Bifunctional flavin adenine dinucleotide synthetase mutants and their applications

Abstract

The present invention discloses a bifunctional flavin adenine dinucleotide synthetase mutant and its application. The bifunctional flavin adenine dinucleotide synthetase is referred to as RFK / FADS, and its amino acid sequence is shown in SEQ ID NO.1. The RFK / FADS mutant is RFK / FADS F55A , RFK / FADS G43Q , RFK / FADS G43Q‑F55H , RFK / FADS G43Q‑F55N or RFK / FADS G43Q‑F55N‑F129Y The genetically engineered bacteria of the present invention produce flavin adenine dinucleotide using glucose as a substrate through a simple fermentation process under aerobic conditions in shake flasks. The yields of strains ZF02, ZF03, ZF04, ZF05, and ZF06 are respectively 120%, 130%, 166%, 246%, and 286% higher than those of ZF01.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a bifunctional flavin adenine dinucleotide synthetase mutant and its application. Background Art

[0002] Flavin adenine dinucleotide (FAD) is the metabolically active form of riboflavin (RF). As an important intracellular cofactor, FAD plays an important role in cellular metabolism, including electron transfer, photosynthesis, and fatty acid oxidation. It has been widely used in medicine and enzyme catalysis. In the pharmaceutical industry, FAD can be used to treat RF deficiency and some genetic diseases through intramuscular and intravenous injections due to its high water solubility and utilization rate. [1] In enzyme-catalyzed reactions, flavin mononucleotide FMN (FMN) and FAD are both essential redox cofactors in most flavoproteins or flavoenzymes in cells. In addition, FAD is also widely used in the development and application of biosensors and microbial fuel cells. [2,3] .

[0003] Currently, FAD is mainly commercially produced by the whole-cell catalytic method, which uses an engineered strain overexpressing FAD synthetase to further catalyze the in vitro added substrates FMN and ATP into FAD and PPi. [4-7] However, when whole-cell catalytic production of FAD is used, the high price of the substrate ATP leads to high production costs. In contrast, microbial fermentation methods that use cheap substrates and do not require high-density culture have greater potential. [8-12] . Researchers have constructed microbial "cell factories" through metabolic engineering strategies. These modified microorganisms can use inexpensive raw materials to produce the required natural products within microbial cells. This advancement not only reduces the production cost of FAD, but also improves the environmental sustainability of its production process. In addition, there are currently few studies on mutations of FAD synthetase. In organisms, riboflavin and ATP generate FMN and ADP under the catalysis of riboflavin kinase (RFK), and FMN and ATP (or AMP) further generate FAD and pyrophosphate under the catalysis of FAD synthetase (FADS). In eukaryotes, these two enzymes exist as monofunctional enzymes, while in prokaryotes, riboflavin kinase and FAD synthetase usually exist as bifunctional enzymes encoded by the same peptide chain. However, in-depth research is limited to a few species such as Corynebacterium ammoniagenes and Candida glabrata. [13-15], and related research has only focused on the enzymatic properties and catalytic mechanisms, without applying it to FAD production. Therefore, it is urgent to construct a bifunctional FAD synthetase mutant from Escherichia coli with improved catalytic performance for the synthesis of flavin adenine dinucleotide. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a bifunctional flavin adenine dinucleotide synthetase mutant.

[0005] The second object of the present invention is to provide a gene encoding the above-mentioned bifunctional flavin adenine dinucleotide synthetase mutant.

[0006] The third object of the present invention is to provide a recombinant expression plasmid containing the above encoding gene.

[0007] The fourth object of the present invention is to provide a genetically engineered bacterium containing the above-mentioned recombinant expression plasmid.

[0008] The fifth object of the present invention is to provide the use of the above genetically engineered bacteria in fermenting and preparing flavin adenine dinucleotide.

