A bifunctional flavin adenine dinucleotide synthetase mutant and its application in fad synthesis

Abstract

This invention discloses a bifunctional flavin adenine dinucleotide synthase mutant and its application in FAD synthesis. The mutant disclosed in this invention is RFK / FADS. C18R or RFK / FADS C18R‑R46H ;RFK / FADS C18R The amino acid sequence is shown in SEQ ID NO.15, RFK / FADS C18R‑R46H The amino acid sequence is shown in SEQ ID NO. 16. Genetically engineered bacteria carrying the bifunctional flavin adenine dinucleotide synthase mutant gene plasmid produced flavin adenine dinucleotide using glucose as a substrate through a simple fermentation process under aerobic conditions in a shake flask. The FAD yields of strains XF02 and XF03 containing the mutant gene were 79.31 mg / L and 167.56 mg / L, respectively, representing increases of 58.62% and 235.12% compared to strain XF01 containing the non-mutated gene. By constructing microbial "cell factories" through metabolic engineering strategies, modified microorganisms can utilize inexpensive raw materials to generate desired natural products within their cells. This advancement not only reduces the production cost of FAD but also improves the environmental sustainability of its production process.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to a bifunctional flavin adenine dinucleotide synthetase mutant and application thereof in FAD synthesis. BACKGROUND

[0002] Flavin adenine dinucleotide (FAD) is the metabolically active form of riboflavin (RF). As an important cofactor in cells, FAD plays an important role in cellular metabolism such as electron transport, photosynthesis, fatty acid oxidation, and has been widely used in the fields of medicine and biological enzyme catalysis. In biological enzyme catalysis, both FMN (Flavin mononucleotide) and FAD are indispensable redox cofactors in most flavoproteins or flavoenzymes in cells. In addition, FAD is also widely used in the research and application of biosensors and microbial fuel cells.

[0003] Currently, FAD is mainly produced by whole-cell catalysis method. The substrate FMN and ATP added in vitro can be further catalyzed to FAD and PPi by using an engineered strain overexpressing FAD synthetase. However, in the whole-cell catalytic production of FAD, the high price of substrate ATP leads to high production cost. In contrast, microbial fermentation method, which has cheap substrate and does not require high-density culture, has greater potential. Researchers have constructed microbial "cell factories" through metabolic engineering strategies. These modified microorganisms can use low-cost raw materials to produce the desired natural products in microbial cells. This progress not only reduces the production cost of FAD, but also improves the environmental sustainability of the production process. In addition, there is little research on the mutation of FAD synthetase. In vivo, riboflavin and ATP are catalyzed by riboflavin kinase (RFK) to generate FMN and ADP, and FMN and ATP (or AMP) are further catalyzed by FAD synthetase (FADS) to generate FAD and pyrophosphate. In eukaryotes, these two enzymes exist in the form of monofunctional enzymes, while in prokaryotes, riboflavin kinase and FAD synthetase usually exist in the form of bifunctional enzymes encoded by the same peptide chain. However, in-depth research is limited to only a few species such as Corynebacterium ammoniagenes and Candida glabrata, and related research has only stayed on the enzymatic properties and catalytic mechanism, and has not been applied to the production of FAD. Therefore, it is urgent to construct bifunctional flavin adenine dinucleotide synthetase mutants from Escherichia coli with improved catalytic performance for the synthesis of FAD.

[0004] The mutant of the advantageous enzyme can be obtained by directed evolution, which simulates the natural evolution process (mutation-screening-iteration) to optimize the function without prior knowledge of the structure of the enzyme, and the function diversity is improved. The error-prone PCR technology in directed evolution is a molecular biology technology that artificially introduces random mutations by adjusting the reaction conditions (such as Mn2+ concentration, dNTP ratio). It is simple to operate and only requires conventional PCR equipment to generate a diversified mutation library, and the mutation rate is controllable. The mutation diversity and function preservation can be balanced by adjusting the reaction parameters. SUMMARY

[0005] To overcome the high cost of substrate ATP in the whole cell catalytic production of FAD in the prior art, resulting in high production cost, the application provides a bifunctional flavin adenine dinucleotide synthetase mutant with improved catalytic performance for the synthesis of FAD, solving the problem of high cost of whole cell catalytic production of FAD in the prior art.

