High-yield acarbose method for enhancing expression of genes acbA and acbB after RNA secondary structure optimization

Abstract

The invention discloses a high-yield acarbose method for enhancing expression of genes acbA and acbB after RNA secondary structure optimization. The method comprises the following steps: optimizing RNA secondary structures coded by acarbose biosynthesis genes acbA and acbB to obtain genes opt-acbA and opt-acbB, and further enhancing expression in actinoplanes to obtain a mutant strain with high yield of acarbose. According to the invention, by optimizing the RNA secondary structure coded by acarbose biosynthetic genes acbA and acbB, the translation level of acbA and acbB coded proteins can be improved, and finally, the yield of acarbose is obviously improved. And compared with the original strain, the fermentation yields can be respectively increased by 1.82 times and 1.32 times. And compared with a sequence-unoptimized gene overexpression mutant strain, the fermentation yields can be respectively improved by 81.4% and 30.4%.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a method for increasing the production of acarbose by enhancing the expression of genes acbA and acbB after RNA secondary structure optimization. Specifically, the method improves the production of acarbose after enhancing the expression by optimizing the RNA secondary structure encoded by the acarbose biosynthesis genes acbA and acbB; in particular, it relates to a method for enhancing the translation level of proteins encoded by the optimized genes opt-acbA and opt-acbB to improve the fermentation level of acarbose; by enhancing the expression of opt-acbA and opt-acbB after RNA secondary structure optimization in Actinoplanes sp.SE50 / 110 using a strong promoter kasOp*, the translation level of proteins encoded by the genes acbA and acbB can be improved, and finally the production of acarbose can be significantly improved. Background Art

[0002] Diabetes mellitus (DM) is a chronic metabolic disease, and its many complications have caused serious damage to human health, such as diabetic ketoacidosis, vascular diseases, and dominant functional lesions. Among them, the number of patients with type 2 diabetes accounts for more than 90% of the total number of patients with diabetes. Since the 1990s, acarbose has been widely used to treat type 2 diabetes. Because acarbose has a strong affinity for amylase and α-glucosidase in the intestine, it can be used as a competitive substrate to slow down the hydrolysis of starch and polysaccharides, thereby delaying the production of glucose to control postprandial blood sugar levels. In 2021, more than 537 million adults (20-79 years old) suffered from this disease. In recent years, with the changes in people's living standards and dietary structure, the number of obese and overweight people has increased, which has also caused the prevalence of type 2 diabetes to rise rapidly worldwide. According to the International Diabetes Federation, by 2030, this number is expected to exceed 643 million, and the demand for acarbose is expected to increase significantly.

[0003] At present, the industry mainly uses the fermentation production of Actinoplanes sp. In the early stage, mutation breeding and fermentation process optimization were mainly used to increase the yield, which had a long cycle and had reached a bottleneck. The rational metabolic engineering transformation achieved good results. In the previous study, the inventor's research group confirmed that the biosynthesis of the deoxyamino sugar unit of acarbose is based on D-glucose-1-phosphate as a substrate, which is catalyzed by AcbA (D-glucose-1-phosphate thymine transferase), AcbB (dTDP-D-glucose-4,6-dehydratase) and AcbV (dTDP-4-keto-6-deoxy-D-glucose transaminase) to generate dTDP-D-glucose and dTDP-4-keto-6-deoxy-D-glucose, respectively. This also shows that the sugar substrate assembled in the core part of acarbose during the synthesis process is dTDP-4-amino-4,6-dideoxy-D-glucose. At the same time, the enzymatic properties of related proteins were studied by exploring the optimal reaction conditions of the enzymes one by one. For dTDP-D-glucose substrate, the kcat / Km value of AcbA protein is 0.355L / (min·mmol), which is lower than the isozymes reported in other bacteria. For example, the kcat / Km of RfbA protein in E. coli and Salmonella enterica LT2 is 173.960L / (min·mmol) and 16.041L / (min·mmol), respectively, indicating that the catalytic ability of AcbA is low. In addition, the kcat / Km value of AcbB protein is 21.390L / (min·mmol), which is close to the kcat / Km of RfbB protein in S. enterica LT2 of 54.861L / (min·mmol). At the same time, we previously measured the transcription amount of SE50 / 110 fermentation for 24h, 48h, and 72h, and found that the transcription of acbA and acbB genes was low. The inventors believe that the low catalytic activity of AcbA and AcbB in the synthesis of deoxyamino sugar units may be the rate-limiting step in the biosynthesis of acarbose. Therefore, increasing the intracellular expression of AcbA and AcbB proteins, or expressing highly active heterologous isoproteins, may be beneficial to the synthesis of acarbose deoxyamino sugar units, thereby achieving the purpose of increasing the yield of acarbose. However, the inventors previously overexpressed acbA and acbB genes and heterologous high-efficiency isozyme encoding genes, and the gene transcription of acbA and acbB increased significantly, but the yield of acarbose in the strain increased to a limited extent, indicating that overexpression of acbA and acbB genes, although the transcription level increased, the translation level was still low. Summary of the invention

