Recombinant bacteria for synthesizing flavin adenine dinucleotide and construction method and application thereof

Abstract

The present application relates to a recombinant bacteria for synthesizing flavin adenine dinucleotide, a construction method and application thereof, and belongs to the field of biotechnology and enzyme engineering. The present application takes Bacillus subtilis 168 (trpC2) as a starting strain, obtains a chassis cell W0 by knocking out yfkN, guaC and purR genes in a purine pathway, simultaneously expresses FAD synthetase mutant from Bacillus subtilis and riboflavin transporter RibM from Streptomyces davawensis in the W0, and the FAD yield reaches 126.4 mg / L in a flask fermentation, which is increased by 73.2% compared with the wild type. After fermentation process optimization, the FAD yield reaches 563 mg / L. The present application effectively solves the technical bottlenecks of insufficient substrate uptake and low synergistic catalytic efficiency of bifunctional enzyme in FAD biosynthesis, and provides an efficient cell factory for green industrial production of FAD.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and enzyme engineering, and in particular to recombinant bacteria that synthesize flavin adenine dinucleotide, their construction methods, and applications. Background Technology

[0002] Flavin adenine dinucleotide (FAD) is a crucial redox coenzyme in living organisms, widely involved in core life processes such as energy metabolism (e.g., the tricarboxylic acid cycle, fatty acid β-oxidation), redox homeostasis maintenance, and the electron transport chain. Due to its unique redox properties and conformation-dependent fluorescence, FAD has demonstrated significant application value in food, medicine, disease diagnosis, and biosensing. However, the persistently high market price of FAD (approximately RMB 260,000 / kg) severely restricts its large-scale application.

[0003] Currently, the production methods of flavin adenine dinucleotide (FAD) are mainly divided into chemical synthesis and biosynthesis. Chemical synthesis suffers from problems such as harsh reaction conditions, expensive raw materials, low yield, and difficulties in separation and purification, making industrial-scale production difficult. Biosynthesis, due to its milder conditions and environmental friendliness, has become a research hotspot. Traditional bio-fermentation produces FAD by modifying the endogenous de novo synthetic pathway of microorganisms. However, its biosynthesis involves dozens of steps, including the pentose phosphate pathway, the purine pathway, and the riboflavin synthesis pathway, making it quite complex. Furthermore, chassis modification requires balancing host growth and production, making gene editing difficult and the de novo synthesis cycle long.

[0004] In recent years, the "one-step" whole-cell catalysis strategy has attracted widespread attention. This strategy uses exogenously added, inexpensive riboflavin (RF) as a substrate and utilizes FAD synthases (FADs) expressed by recombinant microorganisms to directly catalyze the conversion of riboflavin to flavin adenine dinucleotide, significantly simplifying the metabolic pathway. However, this method still faces two major bottlenecks: first, the low transmembrane uptake efficiency of the substrate RF leads to insufficient utilization of exogenous RF; second, the catalytic efficiency of bifunctional FAD synthases in prokaryotes is not high, and there is a complex conformational coupling between the two domains, making it difficult to achieve a synergistic enhancement of the two catalytic activities through traditional enzyme engineering. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a recombinant bacterium that synthesizes flavin adenine dinucleotide, its construction method, and its application. Specifically, it involves metabolic engineering of chassis cells, co-expression of FAD synthase and riboflavin transporter protein, and construction of a high-yield FAD cell factory by combining enzyme molecule site-directed mutagenesis.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a recombinant bacterium that synthesizes flavin adenine dinucleotide, wherein the recombinant bacterium is based on Bacillus subtilis 168 (trpC2) as the starting strain; the yfkN gene, guaC gene and purR gene in the genome of the starting strain are knocked out; and the gene BsFADs, which expresses a mutant of FAD synthase, and the gene ribM, which expresses riboflavin transporter, are integrated.

