution at the same time, so that the final concentrations in the culture medium are: isopropyl thiogalactoside solution (0.2mM), L-Cys (10mM), cadmium ion final concentration (0.1mM). The mixed solution is cultured in a constant temperature shaking fermentation shaker at 37°C and 220rpm for a total of 4 days to obtain a mixed fermentation liquid of cadmium sulfide-Bacillus subtilis hybrid material.

[0133] (3) When the mixed fermentation liquid in step (2) is cultured for 12 hours, the shaking table light source is turned on for photocatalysis, and the light intensity is adjusted to 4 mW / cm 2 , and the cells were treated alternately with 12 hours of light and 12 hours of darkness. After repeating twice, the light source was turned off and the fermentation continued. One group was cultured in total darkness, and the other group was cultured under the above light conditions.

[0134] (4) During steps (2) and (3), samples were taken every 12 hours and the cell density was measured using an ultraviolet spectrophotometer.

[0135] (5) Centrifuge at 12000 rpm for 10 min to collect the precipitate and supernatant. The precipitate is freeze-dried and stored for later use. The 2′-FL content in the supernatant is detected by high performance liquid chromatography. Liquid phase conditions: Model: Rezex TM ROA-Organic Acid H + (8%) chromatographic column; temperature: 60°C; detector: differential detector; mobile phase: 5 mM diluted H 2 SO 4 Flow rate: 0.6mL / min

[0136] By analyzing the liquid phase results, it was found that the synthetic semi-artificial photosynthesis biohybrid system had a significant increase in yield under light catalysis, increasing to about 2.59g / L, which was about 2.1 times higher than the control group, and could greatly enhance the metabolic production of 2′-FL. This shows that the metabolically engineered Bacillus subtilis successfully achieved the self-assembly of CdS semiconductor materials, and with the help of its photoelectric properties, the non-photosynthetic Bacillus subtilis can effectively utilize light energy and promote the production of 2′-FL. ( Fig.11 )

[0137] Comparative Example 1

[0138] Compared with Example 6, the difference between this comparative example 1 and Example 6 is that the cadmium chloride solution in step (2) is changed to a cadmium nitrate solution, and the influence of different cadmium sources on the formation of cadmium sulfide-Bacillus subtilis hybrids is explored. The other methods are the same as above.

[0139] Compared with the cell density after adding cadmium chloride, the bacterial activity decreased significantly after adding cadmium nitrate ( Fig.12 ).

[0140] Comparative Example 2

[0141] Compared with Example 6, the difference between Comparative Example 2 and Example 6 is that the cadmium chloride solution in step (2) is changed to a cadmium sulfate solution, and the influence of different cadmium sources on the formation of cadmium sulfide-Bacillus subtilis hybrids is explored. Other methods are the same as above.

[0142] Compared with the cell density after adding cadmium chloride, the bacterial activity decreased significantly after adding cadmium sulfate, which was similar to the growth after adding cadmium nitrate ( Fig.12 ).

[0143] Comparative Examples 1 and 2 illustrate that the optimal cadmium-containing metal salt used in the present invention is cadmium chloride.

[0144] Example 7

[0145] The method of Example 7 is the same as that of Example 6, except that the heavy metal ion source added in step (2) is replaced by lead acetate and mercuric nitrate respectively instead of cadmium chloride.

[0146] and Figure 1-3 Compared with the above, Pb 2+ , Hg 2+ It has a great influence on bacterial growth activity; Figure 13-18 As shown, the semiconductor biohybrid system constructed by the present invention is also suitable for removing Cd 2+ Other heavy metals, such as Pb 2+ , Hg 2+ wait.

[0147] Example 8

[0148] The method of Example 8 is the same as that of Example 6, except that the heavy metal ion source added in step (2) is replaced by lead acetate, mercuric nitrate, and a metal liquid mixed with lead acetate, mercuric nitrate, and the three in equal proportions, and the yield of 2′-FL after illumination is detected by high performance liquid chromatography.

[0149] Depend on Fig.19 As shown, the semiconductor-biohybrid system constructed by the present invention is applicable to CdS system as well as PbS and HgS system. Under illumination conditions, the increase in 2′-FL yield is compared with the blank group (other conditions are the same, only L-Cys and metal ions are not added during fermentation induction in the culture medium). The addition of the three metals alone and the equal-ratio mixture can increase the 2′-FL yield, which proves that the system is also applicable to other metal sulfides. This lays a theoretical foundation for the feasibility of the semi-artificial photosynthesis biohybrid in complex environments such as multiple heavy metal pollution.

[0150] Comparative Example 3

[0151] The method of Comparative Example 3 is the same as that of Example 6, except that the addition of L-Cys and cadmium chloride in step (2) is replaced by exogenous direct addition of successfully prepared cadmium sulfide nanoparticles, so that the final concentration of cadmium sulfide semiconductor in the culture medium is 0.1 mM; at the same time, compared with the in situ self-synthesis adopted by the present invention, the yield of 2′-FL under light is detected by high performance liquid chromatography.