[0004] The object of the present invention is to provide a method for high-yield acarbose by enhancing the expression of genes acbA and acbB after RNA secondary structure optimization; the present invention improves the fermentation level of acarbose by enhancing the translation level of proteins encoded by genes acbA and acbB after RNA secondary structure optimization, and the high-yield acarbose mutant strain (denoted as: SE50 / 110::kasOp*-opt-acbA and SE50 / 110::kasOp*-opt-acbB) obtained by the present invention, based on the enhanced expression of genes opt-acbA and opt-acbB after RNA secondary structure optimization, ultimately improves the yield of acarbose.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for improving the fermentation level of acarbose, optimizing the secondary structure of RNA encoded by acarbose biosynthesis genes acbA and acbB; enhancing the expression of at least one of the acarbose biosynthesis genes opt-acbA and opt-acbB after the RNA secondary structure is optimized in actinomycetes, obtaining an acarbose high-yield strain, and fermenting to obtain acarbose. The present invention optimizes the secondary structure of acbA and acbB genes to obtain opt-acbA and opt-acbB, improves the gene transcription amount of acbA and acbB, and also improves their translation level, thereby improving the yield of acarbose.

[0007] As one embodiment of the present invention, the sequences of the acarbose biosynthesis genes acbA and acbB are SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0008] As one embodiment of the present invention, the sequence of the acarbose biosynthesis gene opt-acbA is shown as SEQ ID NO.4, and the sequence of the acarbose biosynthesis gene opt-acbB is shown as SEQ ID NO.5.

[0009] In some embodiments, in Actinoplanes sp.SE50 / 110, the strong promoter kasOp* is used to enhance the expression of acarbose biosynthesis genes opt-acbA and opt-acbB, respectively, to obtain high-yield acarbose mutants SE50 / 110::kasOp*-opt-acbA and SE50 / 110::kasOp*-opt-acbB. The sequence of kasOp* is SEQ ID NO.1, and kasOp* is located before opt-acbA and opt-acbB.

[0010] As one embodiment of the present invention, the actinomycetes include Actinoplanes sp. SE50 / 110, SE50 / 110::kasOp*-acbA and SE50 / 110::kasOp*-acbB.

[0011] As one embodiment of the present invention, enhancing the expression of acarbose biosynthesis gene with optimized RNA secondary structure in actinomycetes comprises the following steps:

[0012] S1, design and construction of integrative plasmid I for enhanced expression of gene acbA;

[0013] S2, design and construction of integrative plasmid II for enhancing the expression of gene acbB;

[0014] S3, design and construction of integrative plasmid III for enhanced expression of gene opt-acbA;

[0015] S4, design and construction of integrative plasmid IV for enhancing the expression of gene opt-acbB;

[0016] S5 introduces the integrative plasmid I, II, III or IV into the recipient strain through conjugation transfer, then verifies the mutant strain for apramycin resistance, picks the mycelium, and screens the gene expression mutant strain through the difference in PCR product fragment size.

[0017] As an embodiment of the present invention, the specific construction method of the integrative plasmid I is to obtain kasOp* (SEQ ID NO.1) by total gene synthesis, obtain the gene acbA fragment from the SE50 / 110 genome by PCR amplification, obtain the acbA fragment with a size of 1092bp with homology arms by overlap PCR amplification, and connect it with the plasmid pRT801 after double digestion with BcuⅠ and BamHⅠ by Gibson assembly method.

[0018] As one embodiment of the present invention, the acbA gene with homology arms was amplified by PCR using primers kasOp-Gib-F / A-Gib-R.

[0019] As an embodiment of the present invention, the specific construction method of the integrative plasmid II is to obtain kasOp* by total gene synthesis, obtain the gene acbB fragment from the SE50 / 110 genome by PCR amplification, obtain the acbB fragment with a size of 1143bp with homology arms by overlap PCR amplification, and connect it with the plasmid pRT801 after double digestion with BcuⅠ and BamHⅠ by Gibson assembly method.

[0020] As one embodiment of the present invention, the acbB gene with homology arms was obtained by PCR amplification using primers kasOp-Gib-F / B-Gib-R.

[0021] As an embodiment of the present invention, the specific construction method of the integrative plasmid III is to obtain the kasOp*-opt-acbA gene fragment containing kasOp* and opt-acbA by total gene synthesis, obtain the opt-acbA fragment with a size of 1092 bp with homology arms by PCR amplification, and connect it with the plasmid pRT801 after double restriction digestion with BcuⅠ and BamHⅠ by the Gibson assembly method.

[0022] As one embodiment of the present invention, the opt-acbA gene with homology arms was amplified by PCR using primers kasOp-Gib-F / opt-A-Gib-R.

