Method for preparing bose through fermentation

Abstract

The invention provides a method for preparing vitrogen through fermentation, and belongs to the technical field of biology, according to the method, an aceE gene is expressed in escherichia coli MG1655 in an enhanced mode, and an escherichia coli genetically engineered bacterium MG1655-aceE is obtained. When the gene engineering strain is used for fermentation preparation of the bose, because the expression of pyruvate dehydrogenase is enhanced, under the aerobic condition, pyruvic acid generated by glucose metabolism is promoted to react to generate acetyl-CoA, then the acetyl-CoA enters tricarboxylic acid cycle, NADH is continuously generated in the whole reaction process, and the yield of the bose is increased. The generated NADH can catalyze the synthesis reaction of a substrate beta-acetone xyloside to finally prepare the botryine, the production method provided by the invention is green and environment-friendly, the conversion rate of the botryine during shake-flask culture reaches 96.37%, and a feasible new way is provided for green production of the botryine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing bosogenin by fermentation. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Bosylvin, also known as hydroxypropyl tetrahydropyrantriol, is a xylose derivative with anti-aging active ingredients. It can promote the synthesis of collagen, make the skin stronger and more elastic, improve fine lines on the neck, and prevent aging. It has a wide range of applications in the technical fields of cosmetics, food, biology, medicine, etc. At present, the main method for producing bosylvin is chemical synthesis, but the chemical synthesis method has a high emission of three wastes during the production process, which seriously pollutes the environment. Microbial fermentation is a process that uses microorganisms to convert raw materials into products needed by humans through specific metabolic pathways under suitable conditions. Compared with chemical synthesis, it is safer, more efficient, and more environmentally friendly, but there are few reports on the method of preparing bosylvin by microbial fermentation.

[0004] Patent CN 117737149 B specification

[0007] discloses that in the preparation process of bosin, when β-acetone xyloside is used as the reaction raw material, it is necessary to add reduced coenzymes to carry out enzyme-catalyzed synthesis reaction to obtain bosin. Patent CN118325755 B discloses an engineering bacterium for producing S-hydroxypropyl tetrahydropyrantriol and its construction method, which realizes the microbial catalytic preparation of bosin. The patent first transforms Rhodosporidium toruloides by genetic engineering means, and at the same time strengthens the expression of nicotinic acid phosphoribosyltransferase, nicotinamide nucleotide adenylate transferase, nicotinamide phosphoribosyltransferase, nicotinamide riboside kinase and ketoreductase in the strain, promoting NAD conversion through the nicotinic acid conversion pathway. + Subsequently, the modified genetically engineered bacteria, glucose, β-acetone xyloside and isopropanol were added to the phosphate buffer to construct a catalytic reaction system. Isopropanol was used as a reducing agent to convert NAD + Reduced to NADH, NADH then catalyzes β-acetone xyloside to obtain bosylcan. Compared with chemical synthesis, this patent has made great improvements in environmental protection and safety, but the genetic engineering bacteria transformation process needs to be carried out step by step, the transformation process is relatively complicated, and the isopropanol added when constructing the catalytic reaction system is a volatile flammable liquid with low toxicity. Therefore, increasing research and continuing to develop a green, environmentally friendly and efficient microbial fermentation method is of great significance for the preparation of bosylcan. Summary of the invention

[0005] In view of the above prior art, the purpose of the present invention is to provide a method for preparing bosogenin by fermentation. By transforming Escherichia coli and improving the fermentation method, green, environmentally friendly and efficient preparation of bosogenin is achieved.

[0006] To achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing bosogenin by fermentation, comprising the following steps: The activated genetically engineered Escherichia coli was inoculated into the culture medium, IPTG was added to induce the reaction, the dissolved oxygen content was controlled at 30%-35%, glucose and β-acetone xyloside were added to the culture medium to construct a fermentation reaction system, and after the reaction was completed, centrifugation was performed, and the supernatant was collected to prepare bosera.

[0007] The Escherichia coli genetically engineered bacteria is constructed by taking Escherichia coli MG1655 as a starting strain and overexpressing the aceE gene.

[0008] The method for constructing the genetically engineered Escherichia coli comprises the following steps: The aceE gene obtained by PCR amplification was inserted into the pCambia2300 plasmid vector to construct a recombinant expression vector, and the recombinant vector was transformed into Escherichia coli MG1655 to obtain genetically engineered Escherichia coli bacteria.

[0009] The genetically engineered Escherichia coli bacteria were inoculated into the culture medium at an inoculation rate of 1×10 6 CFU / mL.