[0008] The FAD synthase mutant uses the FAD synthase with the amino acid sequence shown in SEQ ID NO.1 as the parent and undergoes the following mutations: proline at position 61 is mutated to tryptophan and / or arginine at position 202 is replaced with tyrosine;

[0009] The FAD synthase is derived from Bacillus subtilis.

[0010] In one embodiment of the present invention, the Gene IDs of the yfkN gene, guaC gene, and purR gene are 936131, 937189, and 937000, respectively.

[0011] In one embodiment of the present invention, the gene copy number ratio of the FAD synthase mutant gene BsFADs to the riboflavin transporter gene ribM is 1:1 to 1:2.

[0012] In one embodiment of the present invention, the riboflavin transporter is derived from Streptomyces davorans; the Gene ID of the riboflavin transporter gene is 14962409, and the amino acid sequence is shown in SEQ ID NO.2.

[0013] A second objective of this invention is to provide a method for constructing the recombinant bacteria that synthesize flavin adenine dinucleotide, comprising the following steps:

[0014] (1) Using Bacillus subtilis 168 (trpC2) as the starting strain, chassis cells were obtained by knocking out the yfkN gene, guaC gene and purR gene;

[0015] (2) The gene BsFADs of the FAD synthase mutant and the gene ribM of the riboflavin transporter were cloned into the expression vector and transformed into the chassis cells obtained in step (1) to obtain the recombinant bacteria that synthesize flavin adenine dinucleotide.

[0016] A third objective of this invention is to provide the application of the recombinant bacteria that synthesize flavin adenine dinucleotide in the production of flavin adenine dinucleotide.

[0017] The fourth objective of this invention is to provide a method for synthesizing flavin adenine dinucleotide, comprising the following steps: using riboflavin as a raw material, and fermenting it with the recombinant bacteria that synthesize flavin adenine dinucleotide.

[0018] In one embodiment of the present invention, the concentration of riboflavin is 100 mg / L-500 mg / L.

[0019] In one embodiment of the present invention, the initial pH value of the fermentation is 5.0-9.0;

[0020] And / or, the fermentation speed is 100 rpm-250 rpm;

[0021] And / or, the fermentation temperature is 30℃-37℃;

[0022] And / or, the fermentation time is 48 h-72 h;

[0023] And / or, the fermentation further includes the addition of IPTG for induction; the concentration of IPTG is 0.1 mM-1.6 mM; the induction time of IPTG is 0-8 h after inoculation.

[0024] In one embodiment of the present invention, the fermentation medium for fermentation includes a carbon source and a nitrogen source; the carbon source includes one or more of glucose, sucrose, maltose, starch and glycerol, with a carbon source concentration of 10 g / L-50 g / L; the nitrogen source includes one or more of yeast extract, tryptone, soybean meal, fish peptone and beef extract, with a nitrogen source concentration of 10 g / L-50 g / L.

[0025] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0026] This invention provides a recombinant bacterium for synthesizing flavin adenine dinucleotide, its construction method, and its applications. First, this invention involves metabolic engineering of Bacillus subtilis 168 by knocking out the yfkN, guaC, and purR genes, resulting in a chassis cell WO with a 5-fold increase in basal FAD production. Based on this, the heterologous RF transporter RibM was introduced, and its copy number ratio with BsFADs was optimized to 1:2, significantly enhancing the transmembrane uptake of substrate RF, increasing FAD production from 37.6 mg / L to 73 mg / L. Furthermore, the isothermal compression coefficient perturbation engineering (ICPE) strategy was applied to rationally design bifunctional enzymes BsFADs, obtaining double-point mutant BsFADs. P61W+R202YThe FAD yield was further increased to 126.4 mg / L. Through systematic optimization of the fermentation process, the final FAD yield in shake flasks reached 563 mg / L, approximately 245 times higher than that of the starting strain W0 (2.3 mg / L). This invention effectively solves the common technical challenges of insufficient substrate uptake and low catalytic efficiency of bifunctional enzymes in FAD biosynthesis, providing a highly efficient cell factory for the green industrial production of FAD. Attached Figure Description