[0023] As an embodiment of the present invention, the specific construction method of the integrative plasmid IV is to obtain the kasOp*-opt-acbB gene fragment containing kasOp* and opt-acbB by total gene synthesis, obtain the opt-acbB fragment with a size of 1143 bp with homology arms by PCR amplification, and connect it with the plasmid pRT801 after double restriction digestion with BcuⅠ and BamHⅠ by the Gibson assembly method.

[0024] As one embodiment of the present invention, the opt-acbB gene with homology arms was obtained by PCR amplification using primers kasOp-Gib-F / opt-B-Gib-R.

[0025] As an embodiment of the present invention, the recipient strain is Actinomycete Actinoplanes sp.SE50 / 110. The gene-enhanced expression mutants are denoted as SE50 / 110::kasOp*-acbA, SE50 / 110::kasOp*-acbB, SE50 / 110::kasOp*-opt-acbA, and SE50 / 110::kasOp*-opt-acbB.

[0026] As an embodiment of the present invention, the fermentation comprises the following steps: activating the control strain SE50 / 110, SE50 / 110::kasOp*-acbA, SE50 / 110::kasOp*-acbB and the mutant strain with enhanced expression of genes opt-acbA and opt-acbB after RNA secondary structure optimization (SE50 / 110::kasOp*-opt-acbA or SE50 / 110::kasOp*-opt-acbB) on solid culture medium.

[0027] As one embodiment of the present invention, the activated mycelium of the acarbose high-yielding strain is cultured in a primary seed culture medium at 30° C. and 220 rpm for 28 to 36 hours; transferred to a secondary seed culture medium at a 10% inoculation rate, and cultured at 30° C. and 220 rpm for 24 to 28 hours; transferred to a fermentation culture medium at a 15% inoculation rate, and fermented at 30° C. and 280 rpm for 3 days, and the fermentation broth is collected and centrifuged to obtain the supernatant.

[0028] As an embodiment of the present invention, the solid culture medium comprises sucrose 3w / v%, peptone 0.5w / v%, yeast powder 0.5w / v%, casein hydrolyzate 0.1w / v%, K2HPO4·3H2O 0.1w / v%, KCl 0.05g, FeSO4 0.005w / v%, and agar powder 2w / v%.

[0029] As an embodiment of the present invention, the primary seed culture medium comprises 1w / v% maltose, 1.5w / v% glucose, 0.5w / v% yeast powder, 0.5w / v% peptone, 1w / v% glycerol, 0.1w / v% K2HPO4·3H2O, 1w / v% malt extract, and 0.1w / v% casein hydrolyzate.

[0030] As an embodiment of the present invention, the secondary seed culture medium includes 4w / v% soybean cake powder, 1.5w / v% maltose, 1w / v% glucose, 1w / v% glycerol, 1w / v% soluble starch, and 0.2w / v% calcium carbonate.

[0031] As an embodiment of the present invention, the fermentation medium includes 5w / v% maltose, 3w / v% glucose, 0.38w / v% sodium glutamate, 0.05w / v% FeCl3, 1-1.5w / v% soybean cake powder, 0.3-0.4w / v% Angel yeast powder, and 0.25w / v% calcium carbonate. As a specific example, the fermentation medium includes 5w / v% maltose, 3w / v% glucose, 0.38w / v% sodium glutamate, 0.05w / v% FeCl3, 1w / v% soybean cake powder, 0.3w / v% Angel yeast powder, and 0.25w / v% calcium carbonate.

[0032] The present invention also relates to an actinomycete with high acarbose production, wherein the expression of a gene with optimized RNA secondary structure is enhanced in the actinomycete to obtain an acarbose high-producing strain; the gene with optimized RNA secondary structure is at least one of opt-acbA and opt-acbB, and the sequences thereof are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0033] The present invention also relates to mutant strains SE50 / 110::kasOp*-opt-acbA and SE50 / 110::kasOp*-opt-acbB with high acarbose production. The technical key points are: optimizing the secondary structure of RNA encoded by the acarbose biosynthesis genes acbA and acbB, and enhancing the expression of the optimized genes opt-acbA and opt-acbB of the strain. Specifically, it relates to a strain of Actinoplanes sp.SE50 / 110::kasOp*-opt-acbA, whose deposit number is CGMCC No.33051. It also relates to a strain of Actinoplanes sp.SE50 / 110::kasOp*-opt-acbB, whose deposit number is CGMCC No.33052.

[0034] The present invention also relates to an integrated plasmid vector for enhancing the expression of genes acbA and acbB with optimized RNA secondary structure, the vector comprising optimized genes opt-acbA and opt-acbB; the sequences thereof are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. The sequence of kasOp* is SEQ ID NO.1, and kasOp* is located before opt-acbA and opt-acbB.

