Bifunctional flavin adenine dinucleotide synthetase mutant and application thereof in FAD synthesis

Abstract

The invention discloses a bifunctional flavin adenine dinucleotide synthetase mutant and an application of the bifunctional flavin adenine dinucleotide synthetase mutant in FAD synthesis. The mutant disclosed by the invention is RFK / FADSC18R or RFK / FADSC18R-R46H, and the structural formula of the mutant is as shown in the specification. The amino acid sequence of the RFK / FADSC18R is as shown in SEQ ID NO. 15, and the amino acid sequence of the RFK / FADSC18R-R46H is as shown in SEQ ID NO. 16. A genetically engineered bacterium carrying the bifunctional flavin adenine dinucleotide synthetase mutant gene plasmid is used for producing flavin adenine dinucleotide by using glucose as a substrate through a simple fermentation process under a shake flask aerobic condition, and the FAD yields of strains XF02 and XF03 containing mutant genes are 79.31 mg / l and 167.56 mg / l in sequence; and compared with a strain XF01 containing a non-mutated gene, the mutant gene is improved by 58.62% and 235.12% respectively. A microbial'cell factory 'is constructed through a metabolic engineering strategy, and the modified microorganisms can generate required natural products in microbial cells by utilizing low-price raw materials. The progress not only reduces the production cost of the FAD, but also improves the environmental sustainability of the production process of the FAD.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a bifunctional flavin adenine dinucleotide synthase mutant and its application in FAD synthesis. Background Art

[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 cell metabolism such as electron transfer, photosynthesis, and fatty acid oxidation, and has currently been widely used in fields such as medicine and biocatalysis. In biocatalytic reactions, both flavin mononucleotide 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 development and application of biosensors and microbial fuel cells.

[0003] Currently, FAD is mainly commercially produced by whole-cell catalysis. An engineered strain overexpressing FAD synthase can further catalyze the in vitro added substrates FMN and ATP into FAD and PPi. However, when producing FAD by whole-cell catalysis, due to the high price of the substrate ATP, the production cost is relatively high. In contrast, the microbial fermentation method with cheap substrates and no need for high-density culture has greater potential. Researchers have constructed microbial "cell factories" through metabolic engineering strategies. These modified microorganisms can generate the required natural products in microbial cells using inexpensive raw materials. This progress not only reduces the production cost of FAD, but also improves the environmental sustainability of its production process. In addition, there are few studies on the mutation of FAD synthase at present. 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 synthase (FADS) to generate FAD and pyrophosphate. In eukaryotes, these two enzymes exist in the form of monofunctional enzymes respectively, while in prokaryotes, riboflavin kinase and FAD synthase usually exist in the form of a bifunctional enzyme encoded by the same peptide chain. However, the in-depth research is limited to only a few species such as Corynebacterium ammoniagenes and Candida glabrata, and the related research only stays on the enzyme properties and catalytic mechanisms, and has not been applied to the production of FAD. Therefore, it is urgent to construct a bifunctional flavin adenine dinucleotide synthase mutant derived from Escherichia coli with improved catalytic performance for FAD synthesis.

[0004] To find advantageous enzyme mutants, directed evolution can be employed. By mimicking the natural evolution process (mutation - screening - iteration), functions can be optimized without prior knowledge of the enzyme's structure information, and functional diversity can be enhanced. By introducing random mutations, mutants with improved catalytic activity, stability, or substrate specificity can be obtained. Error - prone PCR technology in directed evolution is a molecular biology technique that artificially introduces random mutations by adjusting reaction conditions (such as Mn2+ concentration, dNTP ratio). It is easy to operate and only requires conventional PCR equipment to generate a diverse mutant library, and the mutation rate can be controlled. By adjusting reaction parameters, the balance between mutation diversity and function retention can be achieved. Summary of the Invention

[0005] To overcome the deficiency in the prior art that the price of substrate ATP is high in the whole - cell catalyzed production of FAD, resulting in high production costs, the present invention provides a bifunctional flavin adenine dinucleotide synthase mutant with improved catalytic performance for the synthesis of FAD, solving the problem of high costs in the whole - cell catalyzed production of FAD in the prior art.