[0009] The technical solution of the present invention is summarized as follows:

[0010] A bifunctional flavin adenine dinucleotide synthetase mutant, the bifunctional flavin adenine dinucleotide synthetase is abbreviated as RFK / FADS, the amino acid sequence of RFK / FADS is shown in SEQ ID NO.1; the RFK / FADS mutant is RFK / FADS F55A , RFK / FADS G43Q , RFK / FADS G43Q-F55H , RFK / FADS G43Q-F55N or RFK / FADS G43Q-F55N-F129Y .

[0011] A gene encoding the above-mentioned bifunctional flavin adenine dinucleotide synthetase mutant.

[0012] A recombinant expression plasmid containing the above encoding gene.

[0013] A genetically engineered bacterium containing the above-mentioned recombinant expression plasmid.

[0014] The application of the above genetically engineered bacteria in fermentation to prepare flavin adenine dinucleotide.

[0015] Advantages of the present invention:

[0016] The genetically engineered bacteria of the present invention produce flavin adenine dinucleotide using glucose as a substrate through a simple fermentation process under aerobic conditions in shake flasks. The FAD yields of strains ZF02, ZF03, ZF04, ZF05, and ZF06 are 109, 115, 133, 158, and 193 mg / l, respectively, which are increases of 120%, 130%, 166%, 246%, and 286% compared to the FAD yield of 50 mg / l of ZF01, respectively; the FAD yield of ZF08 is 468 mg / l, which is 41.39% higher than the 331 mg / l of strain ZF07; and the FAD yield of ZF10 is 727 mg / l, which is 57% higher than the 463 mg / l of ZF09. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the plasmid map of the mutant plasmid pF-G43Q-F55N-F129Y. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to specific examples. The following examples are provided to enable those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0019] 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 of the following article: Liu Shuang. Metabolic engineering of Escherichia coli LS02T to high-yield riboflavin and flavin coenzyme [D]. Tianjin University, 2022. DOI: 10.27356 / d.cnki.gtjdu.2022.000045.

[0020] The high-riboflavin-producing strains PH08T and PH20T used in the present invention were constructed according to the steps of the following article: Hu Wenya. Metabolic engineering of Escherichia coli to produce riboflavin and flavin mononucleotides [D]. Tianjin University, 2021. DOI: 10.27356 / d.cnki.gtjdu.2021.003367.

[0021] 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.

[0022] The CPEC plasmid construction method was 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).

[0023] The flavin adenine dinucleotide standard was purchased from TCI (https: / / www.tcichemicals.com).

[0024] The primers used were synthesized by Genewiz (https: / / www.genewiz.com.cn / ).

[0025] The seamless cloning reagents used were purchased from Abiotech (https: / / abclonal.com.cn / ).

[0026] All other biochemical reagents used were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0027] The website used for simulation prediction is https: / / github.com / deepmind / alphafold.

[0028] The molecular docking method used was implemented from AutoDock software (https: / / autodock.scripps.edu / ).

[0029] Example 1: Screening of key amino acids of Escherichia coli bifunctional flavin adenine dinucleotide synthetase, comprising the following steps

[0030] (1) The bifunctional flavin adenine dinucleotide synthetase RFK / FADS (SEQ ID NO. 1) from Escherichia coli was simulated and predicted by molecular docking to obtain a simulated crystal structure.

[0031] (2) For the simulated crystal structure obtained in step (1), the substrate is Alanine scanning and multiple sequence alignment were performed on the 46 amino acid residues within the gene to construct a gene encoding the mutant; the obtained gene was overexpressed in the plasmid p20C to obtain a series of plasmids carrying the mutant genes;

[0032] (3) The plasmids in step (2) were introduced into strain LS21 to obtain strains and fermented in shake flasks to test the enzyme; the key amino acid residues of the enzyme were determined to be G43, F55, and F129;

[0033] (4) Constructing a saturation mutation library of the three key amino acid residues in step (3) and the multi-site combination effect; overexpressing the obtained genes in plasmid p20C to obtain a series of plasmids carrying mutant genes and introducing them into strain LS21 respectively, obtaining strains and conducting fermentation tests in shake flasks, and selecting enzyme mutants after mutation combination based on the fermentation data.