[0035] The present invention has found that D-glucose-1-phosphate thymine transferase transcribed and translated by gene acbA and dTDP-D-glucose-4,6-dehydratase transcribed and translated by gene acbB are the rate-limiting enzymes for the biosynthesis of acarbose. According to the analysis of the transcriptome and proteome data of actinomycetes, the transcription amount of genes acbA and acbB is high, but the protein translation level is low, which limits the biosynthesis of acarbose. The present invention tested related genes and strategies, paid a lot of creative work, and finally confirmed that through the scheme of optimizing the secondary structure of RNA encoded by acbA and acbB of the present invention, the expression of genes opt-acbA and opt-acbB after optimization can be enhanced, the translation level of proteins encoded by genes acbA and acbB can be improved, and finally the yield of acarbose can be significantly increased.

[0036] In the present invention, Actinoplanes sp.SE50 / 110::kasOp*-opt-acbA, the deposit number is CGMCC No.33051, the deposit date is: December 12, 2024, the deposit unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0037] Actinoplanes sp.SE50 / 110::kasOp*-opt-acbB, the deposit number is CGMCCNo.33052, the deposit date is December 12, 2024, the deposit unit is the General Microbiology Center of China Culture Collection Administration, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1) By enhancing the expression of acbA and acbB after RNA secondary structure optimization in actinomycetes, the translation level of the proteins encoded by acbA and acbB can be improved, and ultimately the production of acarbose is increased.

[0040] 2) In the present invention, the genes opt-acbA and opt-acbB after RNA secondary structure optimization are enhanced in actinomycetes to obtain a high-yield strain; compared with the starting strain SE50 / 110, the fermentation yields of the high-yield strains SE50 / 110::kasOp*-opt-acbA and SE50 / 110::kasOp*-opt-acbB obtained by the present invention are increased by 1.82 times and 1.32 times, respectively; compared with the sequence-unoptimized gene overexpression mutants SE50 / 110::kasOp*-acbA and SE50 / 110::kasOp*-acbB, the fermentation yields of the high-yield strains SE50 / 110::kasOp*-opt-acbA and SE50 / 110::kasOp*-opt-acbB obtained by the present invention are increased by 81.4% and 30.4%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0042] Figure 1 Schematic diagram of plasmid construction for enhanced expression of gene acbA;

[0043] Figure 2 Schematic diagram of plasmid construction for enhanced expression of gene acbB;

[0044] Figure 3 Schematic diagram of plasmid construction for enhanced expression of gene opt-acbA;

[0045] Figure 4 Schematic diagram of plasmid construction for enhanced expression of gene opt-acbB;

[0046] Figure 5 Schematic diagram of acarbose fermentation yield of the gene mutant strain and the control strain after RNA secondary structure optimization. DETAILED DESCRIPTION

[0047] The present invention is further described below by way of examples. This example is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and processes are given, but the protection scope of the present invention is not limited to the following examples. The experimental methods in the following examples without specifying specific conditions are based on conventional conditions or the conditions recommended by the manufacturer.

[0048] The plasmid pRT801 involved in the present invention has been recorded in the SCI database document "Gren T, Ortseifen V, Wibberg D, Schneiker-Bekel S, Bednarz H, Niehaus K, Zemke T, Persicke M, Pühler A, Kalinowski J. Genetic engineering in Actinoplanes sp. SE50 / 110-development of an intergeneric conjugation system for the introduction of actinophage-based integrative vectors. J Biotechnol. 2016 Aug 20, 232: 79-88".

[0049] The strain SE50 / 110 involved in the present invention has been recorded in the document "Qinqin Zhao, Yuchang Luo, Xin Zhang, Qianjin Kang, Dan Zang, Linquan Bai*&Zixin Deng: A severe leakage of intermediates to shunt products in acarbose biosynthesis. Nature Communications 2020, 11(1): 1468.".

[0050] Example

[0051] This example is a specific process for obtaining the control strains SE50 / 110::kasOp*-acbA, SE50 / 110::kasOp*-acbB and the mutant strains SE50 / 110::kasOp*-opt-acbA, SE50 / 110::kasOp*-opt-acbB with enhanced expression of genes opt-acbA and opt-acbB after RNA secondary structure optimization. The specific steps are as follows:

[0052] Step 1: Construction of plasmid pRT801-kasOp*-acbA

[0053] The gene kasOp* fragment was artificially synthesized, and the genome of Actinomycetes SE50 / 110 was used as a template to obtain the acbA fragment by PCR amplification. The kasOp-acbA fragment was obtained by overlap PCR amplification. The primers kasOp-Gib-F (SEQ ID NO.6) / A-Gib-R (SEQ ID NO.7) with homology arm sequences to the plasmid were used to obtain the acbA gene fragment (1092bp) with homology arm fragments by PCR amplification. The sequence included the strong promoter kasOp* and the gene acbA. The promoter kasOp* was located before the gene acbA. Then the fragment was ligated with the plasmid pRT801 double-digested with BcuⅠ and BamHⅠ, and ligated by Gibson assembly to obtain the plasmid pRT801-kasOp*-acbA.