[0006] To achieve the above-mentioned purposes, the technical scheme adopted by the application comprises:

[0007] A bifunctional flavin adenine dinucleotide synthetase mutant, wherein the mutant is RFK / FADS C18R or RFK / FADS C18R-R46H ; the amino acid sequence of RFK / FADS C18R is shown in SEQ ID NO. 15, and the amino acid sequence of RFK / FADS C18R-R46H is shown in SEQ ID NO. 16.

[0008] A gene encoding the bifunctional flavin adenine dinucleotide synthetase mutant, wherein the nucleotide sequence of the gene encoding RFK / FADS C18R is shown in SEQ ID NO. 5, and the nucleotide sequence of the gene encoding RFK / FADS C18R-R46H is shown in SEQ ID NO. 6.

[0009] A recombinant plasmid comprising the gene encoding the bifunctional flavin adenine dinucleotide synthetase mutant.

[0010] A genetically engineered bacterium comprising the recombinant plasmid.

[0011] The synthetase mutant expression strain is selected from Escherichia coli.

[0012] The genetically engineered bacterium is used for the fermentation preparation of flavin adenine dinucleotide.

[0013] Compared with the prior art, the application has the beneficial effects that:

[0014] The application screens a bifunctional flavin adenine dinucleotide synthetase mutant with improved catalytic performance through an error-prone PCR method in directed evolution, and produces flavin adenine dinucleotide through a simple fermentation process under aerobic conditions in a shake flask with glucose as a substrate by using a genetically engineered bacterium carrying a plasmid of the gene of the bifunctional flavin adenine dinucleotide synthetase mutant. The FAD production of the strains XF02 and XF03 containing the mutant gene is 79.31 mg / l and 167.56 mg / l, respectively, which is increased by 58.62% and 235.12% compared with the strain XF01 containing the non-mutant gene. Through metabolic engineering strategies, a microbial “cell factory” is constructed, and the modified microorganism can generate the required natural product in the microbial cell by using low-cost raw materials. This progress not only reduces the production cost of FAD, but also improves the environmental sustainability of the production process. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Plasmid map of p20c-ribF. DETAILED DESCRIPTION

[0016] The application will be further described below in combination with specific examples, which are only exemplary and do not limit the protection scope of the application. Unless defined, the technical terms used in the following examples have the same meaning as generally understood by those skilled in the art to which the application belongs. The experimental reagents used in the following examples are conventional biochemical reagents unless otherwise specified; and the experimental methods used in the following examples are conventional methods unless otherwise specified.

[0017] The riboflavin-producing strain LS21 (its original strain Escherichia coli MG1655 is commercially available) used in the application is constructed according to the steps of the following article: Liu S. Metabolic engineering of Escherichia coli LS02T for high-yield riboflavin production[D]. Tianjin University, 2022. DOI:10.27356 / d.cnki.gtjdu.2022.000045.

[0018] The plasmid p20C was derived from the following article: LIU S, DIAO N, WANG Z, et al. Modular Engineering of the Flavin Pathway in Escherichia coli for Improved Flavin Mononucleotide and Flavin Adenine Dinucleotide Production [J]. Journal of Agricultural and Food Chemistry, 2019, 67(23): 6532-40.

[0019] The method of CPEC plasmid construction was derived from the following article: QUAN JY, TIAN J D. Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways [J]. Plos One, 2009, 4(7).

[0020] The flavin adenine dinucleotide standard used was purchased from TCI company (https: / / www.tcichemicals.com).

[0021] The primers used were synthesized by Genewiz company (https: / / www.genewiz.com.cn / ).

[0022] The seamless cloning reagent used was purchased from Abclonal company (https: / / abclonal.com.cn / ).

[0023] The other biochemical reagents used were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0024] The error-prone PCR kit used was purchased from Beyotime Biotechnology (https: / / www.beyotime.com / index.htm).

[0025] Terminology

[0026] The bifunctional flavin adenine dinucleotide synthetase, abbreviated as RFK / FADS, has an amino acid sequence as shown in SEQ ID NO. 1.