[0010] The concentration of the IPTG is (0.5-1) mmol / L.

[0011] After the IPTG was added, the reaction was induced at 37°C for 2-3 hours.

[0012] The mass ratio of glucose to β-acetone xyloside is (2-4):1.

[0013] The fermentation reaction system reacts at 37° C. and 200 rpm for 70-72 hours.

[0014] The beneficial effects of the present invention include: The present invention enhances the expression of the aceE gene in Escherichia coli MG1655 to obtain the genetically engineered Escherichia coli MG1655-aceE. The Escherichia coli aceE gene is one of the key enzymes encoding the pyruvate dehydrogenase complex. After the aceE gene of Escherichia coli MG1655 is overexpressed by genetic engineering means, the pyruvate produced by glucose metabolism will be enhanced to flow to the TCA cycle. When the genetically engineered strain is used to ferment and prepare bosensein, the expression of pyruvate dehydrogenase is enhanced due to the enhanced expression of the aceE gene. Under aerobic conditions, the pyruvate produced by glucose through glycolysis metabolism is promoted to produce acetyl-CoA, and then acetyl-CoA enters the tricarboxylic acid cycle. NADH will be continuously generated during the entire reaction process. The generated NADH will catalyze the synthesis reaction of the substrate β-acetone xyloside, and finally bosensein is prepared. The byproducts produced by this production route are carbon dioxide and water. The production method provided by the present invention is green and environmentally friendly. The conversion rate of bosensein during shake flask culture reaches 96.37%, which provides a feasible new way for the green production of bosensein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Schematic diagram of metabolic regulation pathways. DETAILED DESCRIPTION

[0016] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0017] As mentioned above, Bossagra is a substance that is widely used in the technical fields of cosmetics, food, biology, medicine, etc. In the preparation process of Bossagra, microbial fermentation is greener and safer than chemical synthesis, and is gradually gaining favor. Increasing research and continuing to develop a green, environmentally friendly and efficient microbial fermentation method is of great significance for the preparation of Bossagra.

[0018] Based on this, the present invention provides a method for preparing bosaiin by fermentation. Escherichia coli is a common industrial strain with a clear genetic background and a mature fermentation process. It is a facultative anaerobic bacterium with a complete tricarboxylic acid cycle. Glucose and the like can be used as carbon sources and energy sources for Escherichia coli. In the metabolic process, NADH is formed in the process of glucose forming pyruvate through the EMP pathway. In Escherichia coli, pyruvate has four main flows: one is reflux to form phosphoenolpyruvate under the action of phosphoenolpyruvate synthase, the second is to directly form acetic acid under the action of pyruvate oxidase, the third is to reduce lactate to form lactic acid under the action of lactate dehydrogenase, and the fourth is to form acetyl-CoA under the action of pyruvate dehydrogenase. Under aerobic conditions, the process of pyruvate decarboxylation oxidation to form acetyl-CoA under the action of pyruvate dehydrogenase complex will produce NADH again, and more NADH will be formed after acetyl-CoA enters the TCA cycle, and the byproducts of this metabolic pathway are carbon dioxide and water.

[0019] The E. coli aceE gene is one of the key enzymes encoding the pyruvate dehydrogenase complex Pdh. After the aceE gene of E. coli MG1655 is overexpressed by genetic engineering, the flow of pyruvate to the TCA cycle is enhanced, thereby continuously producing NADH. The full genome nucleotide sequence of the aceE gene is shown in SEQ ID NO.1, as follows: SEQ ID NO.1:

[0020] Therefore, the inventors of the present application used Escherichia coli MG1655 as the starting strain and constructed the Escherichia coli genetically engineered bacteria MG1655-aceE that efficiently expresses the aceE gene. A new fermentation production method for preparing bosense was developed using the Escherichia coli genetically engineered bacteria MG1655-aceE. During the preparation process, the dissolved oxygen content was controlled at 30%-35%, which can ensure the smooth progress of the metabolic reaction and will not inhibit the metabolic reaction due to excessive accumulation of intermediates. The reductive coenzyme I generated during the metabolic process, namely NADH, catalyzes the synthesis of the substrate β-acetone xyloside to prepare bosense. This reaction method does not add toxic substances, the reaction process is green and safe, and is suitable for the industrial production of bosense.

[0021] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

[0022] The Escherichia coli MG1655 used in this example was purchased from Shanghai Kanglang Biotechnology Co., Ltd.

[0023] The standard product of hydroxypropyl tetrahydropyrantriol used in the examples was purchased from Chengdu Purifa Technology Development Co., Ltd. with a purity of 99%.