[0054] Step 2: Construction of plasmid pRT801-kasOp*-acbB

[0055] The gene kasOp* fragment was artificially synthesized, and the genome of Actinomycetes SE50 / 110 was used as a template to obtain the acbB fragment by PCR amplification. The kasOp-acbB fragment was obtained by overlap PCR amplification. The primers kasOp-Gib-F (SEQ ID NO.6) / B-Gib-R (SEQ ID NO.8) with homology arm sequences to the plasmid were used to obtain the acbB gene fragment (1143bp) with homology arm fragments by PCR amplification. The sequence included the strong promoter kasOp* and the gene acbB. The promoter kasOp* was located before the gene acbB. Then the fragment was ligated with the plasmid pRT801 double-digested with BcuⅠ and BamHI, and ligated by Gibson assembly to obtain the plasmid pRT801-kasOp*-acbB.

[0056] Step 3: Construction of plasmid pRT801-kasOp*-opt-acbA

[0057] The artificially synthesized gene kasOp*-opt-acbA was used as a template, and the primers kasOp-Gib-F (SEQ ID NO.6) / opt-A-Gib-R (SEQ ID NO.9) with homology arm sequences to the plasmid were used to obtain the opt-acbA gene fragment (1092 bp) with homology arm fragments by PCR amplification. The sequence contained the strong promoter kasOp* and the gene opt-acbA, and the promoter kasOp* was located before the gene opt-acbA. The fragment was then connected with the plasmid pRT801 after double digestion with BcuⅠ and BamHI, and connected by Gibson assembly to obtain the plasmid pRT801-kasOp*-opt-acbA.

[0058] Step 4: Construction of plasmid pRT801-kasOp*-opt-acbB

[0059] The artificially synthesized gene kasOp*-opt-acbB was used as a template, and the primers kasOp-opt-Gib-F (SEQ ID NO.6) / opt-B-Gib-R (SEQ ID NO.10) with homology arm sequences to the plasmid were used to obtain the opt-acbB gene fragment (1143 bp) with homology arm fragments by PCR amplification. The sequence contained the strong promoter kasOp* and the gene opt-acbB, and the promoter kasOp* was located before the gene opt-acbB. The fragment was then connected with the plasmid pRT801- after double digestion with BcuⅠ and BamHI, and connected by Gibson assembly to obtain the plasmid pRT801-kasOp*-opt-acbB.

[0060] Figure 1 The process of inserting the target gene acbA into pRT801 is illustrated. The specific operation is as follows: The constructed gene-enhanced expression plasmid pRT801-kasOp*-acbA is transformed into the host E. coli ET12567 (pUZ8002). The E. coli ET12567 (pUZ8002) containing the pRT801-kasOp*-acbA plasmid is cultured overnight at 37°C in LB medium containing 50 mg / L, 25 mg / L chloramphenicol and 50 mg / L kanamycin. Then, the overnight cultured E. coli ET12567 (pUZ8002) is transferred to a new LB medium at a ratio of 1-3% using LB medium and cultured for 4-5 hours until the OD 600Reach 0.6-0.8, then rinse the bacteria with sterile distilled water to remove the antibiotics in the culture. At the same time, the SE50 / 110 activated on the solid STY plate 2-3 days in advance was cultured with the primary seed culture medium for 32 hours, and then transferred to the TSB culture medium according to the inoculation amount of 10%, a certain amount of bacteria was centrifuged to remove the supernatant, and after rinsing with sterile distilled water for 3 times, it was mixed with the previously prepared receptor E.coli ET12567 (pUZ8002) (the ratio of receptor bacteria to donor bacteria is about 1:30) and evenly spread on the SFM solid culture medium (containing 20m MgCl2), and inverted cultured in a 30℃ incubator for 32 hours. Then, apramycin with a final concentration of 50 mg / L and trimethoprim with a final concentration of 50 mg / L were added to an EP tube containing 1 mL of sterile water, mixed evenly and applied to an SFM plate that had been cultured for 32-36 hours. After the plate was dried, it was inverted in a 30°C constant temperature incubator and cultured for 5-7 days. After the colonies visible to the naked eye appeared, a single colony was picked with a toothpick and expanded on a solid STY medium containing the above concentrations of apramycin and trimethoprim. Finally, the mycelium on the STY solid medium was picked, the genome was extracted, and 801-YZ-F (SEQ ID NO.11) and 801-YZ-R (SEQ ID NO.12) were used as primers to obtain the correct SE50 / 110::kasOp*-acbA mutant strain by PCR.

