3, 4-dihydroxy-2-butanone-4-phosphate synthase mutant and application thereof

Abstract

The invention discloses a 3, 4-dihydroxy-2-butanone-4-phosphate synthase mutant and an application of the 3, 4-dihydroxy-2-butanone-4-phosphate synthase mutant. The mutant is DHBPsG151N, DHBPsP46I, DHBPsT58E or DHBPsT58E-G151N, and the mutant can be used for preparing a recombinant plasmid. According to the invention, a key enzyme 3, 4-dihydroxy-2-butanone-4-phosphate synthase in a riboflavin synthesis route is subjected to semi-rational design transformation to obtain a 3, 4-dihydroxy-2-butanone-4-phosphate synthase mutant with improved catalytic performance, and a genetically engineered bacterium carrying a gene plasmid of the mutant has the advantages that the catalytic performance of the 3, 4-dihydroxy-2-butanone-4-phosphate synthase mutant is improved, and the catalytic performance of the 3, 4-dihydroxy-2-butanone-4-phosphate synthase mutant is improved; the riboflavin is produced through a simple fermentation process by taking glucose as a substrate under a shake flask aerobic condition, and the riboflavin yields of strains RFB02, RFB03, RFB04 and RFB05 containing mutant genes are 419.18 mg / L, 430.01 mg / L, 471.01 mg / L and 490.78 mg / L in sequence, which are respectively improved by 37.81%, 41.37%, 54.85% and 61.35% compared with those of strains RFB01 containing non-mutant genes.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to mutants of 3,4-dihydroxy-2-butanone-4-phosphate synthase and their applications. Background Technology

[0002] Riboflavin, also known as vitamin B2, is a yellow, heat-stable, naturally water-soluble B vitamin. The molecular formula of riboflavin is C2. 17 H 20 N4O6, with a molecular weight of 376.4 g / mol, is slightly soluble in water and readily soluble in alkaline solutions. Riboflavin is usually phosphorylated to flavin mononucleotide (FMN), and then further adenylated to flavin adenine dinucleotide (FAD). FMN and FAD are the active forms of riboflavin, which participate in the redox reactions of body tissues and electron transport in the respiratory chain as flavin coenzymes (cofactors), and are important biochemical reagents [1].

[0003] Riboflavin is an essential nutrient for maintaining normal metabolism in humans and animals. However, humans and animals cannot synthesize riboflavin themselves and must obtain sufficient riboflavin from food to meet their physiological needs. Riboflavin has important applications in nutrition, disease prevention and treatment [2, 3]. It is used as a food additive in infant food, sauces, processed cheese, fruit juices, vitamin-rich dairy products and some energy drinks. At the same time, riboflavin is also used in feed additives, pharmaceuticals, cosmetics and microbial fuel cells [4]. Riboflavin supplementation can not only enhance the function of macrophages, but also reduce inflammation caused by Staphylococcus aureus infection [2].

[0004] Currently, microbial fermentation is the main method for riboflavin production. Bacillus subtilis[5] and Ashus kosinae[6] are the most commonly used riboflavin production strains in industry, but the yield of riboflavin is generally low. Bacillus subtilis is also used for riboflavin production because the spores are difficult to inactivate, and its application in feed can easily cause the spread of genetically engineered bacteria. At the same time, most traditional production strains are modified by mutagenesis combined with rational metabolic engineering, resulting in complex genetic backgrounds. In addition, there is little research on the key enzymes in the riboflavin synthesis pathway, GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPs). Currently, there are reports that the bifunctional enzyme gene ribA (which encodes both DHBPs and GTP cyclohydrolase II) from Bacillus subtilis can be significantly improved by error-prone PCR-based directional modification[7]. In Escherichia coli, these two enzymes are encoded by two genes, which are monofunctional genes. A search revealed that there is currently no research on the rational design or directed evolution of Escherichia coli 3,4-dihydroxy-2-butanone-4-phosphate synthase and its application in riboflavin production. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a mutant of 3,4-dihydroxy-2-butanone-4-phosphate synthase.

[0006] A second objective of this invention is to provide a gene encoding the above-mentioned 3,4-dihydroxy-2-butanone-4-phosphate synthase mutant.

[0007] A third objective of this invention is to provide a recombinant expression plasmid containing the above-described encoding gene.

[0008] A fourth objective of this invention is to provide genetically engineered bacteria containing the above-described recombinant expression plasmid.

[0009] The fifth objective of this invention is to provide an application for the fermentation of riboflavin by the above-mentioned genetically engineered bacteria.

[0010] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: A 3,4-dihydroxy-2-butanone-4-phosphate synthase mutant, said mutant being DHBPs G151N DHBPs P46I DHBPs T58E or DHBPs T58E-G151N DHBPs G151N The amino acid sequence is shown in SEQ ID NO.2, DHBPs P46I The amino acid sequence is shown in SEQ ID NO.3, DHBPs T58E The amino acid sequence is shown in SEQ ID NO.4, DHBPsT58E -G151N The amino acid sequence is shown in SEQ ID NO.5.