[0006] To achieve the aforementioned invention objective, the technical solutions adopted by the present invention include:

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

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

[0009] A recombinant plasmid, which contains the gene encoding the bifunctional flavin adenine dinucleotide synthase mutant.

[0010] A genetically engineered bacterium, which contains the recombinant plasmid described above.

[0011] The expression strain of the synthase mutant is selected from Escherichia coli.

[0012] Use of the genetically engineered bacterium in the fermentation preparation of flavin adenine dinucleotide.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] By using the error-prone PCR method in directed evolution, the present invention screens out a mutant bifunctional flavin adenine dinucleotide synthase with improved catalytic performance. A genetically engineered bacterium carrying a plasmid gene of the mutant bifunctional flavin adenine dinucleotide synthase produces flavin adenine dinucleotide through a simple fermentation process using glucose as a substrate under aerobic conditions in a shake flask. The FAD yields of the mutant gene-containing strains XF02 and XF03 are 79.31 mg / l and 167.56 mg / l respectively, which are 58.62% and 235.12% higher than that of the strain XF01 containing the unmutated gene. By constructing a microbial "cell factory" through metabolic engineering strategies, the modified microorganisms can generate the required natural products within microbial cells using inexpensive raw materials. This progress not only reduces the production cost of FAD, but also improves the environmental sustainability of its production process. Brief Description of the Drawings

[0015] Figure 1 It is the plasmid map of p20c-ribF. Specific Embodiments

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

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

[0018] Plasmid p20C is 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 for constructing CPEC plasmids is derived from the following article: QUAN JY, TIAN JD. 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 (https: / / www.tcichemicals.com).

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

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

[0023] 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 Co., Ltd. (https: / / www.beyotime.com / index.htm).

[0025] Explanation of Terms

[0026] Bifunctional flavin adenine dinucleotide synthase, abbreviated as: RFK / FADS, whose amino acid sequence is shown in SEQ ID NO.1.

[0027] Example 1 Construction and Directed Evolution of Plasmids Carrying the Bifunctional Flavin Adenine Dinucleotide Synthase Gene

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

[0029] (2) Using the plasmid p20c-ribF as a template, and the error-prone PCR kit from Beyotime, and QM-F (SEQ ID NO.11) and QM-R (SEQ ID NO.12) primers, perform error-prone PCR amplification on the first 230 amino acids of the enzyme to obtain a mixed fragment containing different mutation sites with a size of 733 bp.

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

[0031] (4) Connect the p20c-ribF vector fragment (SEQ ID NO.4) and the mixed fragment obtained in step (2) into a tube of mixed plasmids by seamless cloning method.

[0032] (5) Electrotransform the mixed plasmids in step (4) into the competent cells of strain MG1655, perform plate coating, pick cells into multiple 96-well plates, after 24 h of fermentation in the wells, detect the yield in the liquid phase after all samplings. The yields of FAD, FMN, and RF are determined by high performance liquid chromatography (HPLC). Dilute the fermentation broth to an appropriate concentration, centrifuge at 12000 g for 10 min, take the supernatant, filter it through a 0.22 μm water membrane into a brown liquid phase vial, use Agilent high performance liquid chromatography, the chromatographic column is a C18 column, the column temperature is 30 °C, the mobile phase is a 30% methanol solution containing 10 mM NaH2PO4·2H2O, the flow rate is 0.8 mL / min, the sample injection volume is 10 μL, the detector is an ultraviolet VWD detector, and the detection wavelength is 444 nm. Select the dominant bacteria according to the fermentation data, obtain the mutation sites of C18R after sequencing, and name the corresponding strain MG1655-PF-C18R;