[0061] Figure 2 The process of inserting the target gene acbB into pRT801 is illustrated. The specific operation is as follows: The constructed gene enhanced expression plasmid pRT801-kasOp*-acbB is transformed into the host E. coli ET12567 (pUZ8002). The E. coli ET12567 (pUZ8002) containing the pRT801-kasOp*-acbB plasmid is cultured overnight at 37°C in LB medium containing 50 mg / L, 25 mg / L chloramphenicol and 50 mg / L kanamycin. Then, the overnight cultured E. coli ET12567 (pUZ8002) is transferred to a new LB medium at a ratio of 1-3% using LB medium and cultured for 4-5 hours until the OD 600Reach 0.6-0.8, then rinse the bacteria with sterile distilled water to remove the antibiotics in the culture. At the same time, the SE50 / 110 activated on the solid STY plate 2-3 days in advance was cultured with the primary seed culture medium for 32 hours, and then transferred to the TSB culture medium according to the inoculation amount of 10%, a certain amount of bacteria was centrifuged to remove the supernatant, and after rinsing with sterile distilled water for 3 times, it was mixed with the previously prepared receptor E.coli ET12567 (pUZ8002) (the ratio of receptor bacteria to donor bacteria is about 1:30) and evenly spread on the SFM solid culture medium (containing 20m MgCl2), and inverted cultured in a 30℃ incubator for 32 hours. Then, add apramycin with a final concentration of 50 mg / L and trimethoprim with a final concentration of 50 mg / L to an EP tube containing 1 mL of sterile water, mix well and apply to the SFM plate that has been cultured for 32-36 hours, and after the plate is dried, invert it in a 30°C constant temperature incubator and culture it for 5-7 days. After the colonies visible to the naked eye appear, pick a single colony with a toothpick and expand it on the STY solid medium containing the above concentrations of apramycin and trimethoprim. Finally, pick the mycelium on the STY solid medium, extract the genome, and use 801-YZ-F (SEQ ID NO.11) and 801-YZ-R (SEQ ID NO.12) as primers to obtain the correct SE50 / 110::kasOp*-acbB mutant strain by PCR.

[0062] Figure 3 The process of inserting the target gene opt-acbA into pRT801 is illustrated. The specific operation is as follows: The constructed gene-enhanced expression plasmid pRT801-kasOp*-opt-acbA is transformed into the host E. coli ET12567 (pUZ8002). The E. coli ET12567 (pUZ8002) containing the pRT801-kasOp*-opt-acbA plasmid is cultured overnight at 37°C in LB medium containing 50 mg / L, 25 mg / L chloramphenicol and 50 mg / L kanamycin. Then, the overnight cultured E. coli ET12567 (pUZ8002) is transferred to a new LB medium at a ratio of 1-3% using LB medium and cultured for 4-5 hours until the OD 600Reach 0.6-0.8, then rinse the bacteria with sterile distilled water to remove the antibiotics in the culture. At the same time, the SE50 / 110 activated on the solid STY plate 2-3 days in advance was cultured with the primary seed culture medium for 32 hours, and then transferred to the TSB culture medium according to the inoculation amount of 10%, a certain amount of bacteria was centrifuged to remove the supernatant, and after rinsing with sterile distilled water for 3 times, it was mixed with the previously prepared receptor E.coli ET12567 (pUZ8002) (the ratio of receptor bacteria to donor bacteria is about 1:30) and evenly spread on the SFM solid culture medium (containing 20m MgCl2), and inverted cultured in a 30℃ incubator for 32 hours. Then, apramycin with a final concentration of 50 mg / L and trimethoprim with a final concentration of 50 mg / L were added to an EP tube containing 1 mL of sterile water, mixed evenly and applied to an SFM plate that had been cultured for 32-36 hours. After the plate dried, it was inverted in a 30°C constant temperature incubator and cultured for 5-7 days. After the colonies visible to the naked eye appeared, a single colony was picked with a toothpick and expanded on a solid STY medium containing the above concentrations of apramycin and trimethoprim. Finally, the mycelium on the STY solid medium was picked, the genome was extracted, and 801-YZ-F (SEQ ID NO.11) and 801-YZ-R (SEQ ID NO.12) were used as primers to obtain the correct SE50 / 110::kasOp*-opt-acbA mutant strain by PCR.