[0027] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0028] Figure 1 FAD production in Bacillus subtilis before and after modification of the 168-purine pathway;

[0029] Figure 2 Comparison of FAD synthesis yields when FAD synthases from different species are overexpressed in strain W0;

[0030] Figure 3 The effect of co-expression of FAD synthase from Bacillus subtilis and transport proteins from different species on FAD production;

[0031] Figure 4 Comparison of FAD yields between single-point mutants and double-point combination mutants of BsFADs;

[0032] Figure 5 BsFADs were carried by strain W4. P61W+R202Y Changes in FAD production at different RF concentrations;

[0033] Figure 6 BsFADs were carried by strain W4. P61W+R202Y Changes in FAD production under different pH (A) and shaking speed (B) conditions;

[0034] Figure 7 BsFADs were carried by strain W4. P61W+R202Y Changes in FAD production under different IPTG concentrations (A) and addition times (B);

[0035] Figure 8 BsFADs were carried by strain W4. P61W+R202Y Changes in FAD production under different carbon source types (A) and maltose concentrations (B);

[0036] Figure 9 BsFADs were carried by strain W4. P61W+R202Y Changes in FAD production under different nitrogen source types (A) and yeast extract (B);

[0037] Figure 10 BsFADs were carried by strain W4. P61W+R202Y FAD production variation under optimal conditions. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0039] The first objective of this invention is to provide a recombinant Bacillus subtilis strain that efficiently synthesizes FAD, expressing FAD synthases (BsFADs) from Bacillus subtilis and riboflavin transporter RibM from Streptomyces davawensis. The amino acid sequence of the BsFADs is shown in SEQ ID NO.1, and the amino acid sequence of the RibM is shown in SEQ ID NO.2.

[0040] Furthermore, the chassis cells of the recombinant Bacillus subtilis are derived strains of Bacillus subtilis 168 obtained through metabolic engineering. Specifically, by knocking out at least one of the yfkN, guaC, and purR genes, bypass metabolic consumption is reduced, resulting in optimized chassis cells W0. Experiments show that the background FAD production of strain W0 is 5 times higher than that of wild-type Bacillus subtilis 168, reaching 2.5 mg / L.

[0041] Furthermore, in the recombinant Bacillus subtilis, the gene copy number ratio of BsFADs to RibM is 1:1 to 1:2. Experiments show that when the ratio is 1:2, the FAD production increases from 37.6 mg / L when BsFADs are expressed alone to 73 mg / L.

[0042] Further, the BsFADs are mutants obtained through isothermal compression coefficient perturbation engineering (ICPE). The mutants have at least one of the following mutations relative to the amino acid sequence shown in SEQ ID NO.1: proline at position 61 is replaced by tryptophan (P61W); arginine at position 202 is replaced by tyrosine (R202Y); or both P61W and R202Y mutations are present. Double mutant BsFADs are also included. P61W+R202Y The best results were achieved, with FAD production reaching 126.4 mg / L in shake-flask fermentation, a 73.2% increase compared to the wild type (73 mg / L).

[0043] A second objective of this invention is to provide a nucleic acid molecule encoding the aforementioned FAD synthase or transport protein.

[0044] A third object of the present invention is to provide an expression vector carrying the nucleic acid molecule. Preferably, the expression vector is pHT01 or a derivative thereof.

[0045] A fourth objective of this invention is to provide a method for constructing the recombinant Bacillus subtilis, comprising the following steps:

[0046] (1) Using Bacillus subtilis 168 as the starting strain, chassis cells were obtained by knocking out at least one of the yfkN, guaC, and purR genes;

[0047] (2) The genes encoding BsFADs and RibM were cloned into expression vectors and transformed into the chassis cells obtained in step (1);

[0048] Optionally, the gene copy number ratio of BsFADs to RibM can be adjusted to 1:1-2:1;

[0049] Optionally, P61W and / or R202Y mutations may be introduced via site-directed mutagenesis.