[0034] RFK / FADS mutant F55A , RFK / FADS G43Q , RFK / FADS G43Q-F55H , RFK / FADS G43Q-F55N or RFK / FADS G43Q-F55N-F129Y ;

[0035] The gene encoding the above-mentioned bifunctional flavin adenine dinucleotide synthetase mutant, the enzyme mutant RFK / FADS F55A , RFK / FADS G43Q , RFK / FADS G43Q-F55H , RFK / FADS G43Q-F55N and RFK / FADS G43Q-F55N-F129Y The nucleotide sequences of the genes are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0036] Example 2: Construction of a recombinant expression plasmid containing the above-mentioned encoding gene, comprising the following steps:

[0037] (1) Using the Escherichia coli MG1655 genome as a template and FADS-F (SEQ ID NO.8) and FADS-R (SEQ ID NO.9) as primers, PCR amplification was performed to obtain a 942 bp RFK / FADS gene fragment; using plasmid p20C as a template and p20C-F (SEQ ID NO.10) and p20C-R (SEQ ID NO.11) as primers, PCR amplification was performed to obtain a 2808 bp p20C vector fragment (SEQ ID NO.7); the RFK / FADS gene fragment and the p20C vector fragment (SEQ ID NO.7) were combined to construct plasmid pF using the CPEC plasmid construction method;

[0038] (2) Using plasmid pF as a template and primers FADS-G43Q-F (SEQ ID NO. 12) and FADS-G43Q-R (SEQ ID NO. 13), PCR amplification was performed to obtain the RFK / FADS-G43Q linear fragment, and plasmid pF-G43Q was constructed by seamless cloning.

[0039] (3) Using plasmid pF as a template and FADS-F55A-F (SEQ ID NO. 14) and FADS-F55A-R (SEQ ID NO. 15) as primers, PCR amplification was performed to obtain the RFK / FADS-F55A linear fragment, and plasmid pF-F55A was constructed by seamless cloning;

[0040] (4) Using plasmid pF-G43Q as a template and primers FADS-G43QF55H-F (SEQ ID NO. 16) and FADS-G43QF55H-R (SEQ ID NO. 17) as primers, PCR amplification was performed to obtain the RFK / FADS-G43Q-F55H linear fragment, and the plasmid pF-G43Q-F55H was constructed by seamless cloning;

[0041] (5) Using plasmid pF-G43Q as a template and primers FADS-G43QF55N-F (SEQ ID NO. 18) and FADS-G43QF55N-R (SEQ ID NO. 19) as primers, PCR amplification was performed to obtain the RFK / FADS-G43Q-F55N linear fragment, and the plasmid pF-G43Q-F55N was constructed by seamless cloning.

[0042] (6) Using plasmid pF-G43Q-F55N as a template and primers FADS-G43QF55NF129Y-F (SEQ ID NO. 20) and FADS-G43QF55NF129Y-R (SEQ ID NO. 21) as primers, PCR amplification was performed to obtain the RFK / FADS-G43Q-F55N-F129Y linear fragment. The plasmid pF-G43Q-F55N-F129Y was constructed by seamless cloning. Figure 1 .

[0043] Example 3: The genetically engineered bacteria containing the above-mentioned recombinant expression plasmid comprises the following steps:

[0044] (1) Plasmid pF and mutant plasmids pF-F55A, pF-G43Q, pF-G43Q-F55H, pF-G43Q-F55N, and pF-G43Q-F55N-F129Y were electroporated into the competent strain LS21 to construct strains ZF01, ZF02, ZF03, ZF04, ZF05, and ZF06.

[0045] (2) Plasmid pF and mutant plasmid pF-G43Q-F55N-F129Y were electroporated into the competent strain PH08T with high riboflavin production, respectively, to construct strains ZF07 and ZF08.