[0063] Figure 4 The process of inserting the target gene opt-acbB into pRT801 is illustrated. The specific operation is as follows: The constructed gene enhanced expression plasmid pRT801-kasOp*-opt-acbB is transformed into the host E. coli ET12567 (pUZ8002). The E. coli ET12567 (pUZ8002) containing the pRT801-kasOp*-opt-acbB plasmid is cultured overnight at 37°C in LB medium containing 50 mg / L, 25 mg / L chloramphenicol and 50 mg / L kanamycin. Then, the overnight cultured E. coli ET12567 (pUZ8002) is transferred to a new LB medium at a ratio of 1-3% using LB medium and cultured for 4-5 hours until the OD 600Reach 0.6-0.8, then rinse the bacteria with sterile distilled water to remove the antibiotics in the culture. At the same time, the SE50 / 110 activated on the solid STY plate 2-3 days in advance was cultured with the primary seed culture medium for 32 hours, and then transferred to the TSB culture medium according to the inoculation amount of 10%, a certain amount of bacteria was centrifuged to remove the supernatant, and after rinsing with sterile distilled water for 3 times, it was mixed with the previously prepared receptor E.coli ET12567 (pUZ8002) (the ratio of receptor bacteria to donor bacteria is about 1:30) and evenly spread on the SFM solid culture medium (containing 20m MgCl2), and inverted cultured in a 30℃ incubator for 32 hours. Then, apramycin with a final concentration of 50 mg / L and trimethoprim with a final concentration of 50 mg / L were added to an EP tube containing 1 mL of sterile water, mixed evenly and applied to an SFM plate that had been cultured for 32-36 hours. After the plate dried, it was inverted in a 30°C constant temperature incubator for 5-7 days. After the colonies visible to the naked eye appeared, a single colony was picked with a toothpick and expanded on a solid STY medium containing the above concentrations of apramycin and trimethoprim. Finally, the mycelium on the STY solid medium was picked, the genome was extracted, and 801-YZ-F (SEQ ID NO.11) and 801-YZ-R (SEQ ID NO.12) were used as primers to obtain the correct SE50 / 110::kasOp*-opt-acbB mutant strain by PCR.

[0064] The endonuclease recognition sites (enzyme cutting sites) involved in the above steps 1, 2, 3 and 4 are shown in Table 1 below:

[0065] Table 1

[0066]

[0067] The primer sequences used in the above steps 1, 2, 3 and 4 are shown in Table 2:

[0068] Table 2

[0069]

[0070] The PCR system and conditions used for the preparation of gene fragments in the above steps 1 and 2 are:

[0071] PCR reaction system: DNA template 20ng, primer 20pmol, 50% DMSO 2μL, 25mM Mg 2+ 2 μL, Buffer 2 μL, pfu DNA polymerase 1 unit, add pure water to make up to 20 μL;

[0072] PCR conditions: 95°C for 5 min; 95°C for 30 s; 62°C for 30 s; 72°C for 1.5 min; 32 cycles; 72°C for 5 min.

[0073] Step 3: Detect the fermentation yield of acarbose using HPLC. Take 100 μL of the fermentation broth supernatant, add 400 μL of ultrapure water, add 500 μL of chloroform, mix well, and centrifuge at 12,000 rpm for 10 min. Use a syringe to draw the supernatant, filter it through an organic filter membrane, and add it to an injection vial. Use Agilent 1260HPLC for chromatographic analysis.

[0074] Among them, the HPLC parameters are as follows:

[0075] Chromatographic column: Agilent ZORBAX NH2 column (4.6mm x 250mm, 5μm).

[0076] Mobile phase: Phase A: phosphate solution (0.6 g KH2PO4, 0.704 g Na2HPO4·12H2O, ultrapure water to 1 L, 100 mL acetonitrile); Phase B: pure acetonitrile. Flow rate was 1.0 mL / min, and the mobile phase ratio was 35% for phase A and 65% for phase B.

[0077] Detection conditions: detection wavelength is 210nm, injection volume is 10μL, column temperature is 30℃, and sample detection time is about 30min.

[0078] Figure 5 The results of acarbose fermentation level detection after enhanced expression of acarbose biosynthesis genes opt-acbA and opt-acbB with optimized RNA secondary structure. The results show that after the enhanced expression of these genes, the fermentation level of acarbose is significantly improved. Compared with the starting strain SE50 / 110, the fermentation yields of the high-yield strains SE50 / 110::kasOp*-opt-acbA and SE50 / 110::kasOp*-opt-acbB obtained by the present invention are increased by 1.82 times and 1.32 times, respectively. Compared with the sequence-unoptimized gene overexpression mutants SE50 / 110::kasOp*-acbA and SE50 / 110::kasOp*-acbB, the fermentation yields of the high-yield strains SE50 / 110::kasOp*-opt-acbA and SE50 / 110::kasOp*-opt-acbB obtained by the present invention are increased by 81.4% and 30.4%, respectively, and the laboratory well plate fermentation levels reach 2.14 g / L and 1.77 g / L, respectively.

[0079] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for improving the fermentation level of acarbose, characterized in that: Optimize the secondary structure of RNA encoded by acarbose biosynthesis genes acbA and acbB; enhance the expression of at least one of the acarbose biosynthesis genes opt-acbA and opt-acbB with optimized RNA secondary structure in actinomycetes to obtain acarbose high-yield strain, and obtain acarbose by fermentation.

2. The method for improving the fermentation level of acarbose according to claim 1, characterized in that: The sequences of acarbose biosynthesis gene acbA and acbB are SEQ ID NO.2 and SEQ ID NO.3 respectively, the sequence of acarbose biosynthesis gene opt-acbA is shown in SEQ ID NO.4, and the sequence of acarbose biosynthesis gene opt-acbB is shown in SEQ ID NO.

5.