[0024] Example 1: Construction of genetically engineered Escherichia coli bacteria that efficiently express the aceE gene Primers were designed according to the aceE full genome nucleotide sequence published in GeneBank (shown in SEQ ID NO.1), and PCR amplification was performed with the primer sequences shown in SEQ ID NO.2-SEQ ID NO.3. The PCR reaction conditions were: denaturation at 94°C for 5 min. 94°C, 1 min; 53°C, 1.5 min; 72°C, 4 min; 35 cycles. Finally, extension at 72°C for 10 min. The PCR product was electrophoresed and purified by gel cutting. The recovered PCR product was inserted into the vector pCambia2300 to construct a recombinant expression vector. The recombinant expression vector with correct sequencing was transformed into Escherichia coli MG1655 competent cells, and a single colony containing the recombinant vector was picked from the plate to obtain the Escherichia coli genetic engineering bacteria MG1655-aceE that efficiently expressed the aceE gene.

[0025] SEQ ID NO.2 (F): 5-ATGTCAGAACGTTTCCCAAAATGACGTGGATCCG-3; SEQ ID NO. 3 (R): 5-TTACGCCAGACGGCGGGTTAACTTTATCTGCATC-3.

[0026] Example 2: Fermentation to prepare bosogenin (1) Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 950 ml of deionized water, adjust the pH to 7.0 with 5 mol / L NaOH, and dilute to 1 L with deionized water to obtain LB liquid medium. Sterilize and set aside. 6 The inoculum size of CFU / mL was inoculated into 100 mL of sterilized LB liquid medium (containing Amp 100 pg·mL), and IPTG with a final concentration of 1 mmol / L was added for induction at 37°C for 3 h. During this stage, the pH was maintained at 7.0 and the dissolved oxygen content was controlled at 30%.

[0027] (2) Add 8 g of glucose and 4 g of β-acetone xyloside to the LB liquid culture medium in step (1) to construct a fermentation reaction system. The reaction system is reacted at 37°C and 200 rpm for 72 h. After the reaction is completed, centrifuge at 12,000 rpm for 10 min, and the supernatant is filtered through a 0.22 μm filter membrane to obtain a supernatant containing bosogenin.

[0028] Example 3: Fermentation to prepare bosogenin (1) Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 950 ml of deionized water, adjust the pH to 7.0 with 5 mol / L NaOH, and dilute to 1 L with deionized water to obtain LB liquid medium. Sterilize and set aside. 6 The inoculum size of CFU / mL was inoculated into 100 mL of sterilized LB liquid medium (containing Amp 100 pg·mL), and IPTG with a final concentration of 1 mmol / L was added for induction at 37°C for 3 h. During this stage, the pH was maintained at 7.0 and the dissolved oxygen content was controlled at 35%.

[0029] (2) Add 8 g of glucose and 2 g of β-acetone xyloside to the LB liquid culture medium in step (1) to construct a fermentation reaction system. The reaction system is reacted at 37°C and 200 rpm for 72 h. After the reaction is completed, centrifuge at 12,000 rpm for 10 min, and the supernatant is filtered through a 0.22 μm filter membrane to obtain a supernatant containing bosogenin.

[0030] Comparative Example 1: Preparation of Bosera by Fermentation (1) Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 950 ml of deionized water, adjust the pH to 7.0 with 5 mol / L NaOH, and dilute to 1 L with deionized water to obtain LB liquid medium. Sterilize and set aside.6 The inoculum size of CFU / mL was inoculated into 100 mL of sterilized LB liquid medium (containing Amp 100 pg·mL), and IPTG with a final concentration of 1 mmol / L was added for induction at 37°C for 3 h. During this stage, the pH was maintained at 7.0 and the dissolved oxygen content was controlled at 30%.

[0031] (2) Add 8 g of glucose and 4 g of β-acetone xyloside to the LB liquid culture medium in step (1) to construct a fermentation reaction system. The reaction system is reacted at 37°C and 200 rpm for 72 h. After the reaction is completed, centrifuge at 12,000 rpm for 10 min, and the supernatant is filtered through a 0.22 μm filter membrane to obtain a supernatant containing bosogenin.

[0032] Comparative Example 2: Preparation of Bosera by Fermentation (1) Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 950 ml of deionized water, adjust the pH to 7.0 with 5 mol / L NaOH, and dilute to 1 L with deionized water to obtain LB liquid medium. Sterilize and set aside. 6 The inoculum size of CFU / mL was inoculated into 100 mL of sterilized LB liquid medium (containing Amp 100 pg·mL), and IPTG with a final concentration of 1 mmol / L was added for induction at 37°C for 3 h. During this stage, the pH was maintained at 7.0 and the dissolved oxygen content was controlled at 10%.