[0050] The fifth objective of this invention is to provide the application of the recombinant Bacillus subtilis in the production of FAD.

[0051] The sixth object of this invention is to provide a method for producing FAD, comprising the following steps:

[0052] 1) The recombinant Bacillus subtilis was inoculated into a fermentation medium containing exogenously added RF (100 mg / L-500 mg / L).

[0053] 2) Incubate at 30℃-37℃ and 150 rpm-250 rpm for 48-72 hours;

[0054] 3) Collect the fermentation broth and extract FAD.

[0055] Furthermore, the fermentation medium contains a carbon source, a nitrogen source, and inorganic salts. The carbon source is selected from one or more of glucose, sucrose, maltose, starch, and glycerol, with a carbon source concentration of 10 g / L-90 g / L; the nitrogen source is selected from one or more of yeast extract, tryptone, soybean meal, fish peptone, and beef extract, with a nitrogen source concentration of 10 g / L-50 g / L; the initial pH of the fermentation medium is 5.0-9.0.

[0056] Preferably, the fermentation medium contains: 40 g / L maltose, 30 g / L yeast extract, initial pH 8.0, and 400 mg / L RF; the culture conditions are 220 rpm and 37°C; the concentration of the inducer IPTG is 0.8 mM, and the induction time is 6 hours after inoculation. Under these preferred conditions, after 72 hours of fermentation, the FAD yield reaches 563 mg / L, and the FAD yield per unit biomass reaches 236 mg / g DCW.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0058] The culture media involved in the following examples are as follows:

[0059] LB liquid medium (1 L): 10 g peptone, 10 g sodium chloride, 5 g yeast extract, pH 7.0-7.2, sterilized at 121℃ for 20 min.

[0060] LB solid medium: Add 16 g / L agar powder to LB liquid medium.

[0061] Fermentation basal medium (1 L): glucose 40 g, yeast extract 20 g, (NH4)2SO4 6 g, K2HPO4 1 g, KH2PO4 1 g, MgSO4·7H2O 2 g, CaCl2 0.06 g, RF added according to the experimental design (100-500 mg), sterilized at 121℃ for 20 min. Initial pH 5.0-9.0.

[0062] Optimized post-fermentation medium (1 L): maltose 40 g, yeast extract 30 g, (NH4)2SO4 6 g, K2HPO4 1 g, KH2PO4 1 g, MgSO4·7H2O 2 g, CaCl2 0.06 g, RF 400 mg, initial pH 8.0.

[0063] GM I culture medium (100 mL): 10× Tbase 10 mL, 100 g / L glucose 5 mL, 50 g / L yeast extract 2 mL, 10 g / L acid hydrolyzed casein 2 mL, 2.5 g / L tryptophan 2 mL, and sterile water to bring the total to 100 mL.

[0064] GM II culture medium (100 mL): 10× Tbase 10 mL, 100 g / L glucose 5 mL, 50 g / L yeast extract 0.04 mL, 10 g / L acid-hydrolyzed casein 0.5 mL, 0.5 mol / L MgCl2 0.5 mL, 0.05 mol / L CaCl2 1 mL, sterile water to 100 mL.

[0065] 10× Tbase (1 L): (NH4)2SO4 20.0 g, K2HPO4 140.0 g, KH2PO4 60.0 g, sodium citrate (Na3C6H5O7·2H2O) 10.0 g, MgSO4·7H2O 2.0 g.

[0066] The strains and plasmids involved in the following examples:

[0067] Expression plasmid pHT01: carries the IPTG-inducible promoter Pgrac, chloramphenicol resistance (for Bacillus subtilis), and ampicillin resistance (for Escherichia coli), purchased from the Miaoling plasmid platform.

[0068] The plasmid pKS2 was donated to South China University of Technology. The construction method can be found in Chinese patent CN102586167A.