[0046] (3) Plasmid pF and mutant plasmid pF-G43Q-F55N-F129Y were electroporated into the competent strain PH20T with high riboflavin production, respectively, to construct strains ZF09 and ZF10.

[0047] Example 4: Preparation of fermentation medium, comprising the following steps

[0048] Take 1 mL of component I, 1 mL of component II, and 1 mL of a mixture of components 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 to obtain a fermentation medium;

[0049] The component I is as follows: 10 g (NH4)2SO4 and 2 g MgSO4 are diluted to 200 mL with distilled water and sterilized at 121°C for 20 min;

[0050] Component II is as follows: 38.3 g Na2HPO4 and 15 g KH2PO4 are diluted to 200 mL with distilled water and sterilized at 121°C for 20 min;

[0051] The component III is as follows: 5g of ammonium ferric citrate, 2g of CaCl2·2H2O, 41.7mL of 12mol / L HCl aqueous solution, and dilute to 1000mL with distilled water;

[0052] The component IV is as follows: 1g ZnSO4·7H2O, 0.3g MnCl2·4H2O, 3g H3BO3, 2g CoCl2·6H2O, 0.1g CuSO4·5H2O, 0.2g NiCl2·6H2O, 0.3g NaMoO4·2H2O, dilute to 1000mL with distilled water;

[0053] The mixture of components III and IV is prepared by taking 100 ml of component III and 1 ml of component IV, making the volume up to 200 ml with distilled water, adjusting the pH to 4.5-5.5 with 5M NaOH aqueous solution, and sterilizing at 121° C. for 20 min.

[0054] Example 5: Shake flask fermentation of the strain, comprising the following steps

[0055] (1) Activation of the strain: Streak the strain obtained in Example 3 on LB solid medium and culture at 37°C for 12-20 hours to rejuvenate the strain;

[0056] (2) Cultivation of seed liquid: inoculate the single colony obtained in step (1) into LB liquid medium and culture at 37°C and 220 rpm for 12-20 h;

[0057] (3) Shake flask fermentation: The seed solution obtained in step (2) was fermented at the initial OD 600 An inoculum size of 0.025 was inoculated into the fermentation medium, and the shake flask volume was 50 mL / 500 mL. Spectinomycin was added to a final concentration of 100 mg / L and cultured at 37°C and 220 rpm. After 24 hours of fermentation, the FAD production of strains ZF02, ZF03, ZF04, ZF05, and ZF06 was 109, 115, 133, 158, and 193 mg / L, respectively, representing increases of 120%, 130%, 166%, 246%, and 286% compared to the FAD production of 50 mg / L of ZF01.

[0058] The seed solution obtained in step (2) was 600 An inoculum size of 0.025 was used to inoculate the fermentation medium, with the shake flask volume of 50 mL / 500 mL. Chloramphenicol was added to a final concentration of 10 mg / L, and spectinomycin was added to a final concentration of 100 mg / L. After 48 hours of fermentation at 220 rpm, strain ZF08 produced 468 mg / L of FAD, a 41.39% increase compared to 331 mg / L of strain ZF07. ZF10 produced 727 mg / L of FAD, a 57% increase compared to 463 mg / L of strain ZF09.

[0059] References

[0060] [1]MEDINA M,FERREIRA P,MARTíNEZ-JúLVEZ M.Flavoproteins andflavoenzymes with biomedical and therapeutic impact[J].Current PharmaceuticalDesign,2013,19(14):2497-8.

[0061] [2]JORNS M S,WANG B,JORDAN S P.DNA Repair Catalyzed by Escherichiacoli DNA Photolyase Containing Only Reduced Flavin:Elimination of the Enzyme's Second Chromophore by Reduction with Sodium Borohydride[J].Biochemistry,1987,26(21):6810-6.

[0062] [3]LIPTON S A,BOSSY-WETZEL E.Dueling activities of AIF in cell deathversus survival:DNA binding and redox activity[J].Cell,2002,111(2):147-50.