[0027] Example 1 Construction and directed evolution of plasmid carrying bifunctional flavin adenine dinucleotide synthetase gene

[0028] (1) Using FADS-F (SEQ ID NO. 7) and FADS-R (SEQ ID NO. 8) as primers, PCR amplification was performed with E. coli MG1655 genome as template to obtain RFK / FADS gene fragment (SEQ ID NO. 2) with a size of 942 bp; using p20C-F (SEQ ID NO. 9) and p20C-R (SEQ ID NO. 10) as primers, PCR amplification was performed with plasmid p20C as template to obtain p20C vector fragment (SEQ ID NO. 3) with a size of 2808 bp; the RFK / FADS gene fragment and the p20C vector fragment (SEQ ID NO. 3) were connected by CPEC plasmid construction method to construct plasmid p20c-ribF;

[0029] (2) Using QM-F (SEQ ID NO. 11) and QM-R (SEQ ID NO. 12) as primers, error-prone PCR amplification was performed on the first 230 amino acids of the enzyme using the error-prone PCR kit of Biyun Tian to obtain a mixed fragment containing different mutation points with a size of 733bq.

[0030] (3) PCR amplification was performed using FQM-F (SEQ ID NO. 13) and FQM-R (SEQ ID NO. 14) as primers to obtain p20c-ribF vector fragment (SEQ ID NO. 4).

[0031] (4) The p20c-ribF vector fragment (SEQ ID NO. 4) and the mixed fragment obtained in step (2) were connected into a mixed plasmid by seamless cloning method.

[0032] (5) The mixed plasmid in step (4) was electroporated into the competent strain MG1655, plated, and the cells were picked into multiple 96-well plates. After fermentation for 24 h, all samples were detected in liquid phase. The yields of FAD, FMN and RF were determined by high performance liquid chromatography (HPLC) method. The fermentation broth was diluted to an appropriate concentration, centrifuged at 12000g for 10 min, and the supernatant was filtered through a 0.22μm water membrane into a brown liquid phase vial. Agilent high performance liquid chromatography was used with a C18 column, column temperature 30℃, mobile phase 30% methanol solution containing 10mM NaH2PO4·2H2O, flow rate 0.8mL / min, sample injection volume 10μL, detector UV VWD detector, detection wavelength 444nm. According to the fermentation data, the dominant strain was selected, and the mutation site of C18R was obtained after sequencing, and the corresponding strain was named MG1655-PF-C18R;

[0033] (6) Pick the strain MG1655-PF-C18R from step (5) in a test tube and culture it for 12 hours. Then, extract the plasmid pF-C18R using a plasmid extraction kit. Using the extracted plasmid pF-C18R as a template, use QM-F (SEQ ID NO.11) and QM-R (SEQ ID NO.12) primers to perform another round of error-prone PCR amplification to obtain a mixed fragment containing different mutation points. This mixed fragment is ligated with the p20c-ribF vector fragment (SEQ ID NO.4) using a seamless cloning method to form a mixed plasmid. The mixed plasmid is electroporated into the competent cells of strain MG1655 and plated. After fermentation in well plates, the dominant strain is selected based on the fermentation data. After sequencing, the mutation site C18R-R46H is obtained, and the corresponding strain is named MG1655-PF-C18R-R46H.

[0034] The RFK / FADS mutant is RFK / FADS. C18R and RFK / FADS C18R-R46H Two types; RFK / FADS C18R The amino acid sequence is shown in SEQ ID NO.15, RFK / FADS C18R-R46H The amino acid sequence is shown in SEQ ID NO.16.

[0035] Encoding the bifunctional flavin adenine dinucleotide synthase mutant RFK / FADS C18R The nucleotide sequence of the gene is shown in SEQ ID NO.5, encoding the bifunctional flavin adenine dinucleotide synthase mutant RFK / FADS. C18R-R46H The nucleotide sequence of the gene is shown in SEQ ID NO.6.

[0036] Example 2: Genetically engineered bacteria containing the above recombinant expression plasmid

[0037] (1) Pick the strain MG1655-PF-C18R-R46H from Example 1 into a test tube, culture it for 12 hours, and then extract the plasmid pF-C18R-R46H using a plasmid extraction kit.