[0069] The primer sequences involved in the following examples are shown in Table 1:

[0070] Table 1. Main primers used in this study

[0071]

[0072] The following examples illustrate the methods for preparing and transforming competent Bacillus subtilis cells:

[0073] (1) Pick a single colony of Bacillus subtilis from a fresh LB plate and inoculate it into 5 mL of GMI culture medium. Incubate overnight (12-14 h) at 30°C and 100 rpm with shaking.

[0074] (2) Take 250 μL of overnight culture and transfer it to 5 mL of GM II culture medium. Incubate at 37℃ and 200 rpm for 3.5 h.

[0075] (3) Take 2 mL of GM II culture medium and transfer it to 20 mL of fresh GM II culture medium. Incubate at 37℃ and 100 rpm for 1.5 h.

[0076] (4) Collect the bacterial cells by centrifuging at 4℃ and 5000 rpm for 7 min. Gently resuspend the bacterial cells in 2 mL of the original culture supernatant to obtain competent cells.

[0077] (5) Add 1-2 μg of the plasmid to be transformed to 200 μL of competent cells, mix gently, and culture at 37℃ and 200 rpm for 1 h.

[0078] (6) Spread the entire transformation solution onto LB plates containing chloramphenicol (10 μg / mL), incubate upside down at 37°C for 12-18 h, and pick transformants for colony PCR verification.

[0079] The HPLC method for detecting flavonoids involved in the following examples:

[0080] After appropriate dilution, the fermentation broth sample or enzyme activity reaction solution was centrifuged at 12,000 rpm for 2 min, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. A Shimadzu LC-20A high-performance liquid chromatography system was used with an Agilent ZORBAX Eclipse Plus C18 column (4.6 × 250 mm, 5 μm) at 30℃. Mobile phase A was 10 mM sodium dihydrogen phosphate solution (pH 6.0), and mobile phase B was methanol; isocratic elution was performed at A:B = 70:30 (v / v); the flow rate was 1 mL / min; the detection wavelength was 445 nm; and the injection volume was 10 μL. Qualitative analysis was performed based on the retention time of the standards, and quantification was performed based on the peak area.

[0081] Example 1: Metabolic Engineering Modification of Chassis Cells

[0082] Starting with Bacillus subtilis 168 (trpC2) as the starting strain, the yfkN (Gene ID 936131), guaC (Gene ID 937189), and purR (Gene ID 937000) genes in the purine pathway were sequentially knocked out using homologous recombination technology to obtain the derived strain W0. Specific steps included: constructing knockout plasmids pKS2 for yfkN, guaC, and purR respectively, transforming them into Bacillus subtilis 168 competent cells, obtaining single-knockout strains through antibiotic screening and PCR verification, and then sequentially stacking the knockouts to finally obtain the three-gene knockout strain W0.

[0083] Wild-type Bacillus subtilis 168 and its derivative strain W0 were inoculated into fermentation basal medium (without exogenous RF) and cultured at 37°C with shaking for 48 h. FAD production was detected by HPLC. The results are as follows: Figure 1 As shown, strain W0 produced a background FAD yield of 2.5 mg / L, which is 5 times higher than that of wild-type 168 (0.5 mg / L), along with an accumulation of approximately 1.3 mg / L RF. Therefore, W0 was selected as the starting chassis strain for subsequent engineering.

[0084] Example 2 Screening of FAD synthase

[0085] FAD synthase genes (Gene IDs 949129, 938110, 3919980, 1129915771, and 77218353, respectively) from *Escherichia coli* (EcFADs), *Bacillus subtilis* (BsFADs), *Staphylococcus aureus* (SaFADs), *Corynebacterium ammoniagenes* (CaFADs), and *Lactobacillus plantarum* (LpFADs) were amplified, cloned into the pHT01 vector, and transformed into strain WO. Fermentation was carried out for 48 h with 100 mg / L exogenous RF. The results are as follows: Figure 2 As shown in the figure, the strain WO-BsFADs, which overexpresses BsFADs (amino acid sequence shown in SEQ ID NO. 1), exhibited the best performance, with a FAD yield of 37.6 mg / L. Therefore, BsFADs were selected as the core catalytic element.