[0033] (2) Add 8 g of glucose and 4 g of β-acetone xyloside to the LB liquid culture medium in step (1) to construct a fermentation reaction system. The reaction system is reacted at 37°C and 200 rpm for 72 h. After the reaction is completed, centrifuge at 12,000 rpm for 10 min, and the supernatant is filtered through a 0.22 μm filter membrane to obtain a supernatant containing bosogenin.

[0034] Comparative Example 3: Preparation of Bosera by Fermentation (1) Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 950 ml of deionized water, adjust the pH to 7.0 with 5 mol / L NaOH, and dilute to 1 L with deionized water to obtain LB liquid medium. Sterilize and set aside. 6 The inoculum size of CFU / mL was inoculated into 100 mL of sterilized LB liquid medium (containing Amp 100 pg·mL), and IPTG with a final concentration of 1 mmol / L was added for induction at 37°C for 3 h. During this stage, the pH was maintained at 7.0 and the dissolved oxygen content was controlled at 10%.

[0035] (2) Add 8 g of glucose and 4 g of β-acetone xyloside to the LB liquid culture medium in step (1) to construct a fermentation reaction system. The reaction system is reacted at 37°C and 200 rpm for 72 h. After the reaction is completed, centrifuge at 12,000 rpm for 10 min, and the supernatant is filtered through a 0.22 μm filter membrane to obtain a supernatant containing bosogenin.

[0036] Test Example 1: Determination of the concentration and mass of bosogenin by high performance liquid chromatography (HPLC) The concentration of bosine in the supernatant prepared in Example 2, Comparative Example 1 and Comparative Example 2 was determined by high performance liquid chromatography (HPLC). The high performance liquid chromatography analysis method is as follows: Weigh 50 mg of bosine standard accurately, prepare it into 50 mg / mL standard solution with ultrapure water, shake well and set aside. The HPLC instrument is Agilent 1260, the detector is a differential detector, the mobile phase is pure water: acetonitrile = 25:75 (v / v), the chromatographic column is OSAKA SODA CAPCELLPAK NH2 liquid chromatography column (250×4.6 / 5μm), the mobile phase flow rate is 1 mL / min, the injection volume is 10 μL, and the detection temperature is 35°C.

[0037] After measurement, the concentrations of bosaiin prepared in Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3 were 38.55 g / L, 12.90 g / L, 32.26 g / L and 9.74 g / L, respectively. The conversion rate was calculated according to the formula: the mass of bosaiin in the supernatant / the mass of β-acetone xyloside input, and the conversion rates were 96.37%, 32.26%, 80.64% and 24.36%, respectively. The test results show that the fermentation method of preparing bosaiin by using the genetically engineered Escherichia coli MG1655-aceE and controlling the dissolved oxygen conditions is green and feasible, which lays a foundation for the industrial production of bosaiin by using this method.

[0038] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principle of the present application shall be deemed to be appropriate. Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. A method for preparing bosogenin by fermentation, characterized in that: The steps include: The activated genetically engineered Escherichia coli was inoculated into the culture medium, IPTG was added to induce the reaction, the dissolved oxygen content was controlled at 30%-35%, glucose and β-acetone xyloside were added to the culture medium to construct a fermentation reaction system, and after the reaction was completed, centrifugation was performed, and the supernatant was collected to prepare bosera.

2. The method according to claim 1, characterized in that The Escherichia coli genetically engineered bacteria is constructed by taking Escherichia coli MG1655 as a starting strain and overexpressing the aceE gene.

3. The method according to claim 1, characterized in that The method for constructing genetically engineered Escherichia coli comprises the following steps: The aceE gene obtained by PCR amplification was inserted into the pCambia2300 plasmid vector to construct a recombinant expression vector, and the recombinant vector was transformed into Escherichia coli MG1655 to obtain genetically engineered Escherichia coli bacteria.

4. The method according to claim 1, characterized in that The genetically engineered Escherichia coli was 1×10 6 CFU / mL was inoculated into the culture medium.

5. The method according to claim 1, characterized in that The concentration of the IPTG is (0.5-1) mmol / L.

6. The method according to claim 1, characterized in that After the IPTG was added, the reaction was induced at 37°C for 2-3 hours.

7. The method according to claim 1, characterized in that The mass ratio of glucose to β-acetone xyloside is (2-4):

1.

8. The method according to claim 1, characterized in that The fermentation reaction system was reacted at 37° C. and 200 rpm for 70-72 hours.

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