[0038] (2) The plasmid p20c-ribF was electroporated into the competent cells of strain LS21 to construct strain XF01.

[0039] (3) The mutant plasmids pF-C18R and pF-C18R-R46H were electroporated into the competent cells of strain LS21 to construct strains XF02 and XF03.

[0040] Example 3: Preparation of Fermentation Culture Medium

[0041] Take 1 mL of component I, 1 mL of component II, 1 mL of mixture of component III and IV, add glucose to a final concentration of 10 g / L, add yeast extract to a final concentration of 5 g / L, add distilled water to 50 mL; obtain fermentation medium;

[0042] The component I is: take 10 g of (NH4)2SO4, 2 g of MgSO4, and make up to 200 mL with distilled water, sterilize at 121℃ for 20 min;

[0043] The component II is: take 38.3 g of Na2HPO4, 15 g of KH2PO4, and make up to 200 mL with distilled water, sterilize at 121℃ for 20 min;

[0044] The component III is: take 5 g of ferric ammonium citrate, 2 g of CaCl2·2H2O, 41.7 mL of 12 mol / L HCl aqueous solution, and make up to 1000 mL with distilled water;

[0045] The component IV is: take 1 g of ZnSO4·7H2O, 0.3 g of MnCl2·4H2O, 3 g of H3BO3, 2 g of CoCl2·6H2O, 0.1 g of CuSO4·5H2O, 0.2 g of NiCl2·6H2O, and 0.3 g of NaMoO4·2H2O, and make up to 1000 mL with distilled water;

[0046] The mixture of component III and IV is: take 100 mL of component III, 1 mL of component IV, make up to 200 mL with distilled water, and adjust the pH value to 4.5-5.5 with 5M NaOH aqueous solution, sterilize at 121℃ for 20 min.

[0047] Example 4: Shake flask fermentation of the strain

[0048] (1) Activate the strain: the strain obtained in Example 2 is streaked on LB solid medium and cultured at 37℃ for 12-20 h to rejuvenate the strain;

[0049] (2) Culture of seed liquid: the single colony obtained in step (1) is inoculated into LB liquid medium and cultured at 37℃, 220 rpm for 12-20 h;

[0050] (3) Shake flask fermentation: the seed liquid obtained in step (2) is measured by ultraviolet spectrophotometer at 600 nm absorbance value, and the initial OD 600The inoculation amount was 0.025, and the fermentation medium was inoculated, and the shake flask liquid volume was 50 mL / 500 mL. The final concentration of spectinomycin was 100 mg / L, and the culture was carried out at 37°C, 220 rpm. After 24 h of fermentation, the FAD yield of strains XF02 and XF03 was 79.31 mg / l and 167.56 mg / l, respectively, which was 58.62% and 235.12% higher than 50.01 mg / l of XF01, respectively.

[0051] The above examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and these modifications and replacements all fall within the protection scope of the present application.

Claims

1. A bifunctional flavin adenine dinucleotide synthase mutant, characterized in that, The bifunctional flavin adenine dinucleotide synthase mutant is RFK / FADS. C18R or RFK / FADS C18R-R46H ;RFK / FADS C18R The amino acid sequence is shown in SEQ ID NO.15, RFK / FADS C18R-R46H The amino acid sequence is shown in SEQ ID NO.

16.

2. A gene encoding a gene, characterized in that, The gene is the gene encoding the bifunctional flavin adenine dinucleotide synthase mutant of claim 1, and the gene encoding RFK / FADS is... C18R The nucleotide sequence of the gene is as shown in SEQ ID NO.5, which encodes RFK / FADS. C18R-R46H The nucleotide sequence of the gene is shown in SEQ ID NO.

6.

3. A recombinant plasmid, characterized in that, The recombinant plasmid contains the gene encoding the bifunctional flavin adenine dinucleotide synthase mutant as described in claim 2.

4. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria comprises the recombinant plasmid as described in claim 3.

5. The bifunctional flavin adenine dinucleotide synthase mutant according to claim 1, characterized in that, The synthase mutant expression strain was selected from Escherichia coli.

6. The use of the genetically engineered bacteria according to claim 4 in the fermentation preparation of flavin adenine dinucleotide.

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