[0086] Example 3: Screening and Copy Number Optimization of Riboflavin Transporter Proteins

[0087] Genes of *Bacillus subtilis* fmnP (Gene ID 938970), *Streptococcus pneumoniae* ribU (Gene ID 1985744587), and *Streptococcus davorense* ribM (Gene ID 14962409) were amplified and tandemly constructed with BsFADs into the pHT01 vector. This vector was then transformed into strain W0 to obtain strains W1 (FmnP), W2 (RibU), and W3 (RibM). Fermentation was carried out in a medium containing 100 mg / L RF for 48 h, and the results are as follows: Figure 3 As shown in the figure, strain W3, which expresses RibM (amino acid sequence shown in SEQ ID NO.2), showed the best results, with a FAD yield of 59 mg / L.

[0088] To further optimize the copy number ratio of BsFADs to ribM, recombinant strains with ratios of 1:1 (W3), 1:2 (W4), 2:1 (W5), and 2:2 (W6) were constructed. The results are as follows... Figure 3 As shown, the highest FAD yield (W4) was achieved at a ratio of 1:2, reaching 73 mg / L. Therefore, the optimal ratio was determined to be BsFADs:RibM = 1:2.

[0089] Example 4: BsFAD Mutation Screening Based on ICPE Strategy

[0090] Candidate mutations were introduced into the pHT01-BsFADs-RibM×2 plasmid (W4 strain plasmid background) via site-directed mutagenesis, transformed into W0 strain, and FAD yield was determined by shake-flask fermentation. Results are as follows: Figure 4As shown, the five single-point mutants (P61W, R202Y, R202H, T205E, and R254D) showed significant improvements, with R202Y showing the best effect, producing 100.7 mg / L of FAD (a 37.9% increase compared to the wild type of 73 mg / L), and P61W producing 94.6 mg / L (a 29.6% increase).

[0091] The five beneficial single-point mutations were combined in pairs to construct nine double-point mutants. Fermentation screening results showed that the double-point mutant P61W+R202Y (amino acid sequence shown in SEQ ID NO.3) performed the best, with a FAD yield of 126.4 mg / L, which was 73.2% higher than that of the wild type.

[0092] Example 5: Fermentation Process Optimization

[0093] To carry BsFADs P61W+R202Y The W4 strain (W4mut) of RibM (1:2) was used as the research object to systematically optimize fermentation parameters.

[0094] RF concentration optimization: Initial RF concentrations were set at 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L. Results ( Figure 5 The results showed that FAD production increased with increasing RF concentration, reaching 245 mg / L at 400 mg / L; however, it only increased slightly to 254 mg / L at 500 mg / L. Therefore, the optimal RF concentration was determined to be 400 mg / L.

[0095] Initial pH and dissolved oxygen optimization: Initial pH was set to 5.0-9.0, and the results were ( Figure 6 The results showed that the highest FAD yield (385 mg / L) was achieved at pH 8.0. With the shaker speed set between 100-250 rpm, the FAD yield reached 417 mg / L at 220 rpm. The optimal pH was determined to be 8.0 and the optimal shaker speed to be 220 rpm.

[0096] IPTG induction conditions optimization: IPTG concentrations were set from 0.1 to 1.6 mM, and the results were as follows ( Figure 7 The results showed that FAD production plateaued at 0.8 mM (417 mg / L). Induction timing was set (0-8 h post-inoculation), and the highest FAD production (432 mg / L) was induced at 6 h post-inoculation (OD600≈4.0). Therefore, IPTG 0.8 mM and 6 h post-inoculation induction were determined.

