Difunctional flavin adenine dinucleotide synthetase mutant and application thereof

Abstract

The invention discloses a double-function flavin adenine dinucleotide synthetase mutant and application, the double-function flavin adenine dinucleotide synthetase is called RFK / FADS for short, and the amino acid sequence of the double-function flavin adenine dinucleotide synthetase is shown as SEQ ID NO.1; and the RFK / FADS mutant is RFK / FADSF55A, RFK / FADSG43Q, RFK / FADSG43Q-F55H, RFK / FADSG43Q-F55N or RFK / FADSG43Q-F55N-F129Y, and the RFK / FADSG43Q-F55N and the FADSG43Q-F55N-F129Y can be used for preparing the According to the genetically engineered bacterium disclosed by the invention, flavine adenine dinucleotide is produced through a simple fermentation process by taking glucose as a substrate under a shake flask aerobic condition, and the yields of the strains ZF02, ZF03, ZF04, ZF05 and ZF06 are respectively increased by 120%, 130%, 166%, 246% and 286% in sequence compared with those of ZF01.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a bifunctional flavin adenine dinucleotide synthetase mutant and application thereof. 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 cell metabolism such as electron transfer, photosynthesis, and fatty acid oxidation. It has been widely used in the fields of medicine and enzyme catalysis. In the pharmaceutical industry, FAD can be injected intramuscularly and intravenously to treat RF deficiency and some hereditary diseases due to its high water solubility and utilization rate. [1] In enzyme catalysis reactions, flavin mononucleotide FMN (FMN) and FAD are both indispensable redox cofactors in most flavin proteins or flavin enzymes in cells. In addition, FAD is also widely used in the development and application of biosensors and microbial fuel cells. [2,3] .

[0003] At present, FAD is mainly commercially produced by the whole-cell catalytic method. The engineered strain overexpressing FAD synthetase can further catalyze the in vitro added substrates FMN and ATP into FAD and PPi. [4-7] However, when whole-cell catalysis produces FAD, the production cost is high due to the high price of the substrate ATP. 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 are able to 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 vivo, 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, respectively, 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 stayed on the enzymatic properties and catalytic mechanism, and has not been applied to the production of FAD. 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 referred to 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 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 by a simple fermentation process with glucose as a substrate under aerobic conditions in a shake flask. The FAD production of strains ZF02, ZF03, ZF04, ZF05 and ZF06 are 109, 115, 133, 158 and 193 mg / l, respectively, which are 120%, 130%, 166%, 246% and 286% higher than the FAD production of 50 mg / l of ZF01, respectively; the FAD production of ZF08 is 468 mg / l, which is 41.39% higher than the 331 mg / l of strain ZF07; the FAD production 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 in conjunction with specific embodiments. The following embodiments are intended to enable those skilled in the art to better understand the present invention, but do not impose any limitation on the present invention.

[0019] The riboflavin-producing strain LS21 used in the present invention (its original strain Escherichia coli MG1655 is commercially available) is 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 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.

[0022] The CPEC plasmid construction method comes 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 used 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] Other biochemical reagents used were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0027] The website for the simulation predictions used 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 and molecular docking was performed to obtain a simulated crystal structure.

[0031] (2) For the simulated crystal structure obtained in step (1), Alanine scanning and multiple sequence alignment were performed on 46 amino acid residues in the 1979 gene to construct a gene encoding the mutant; the obtained gene was overexpressed in plasmid p20C to obtain a series of plasmids carrying mutant genes;

[0032] (3) introducing a series of plasmids in step (2) into strain LS21 respectively, obtaining strains and conducting fermentation tests in shake flasks; determining that the key amino acid residues of the enzyme are G43, F55, and F129;

[0033] (4) constructing a saturated 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 the strain 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] A 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 encoding gene, comprising the following steps

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

[0038] (2) Using plasmid pF as a template and FADS-G43Q-F (SEQ ID NO. 12) and FADS-G43Q-R (SEQ ID NO. 13) as primers, 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 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 plasmid pF-G43Q-F55H was constructed by seamless cloning;

[0041] (5) Using plasmid pF-G43Q as a template and 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 plasmid pF-G43Q-F55N was constructed by seamless cloning method;

[0042] (6) Using plasmid pF-G43Q-F55N as a template and 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, and the plasmid pF-G43Q-F55N-F129Y was constructed by seamless cloning method, see 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 respectively electroporated into the competent strain LS21 to construct strains ZF01, ZF02, ZF03, ZF04, ZF05, and ZF06.

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

[0046] (3) The plasmid pF and mutant plasmid pF-G43Q-F55N-F129Y were respectively electroporated into the competent strain PH20T with high riboflavin production 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; obtain a fermentation medium;

[0049] The component I is: take 10g (NH 4 ) 2 SO 4 , 2g MgSO 4 , dilute to 200 mL with distilled water, sterilize at 121°C for 20 min;

[0050] The component II is: take 38.3g Na 2 HPO 4 , 15g KH 2 PO 4 , dilute to 200 mL with distilled water, sterilize at 121°C for 20 min;

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

[0052] The component IV is: take 1g ZnSO 4 7H 2 O, 0.3 g MnCl 2 ·4H 2 O, 3g H 3 BO 3 , 2g CoCl 2 6H 2 O, 0.1 g CuSO 4 ·5H 2 O, 0.2 g NiCl 2 6H 2 O, 0.3 g NaMoO 4 ·2H 2 O, dilute to 1000 mL with distilled water;

[0053] The mixed solution of components III and IV is as follows: 100 ml of component III and 1 ml of component IV are taken, the volume is adjusted to 200 ml with distilled water, the pH value is adjusted to 4.5-5.5 with 5M NaOH aqueous solution, and sterilized at 121° C. for 20 min.

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

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

[0056] (2) Cultivation of seed solution: inoculate the single colony obtained in step (1) into LB liquid culture 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 heated to 40 °C at an initial OD 600 The inoculum size was 0.025, inoculated into the fermentation medium, and the volume of the shake flask was 50mL / 500mL. Spectinomycin was added to a final concentration of 100mg / L, and cultured at 37℃ and 220rpm. After 24h of fermentation, the FAD production of strains ZF02, ZF03, ZF04, ZF05, and ZF06 was 109, 115, 133, 158, and 193mg / l, respectively, which was 120%, 130%, 166%, 246%, and 286% higher than the FAD production of ZF01 of 50mg / l, respectively;

[0058] The seed solution obtained in step (2) was added according to the initial OD 600 The inoculum size was 0.025, inoculated into the fermentation medium, and the volume of the shake flask was 50mL / 500mL. Chloramphenicol was added to a final concentration of 10mg / L, and spectinomycin was added to a final concentration of 100mg / L. After 48h of fermentation, the FAD production of strain ZF08 was 468mg / l, which was 41.39% higher than that of strain ZF07 (331mg / l); the FAD production of ZF10 was 727mg / l, which was 57% higher than that of ZF09 (463mg / l).

[0059] References

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Claims

1. A bifunctional flavin adenine dinucleotide synthetase mutant, wherein the bifunctional flavin adenine dinucleotide synthetase is referred to 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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