[0033] (6) Pick the strain of MG1655-PF-C18R in step (5) into a test tube. After culturing for 12 h, extract plasmid pF-C18R using a plasmid extraction kit. Using the extracted plasmid pF-C18R as a template, with primers QM-F (SEQ ID NO.11) and QM-R (SEQ ID NO.12), perform another round of error-prone PCR amplification to obtain a mixed fragment containing different mutation sites. Connect the mixed fragment with the p20c-ribF vector fragment (SEQ ID NO.4) into a tube of mixed plasmid by seamless cloning method. Electrotransform the mixed plasmid into the competent cells of strain MG1655, perform plate coating, and select the dominant bacteria according to the fermentation data after fermentation in the well plate. After sequencing, obtain the mutation site of C18R-R46H, and name the corresponding strain MG1655-PF-C18R-R46H;

[0034] The RFK / FADS mutants are RFK / FADS C18R and RFK / FADS C18R-R46H There are two types; RFK / FADS C18R The amino acid sequence of RFK / FADS is shown in SEQ ID NO.15, and the amino acid sequence of RFK / FADS C18R-R46H is shown in SEQ ID NO.16.

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

[0036] Example 2 Genetic engineering bacteria containing the above recombinant expression plasmid

[0037] (1) Pick the strain of MG1655-PF-C18R-R46H in Example 1 into a test tube. After culturing for 12 h, extract plasmid pF-C18R-R46H using a plasmid extraction kit.

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

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

[0040] Example 3 Preparation of fermentation medium

[0041] Take 1 mL of Component I, 1 mL of Component II, 1 mL of the mixed solution 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, and add distilled water to 50 mL; obtain the fermentation medium;

[0042] The Component I is: Take 10 g of (NH4)2SO4, 2 g of MgSO4, make up the volume to 200 mL with distilled water, and sterilize at 121 °C for 20 min;

[0043] The Component II is: Take 38.3 g of Na2HPO4, 15 g of KH2PO4, make up the volume to 200 mL with distilled water, and sterilize at 121 °C for 20 min;

[0044] The Component III is: Take 5 g of ammonium ferric citrate, 2 g of CaCl2·2H2O, 41.7 mL of 12 mol / L HCl aqueous solution, make up the volume 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, 0.3 g of NaMoO4·2H2O, make up the volume to 1000 mL with distilled water;

[0046] The mixed solution of Component III and IV is: Take 100 ml of Component III, 1 ml of Component IV, make up the volume to 200 mL with distilled water, and adjust the pH value to 4.5 - 5.5 with 5 M NaOH aqueous solution, and sterilize at 121 °C for 20 min.

[0047] Flask fermentation of the strain in Example 4

[0048] (1) Activate the strain: Streak the strain obtained in Example 2 on the LB solid medium and culture at 37 °C for 12 - 20 h to rejuvenate the strain;

[0049] (2) Culture of the seed liquid: Inoculate the single colony obtained in step (1) into the LB liquid medium and culture at 37 °C and 220 rpm for 12 - 20 h;

[0050] (3) Flask fermentation: Measure the absorbance value of the seed liquid obtained in step (2) at 600 nm through an ultraviolet spectrophotometer, according to the initial OD 600Inoculum size was 0.025, inoculated into the fermentation medium, the liquid volume in the shake flask was 50 mL / 500 mL. Kitasamycin was added to make the final concentration 100 mg / L, cultured at 37 °C and 220 rpm. After 24 h of fermentation, the FAD yields of strains XF02 and XF03 were 79.31 mg / L and 167.56 mg / L respectively, which were increased by 58.62% and 235.12% respectively compared with 50.01 mg / L of XF01.

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

Claims

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

15. C18R-R46H The amino acid sequence is shown in SEQ ID NO.

16.

2. A coding gene, characterized in that The gene encodes the bifunctional flavin adenine dinucleotide synthetase mutant of claim 1, and the gene encoding RFK / FADS 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 comprises the gene encoding the bifunctional flavin adenine dinucleotide synthetase mutant according to claim 2.

4. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria comprises the recombinant plasmid according to claim 3.

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

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

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