[0097] Carbon source optimization: comparison of glucose, sucrose, maltose, starch, and glycerol (40 g / L), results ( Figure 8The results showed that the maltose group had the highest FAD yield (526 mg / L) and the highest cell count. Optimizing the maltose concentration (10-50 g / L), a 40 g / L concentration resulted in a FAD yield of 560 mg / L after 72 hours of fermentation. The optimal maltose concentration was determined to be 40 g / L.

[0098] Nitrogen source optimization: Comparison of soybean meal, tryptone, fish peptone, yeast extract, and beef extract (20 g / L), results ( Figure 9 The results showed that the yeast extract group had the highest FAD yield (554 mg / L). Optimizing the yeast extract concentration (10-50 g / L), a FAD yield of 563 mg / L was achieved at 30 g / L after 72 h of fermentation. The optimal yeast extract concentration of 30 g / L was determined.

[0099] Under optimal conditions (maltose 40 g / L, yeast extract 30 g / L, initial pH 8.0, RF 400 mg / L, 220 rpm, 37℃, IPTG 0.8 mM induced 6 h after inoculation), fermentation for 72 h yielded a FAD production of 563 mg / L. Figure 10 ).

[0100] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant bacterium that synthesizes flavin adenine dinucleotide, characterized in that, The recombinant strain was Bacillus subtilis 168 (trpC2) as the starting strain; the yfkN, guaC and purR genes in the genome of the starting strain were knocked out; and the gene BsFADs, which expresses the FAD synthase mutant and the riboflavin transporter gene ribM, were integrated. The FAD synthase mutant uses the FAD synthase with the amino acid sequence shown in SEQ ID NO.1 as the parent and undergoes the following mutations: proline at position 61 is mutated to tryptophan and / or arginine at position 202 is replaced with tyrosine.

2. The recombinant bacteria according to claim 1, characterized in that, The gene IDs of the yfkN gene, guaC gene, and purR gene are 936131, 937189, and 937000, respectively.

3. The recombinant bacteria according to claim 1, characterized in that, The copy number ratio of the FAD synthase mutant gene BsFADs to the riboflavin transporter gene ribM is 1:1 to 1:

2.

4. The recombinant bacteria according to claim 1, characterized in that, The riboflavin transporter is derived from *Streptomyces davorense*; the gene ID of the riboflavin transporter gene is 14962409, and the amino acid sequence is shown in SEQ ID NO.

2.

5. A method for constructing a recombinant bacterium that synthesizes flavin adenine dinucleotide as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Using Bacillus subtilis 168 (trpC2) as the starting strain, chassis cells were obtained by knocking out the yfkN gene, guaC gene and purR gene; (2) The gene BsFADs of the FAD synthase mutant and the gene ribM of the riboflavin transporter were cloned into the expression vector and transformed into the chassis cells obtained in step (1) to obtain the recombinant bacteria that synthesize flavin adenine dinucleotide.

6. The recombinant bacteria for synthesizing flavin adenine dinucleotide as described in any one of claims 1-4, used in the production of flavin adenine dinucleotide.

7. A method for synthesizing flavin adenine dinucleotide, characterized in that, Includes the following steps: Riboflavin is used as a raw material and is produced by fermentation using recombinant bacteria that synthesize flavin adenine dinucleotide as described in any one of claims 1-4.

8. The method according to claim 7, characterized in that, The concentration of riboflavin is 100 mg / L-500 mg / L.

9. The method according to claim 7, characterized in that, The initial pH value of the fermentation is 5.0-9.0; And / or, the fermentation speed is 100 rpm-250 rpm; And / or, the fermentation temperature is 30℃-37℃; And / or, the fermentation time is 48 h-72 h; And / or, the fermentation may further include the addition of IPTG for induction.

10. The method according to claim 7, characterized in that, The fermentation medium for fermentation includes a carbon source and a nitrogen source; the carbon source includes one or more of glucose, sucrose, maltose, starch, and glycerol; the nitrogen source includes one or more of yeast extract, tryptone, soybean meal, fish peptone, and beef extract.

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