[0063] [4]HAGIHARA T,FUJIO T,AISAKA K.Cloning of FAD synthetase gene fromCorynebacterium ammoniagenes and its application to FAD and FMN production[J].Applied Microbiology and Biotechnology,1995,42(5):724-9.

[0064] [5]KITATSUJI K,ISHINO S,TESHIBA S,ARIMOTO M.Method of producingflavine nucleotides[J].1996.

[0065] [6]WATANABE T,UCHIDA T,KATO J,CHIBATAI.Production of flavine adeninedinucleotide from riboflavine by a mutant of Sarcina lutea[J].Journal ofApplied Microbiology,1974,27(3):531-6.

[0066] [7]SAKAI T,WATANABE T,CHIBATAI.Selection of Microorganisms ProducingFlavin-Adenine Dinucleotide from FMN and Adenine(AMP)and Production ofFlavin-Adenine Dinucleotide by Sarcina lutea[J].Agricultural and BiologicalChemistry,1973,37(4):849-56.

[0067] [8]YATSYSHYN V Y,FEDOROVYCH D V,SIBIRNY AA.Metabolic and bioprocessengineering of the yeast Candida famata for FAD production[J].Journal ofIndustrial Microbiology and Biotechnology,2014,41(5):823-35.

[0068] [9]LIU S,DIAO N,WANG Z,et al.Modular Engineering of the FlavinPathway in Escherichia coli for Improved Flavin Mononucleotide and FlavinAdenine Dinucleotide Production[J].Journal of Agricultural and FoodChemistry,2019,67(23):6532-40.

[0069]

[10] PATEL M V,SAINATHAN C.Enhanced FAD Production in Eremotheciumashbyi with Statistically Optimized Oxidative Stress Modulators[J].International Journal of Microbiology and Biotechnology,2020,(1).

[0070]

[11] LIU S,HU W,WANG Z,CHEN T.Production of riboflavin and relatedcofactors by biotechnological processes[J].Microbial Cell Factories,2020,19(1).

[0071]

[12] PATEL M V,T.S C.Metabolic engineering of Ashbya gossypii forenhanced FAD production through promoter replacement of FMN1 gene[J].Enzymeand Microbial Technology,2020,133.

[0072]

[13] SERRANO A,FRAGO S,HERGUEDAS B,et al.Key Residues at theRiboflavin Kinase Catalytic Site of the Bifunctional Riboflavin Kinase / FMNAdenylyltransferase From Corynebacterium ammoniagenes[J].Cell Biochemistryand Biophysics,2013,65(1):57-68.

[0073]

[14] HUERTAC,GRISHIN N V,ZHANG H.The"super mutant"of yeast FMNadenylyltransferase enhances the enzyme turnover rate by attenuating productinhibition[J].Biochemistry,2013,52(21):3615-7.

[0074]

[15] SERRANO A,FRAGO S,VELáZQUEZ-CAMPOY A,MEDINAM.Role of key residuesat the flavin mononucleotide(FMN):Adenylyltransferase catalytic Site of thebifunctional Riboflavin kinase / flavin adenine dinucleotide(FAD)synthetasefrom Corynebacterium ammoniagenes[J].International Journal of MolecularSciences,2012,13(11):14492-517。

Claims

1. A bifunctional flavin adenine dinucleotide synthetase mutant, wherein the bifunctional flavin adenine dinucleotide synthetase is abbreviated as RFK / FADS, and the amino acid sequence of RFK / FADS is shown in SEQ ID NO. 1; characterized in that: RFK / FADS mutant F55A , RFK / FADS G43Q , RFK / FADS G43Q-F55H , RFK / FADS G43Q-F55N or RFK / FADS G43Q -F55N-F129Y .

2. A gene encoding the bifunctional flavin adenine dinucleotide synthetase mutant according to claim 1.

3. A recombinant expression plasmid containing the coding gene according to claim 2.

4. A genetically engineered bacterium containing the recombinant expression plasmid according to claim 3.

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

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