3. The method for improving the fermentation level of acarbose according to claim 1, characterized in that: The method of enhancing the expression of acarbose biosynthesis gene with optimized RNA secondary structure in Actinoplanes sp.SE50 / 110 specifically comprises the following steps: S1, design and construction of integrative plasmid I for enhanced expression of gene acbA; S2, design and construction of integrative plasmid II for enhanced expression of gene acbB; S3, design and construction of integrative plasmid III for enhanced expression of gene opt-acbA; S4, design and construction of integrative plasmid IV for enhanced expression of gene opt-acbB; S5, the integrative plasmid I, II, III or IV is introduced into the recipient strain by conjugation transfer, and then the mutant strain is verified for apramycin resistance, and the mycelium is picked, and the gene-enhanced expression mutant strain is screened by the difference in PCR product fragment size.

4. The method for improving the fermentation level of acarbose according to claim 3, characterized in that: Include at least one of the following technical features: Construction of integrative plasmid I: The kasOp* sequence shown in SEQ ID NO.1 was obtained by total gene synthesis, and the acbA gene was obtained by PCR using the genome of Actinoplanes sp.SE50 / 110 as a template. The kasOp*-acbA fragment with homology arms was then obtained by overlap PCR amplification, and then connected with the plasmid pRT801 double-digested with BcuⅠ and BamHⅠ by Gibson assembly method; Construction of integrative plasmid II: The kasOp* sequence shown in SEQ ID NO.1 was obtained by total gene synthesis, and the acbB gene was obtained by PCR using the genome of Actinoplanes sp.SE50 / 110 as a template. The kasOp*-acbB fragment with homology arms was then obtained by overlap PCR amplification, and then connected with the plasmid pRT801 after double digestion with BcuⅠ and BamHⅠ by Gibson assembly method; Construction of integrative plasmid III: The kasOp*-opt-acbA gene fragment containing kasOp* and opt-acbA was obtained by total gene synthesis, and the opt-acbA fragment with homology arms was obtained by PCR amplification, and then connected with the plasmid pRT801 after double digestion with BcuⅠ and BamHⅠ by the Gibson assembly method; Construction of integrative plasmid IV: The kasOp*-opt-acbB gene fragment containing kasOp* and opt-acbB was obtained by total gene synthesis, and the opt-acbB fragment with homology arms was obtained by PCR amplification, which was then connected with the plasmid pRT801 after double digestion with BcuⅠ and BamHⅠ by the Gibson assembly method.

5. The method for improving the fermentation level of acarbose according to claim 1, characterized in that: The fermentation comprises the following steps: inoculating the mycelium of the acarbose high-yielding strain into a primary seed culture medium, and culturing at 30°C and 220rpm for 28 to 36 hours; transferring the inoculation amount to a secondary seed culture medium at 10%, and culturing at 30°C and 220rpm for 24 to 28 hours; transferring the inoculation amount to a fermentation culture medium at 15%, and fermenting at 30°C and 280rpm for 3 days, collecting the fermentation liquid, and centrifuging to obtain the supernatant.

6. The method for improving the fermentation level of acarbose according to claim 5, characterized in that: Include at least one of the following technical features: The primary seed culture medium comprises 1w / v% maltose, 1.5w / v% glucose, 0.5w / v% yeast powder, 0.5w / v% peptone, 1w / v% glycerol, 0.1w / v% K2HPO4·3H2O, 1w / v% malt extract, and 0.1w / v% casein hydrolyzate; The secondary seed culture medium comprises 4w / v% soybean cake powder, 1.5w / v% maltose, 1w / v% glucose, 1w / v% glycerol, 1w / v% soluble starch, and 0.2w / v% calcium carbonate; The fermentation medium comprises 5w / v% maltose, 3w / v% glucose, 0.38w / v% sodium glutamate, 0.05w / v% FeCl3, 1-1.5w / v% soybean cake powder, 0.3-0.4w / v% Angel yeast powder, and 0.25w / v% calcium carbonate.

7. A high-yield acarbose actinomycete, characterized in that: The expression of at least one of the acarbose biosynthesis genes opt-acbA and opt-acbB with optimized RNA secondary structure is enhanced in Actinomycetes SE50 / 110 to obtain an acarbose high-yielding strain; the sequence of the gene opt-acbA is shown in SEQ ID NO.4, and the sequence of the gene opt-acbB is shown in SEQ ID NO.

5.

8. An integrative plasmid vector for enhancing the expression of acarbose biosynthesis gene, characterized in that: The vector comprises the acarbose biosynthesis gene opt-acbA or opt-acbB with optimized RNA secondary structure of the acarbose biosynthesis gene acbA or acbB, the sequence of the gene opt-acbA is shown in SEQ ID NO.4, and the sequence of the gene opt-acbB is shown in SEQ ID NO.

5.

9. A strain of Actinoplanes sp.SE50 / 110::kasOp*-opt-acbA, whose deposit number is CGMCC No.33051.

10. A strain of Actinoplanes sp.SE50 / 110::kasOp*-opt-acbB, whose deposit number is CGMCC No.33052.

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