[0152] The preparation method of the cadmium sulfide nanoparticles comprises the following steps: 3.2 g of thiourea and 2.3 g of cadmium sulfate are mixed with 70 mL of deionized water, magnetically stirred to dissolve the mixture to make it completely dissolved, and then the mixture is transferred to a high-pressure reactor lined with polytetrafluoroethylene, and then the mixture is placed in an oven at 160° C. to react at a constant temperature for 12 hours, and then taken out and naturally cooled. The reaction solution is alternately washed with deionized water and ethanol, centrifuged at 8000 rpm for 5 minutes, and freeze-dried to obtain the cadmium sulfide nanoparticles.

[0153] like Fig.21 As shown in the figure, the XRD spectrum shows that the single CdS nanoparticles were successfully prepared according to the above method; and compared with the in-situ self-synthesis of CdS by microorganisms, the yield of exogenous addition of CdS nanoparticles for photocatalytic production is much lower than that of the former ( Fig. 20 ).

[0154] In summary, the biohybrid material based on semi-artificial photosynthesis prepared by the present invention can tolerate high concentrations of heavy metal ions under the action of cysteine ​​(Example 1), which is very important for industrial applications. Compared with the currently commonly used methods such as direct biological adsorption of exogenously added semiconductor materials such as cadmium sulfide, it avoids the incompatibility of semiconductor materials and direct biological adsorption, and has a higher photocatalyst time and space efficiency (Comparative Example 3). At the same time, the present invention utilizes the excellent intracellular catalytic ability of Bacillus subtilis to efficiently convert the photogenerated electrons generated by semiconductor absorption of light energy into bioenergy available to bacteria, potentially greatly improving the efficiency of artificial photosynthesis and the ability to specifically produce complex high-value-added chemicals, greatly reducing energy loss and environmental pollution, and reducing costs, and has high economic, energy, and environmental benefits and technical potential for large-scale industrial applications.

Claims

1. A biohybrid based on semi-artificial photosynthesis, characterized in that: The biohybrid comprises metal sulfide nanoparticles synthesized in situ by microorganisms and Bacillus subtilis. The metal sulfide nanoparticles are loaded on the surface and periplasm of the Bacillus subtilis.

2. The biohybrid based on semi-artificial photosynthesis according to claim 1, characterized in that: The biohybrid utilizes the sulfate reduction metabolic pathway of Bacillus subtilis to generate H2S, which is directly combined with metal ions, thereby in situ self-synthesizing and loading nano-scale metal sulfide nanoparticles on the cell surface and its periplasmic space to form a semiconductor material biohybrid. The Bacillus subtilis is a conventional wild-type Bacillus subtilis or a 2′-FL-producing Bacillus subtilis.

3. A method for self-synthesis of the semi-artificial photosynthesis biohybrid according to claim 1, characterized in that: The steps include: (1) culturing the activated Bacillus subtilis and inducing expression; (2) When the induction expression begins, a salt containing heavy metal ions and a sulfur source are added to the Bacillus subtilis in step (1), and the culture is continued to obtain the metal sulfide-Bacillus subtilis biohybrid, i.e., the semi-artificial photosynthesis biohybrid.

4. The self-synthesis method according to claim 3, characterized in that The activated Bacillus subtilis in step (1) is cultured by inoculating it in a sugar-containing TB liquid culture medium for aerobic expansion culture, and adding isopropylthiogalactoside inducer to induce expression.

5. The self-synthesis method according to claim 4, characterized in that The isopropylthiogalactoside inducer has a concentration of 0.2-1.0 mM; the OD of the bacterial solution after fermentation of Bacillus subtilis in TB expansion medium is 600 =8~20.

6. The self-synthesis method according to claim 3, characterized in that: The heavy metal ion-containing substance in step (2) comprises one or more combinations of anhydrous cadmium chloride, lead acetate trihydrate, and mercuric nitrate, and the heavy metal ion concentration is 0.1 to 1.0 mM; the sulfur source is preferably L-Cys, and the concentration is 1.0 to 100 mM.

7. The preparation method according to claim 3, characterized in that: The fermentation temperature for induced expression is 30-37°C, the rotation speed is 200-220rpm, and the fermentation time is 4-5 days.

8. Application of a semi-artificial photosynthesis biohybrid in increasing the production of human milk oligosaccharide 2′-FL, the biohybrid comprising metal sulfide nanoparticles synthesized in situ by microorganisms and Bacillus subtilis, the metal sulfide nanoparticles being loaded on the surface and periplasm of the Bacillus subtilis, the Bacillus subtilis being a 2′-FL producing Bacillus subtilis.

9. The use according to claim 8, characterized in that: The mixed fermentation liquid based on the semi-artificial photosynthesis biohybrid is subjected to visible light catalytic reaction to obtain the required solar energy and achieve the increased production of the target product 2′-FL.

10. The use according to claim 9, characterized in that: The light intensity of the photocatalysis is 0 to 14 mW / cm 2 , the lighting time is 3 to 24 hours; the pH of the environmental conditions is 6 to 9; the ambient temperature is 30 to 37°C.

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