[0011] The gene encoding the 3,4-dihydroxy-2-butanone-4-phosphate synthase mutant of claim 1 encodes DHBPs. G151N The nucleotide sequence of the gene, as shown in SEQ ID NO.6, encodes DHBPs. P46I The nucleotide sequence of the gene is shown in SEQ ID NO.7, encoding DHBPs. T58E The nucleotide sequence of the gene, as shown in SEQ ID NO.8, encodes DHBPs. T58E-G151N The nucleotide sequence of the gene is shown in SEQ ID NO.9.

[0012] A recombinant expression plasmid containing the encoded gene.

[0013] Genetically engineered bacteria containing the recombinant expression plasmid.

[0014] Application of the genetically engineered bacteria in the fermentation preparation of riboflavin.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes semi-rational design to modify 3,4-dihydroxy-2-butanone-4-phosphate synthase, a key enzyme in the riboflavin synthesis pathway, to obtain a mutant 3,4-dihydroxy-2-butanone-4-phosphate synthase with improved catalytic performance. Genetically engineered bacteria carrying the mutant gene plasmid produce riboflavin using glucose as a substrate through a simple fermentation process under aerobic conditions in a shake flask. The riboflavin yields of the mutant strains RFB02, RFB03, RFB04, and RFB05 were 419.18 mg / L, 430.01 mg / L, 471.01 mg / L, and 490.78 mg / L, respectively, representing increases of 37.81%, 41.37%, 54.85%, and 61.35% compared to the strain RFB01 without the mutant gene. Attached Figure Description

[0016] Figure 1 This is the plasmid map of the mutant plasmid pB-T58E-G151N. Detailed Implementation

[0017] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all the accompanying drawings and corresponding descriptions merely illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0018] 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 this invention pertains.

[0019] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0020] The original strain of Escherichia coli MG1655 used in this invention was obtained from CGSC (Coli Genetic Stock Center, http: / / cgsc.biology.yale.edu / ).

[0021] The original strain of Escherichia coli SN02 used in this invention was constructed according to the steps of the following patent: Chen Tao, Hou Junyuan. (2025). High-riboflavin-producing Escherichia coli strain and its construction method and application (CN Patent No.2026100044322).

[0022] The plasmids pTKRED and pTKS / CS were obtained from Addgene (https: / / www.addgene.org / ).

[0023] The methods for λ-Red gene recombination and shake-flask fermentation of the strain are derived from the following article: LIN ZQ, XU ZB, LI YF, et al. Metabolic engineering of Escherichia coli for the production of riboflavin [J]. Microbial Cell Factories, 2014, 13. Plasmids pZY48 and pLS01 are derived from the following article: LIU S, KANG P, CUI ZZ, et al. Increased riboflavin production by knockout of 6-phosphofructokinase I and blocking the Entner-Doudoroff pathway in Escherichia coli [J]. Biotechnology Letters, 2016, 38(8): 1307-14. The riboflavin standard used was purchased from Sigma-Aldrich (https: / / www.sigmaaldrich.cn / CN / zh). The primers used were synthesized by Genewiz (https: / / www.genewiz.com.cn / ). The seamless cloning reagent used was purchased from Abclonal (https: / / abclonal.com.cn / ). The DNA polymerase and other molecular biology reagents used were purchased from Novizan (https: / / bio.vazyme.com / ). Other biochemical reagents used were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ). The molecular docking method used was implemented using AutoDock software (https: / / autodock.scripps.edu / ).

[0024] Example 1: Construction of riboflavin-producing strain RSN01, including the following steps Step 1: Prepare the RF-yajI-tet nucleic acid fragment, and integrate the artificial riboflavin operon into Escherichia coli SN02 by λ-red gene recombination to construct strain RSN01. The nucleotide sequence encoding the artificial riboflavin operon is shown in SEQ ID NO.10.

[0025] (1) Using the genome of *Escherichia coli* MG1655 as a template, PCR amplification was performed using primers yajI UF (SEQ ID NO. 39) and yajI UR (SEQ ID NO. 20), yajI LF (SEQ ID NO. 21) and yajI LR (SEQ ID NO. 22) to obtain yajI-U (SEQ ID NO. 11) and yajI-L (SEQ ID NO. 12), with sizes of 595 bp and 545 bp, respectively. Using plasmid pTKS / CS as a template, PCR amplification was performed using primers TF (SEQ ID NO. 23) and tet-R (SEQ ID NO. 24), tet-F (SEQ ID NO. 25) and TR (SEQ ID NO. 26) to obtain T1 (SEQ ID NO. 13) and T2 (SEQ ID NO. 14), with sizes of 774 bp and 728 bp, respectively. Using plasmid pLS01 as a template, PCR amplification was performed using primers RF-F (SEQ ID NO. 26) and RF-F (SEQ ID NO. 27) to obtain yajI-U (SEQ ID NO. 11) and yajI-L (SEQ ID NO. 12), respectively. Using primers NO.27 and RF-R (SEQ ID NO.28), PCR amplification was performed to obtain GENE-RF (SEQ ID NO.15), which was 4091 bp in size; (2) Using three fragments, yajI-U (SEQ ID NO.11), GENE-RF (SEQ ID NO.15), and T1 (SEQ ID NO.13), as templates, and yajI UF (SEQ ID NO.39) and tet-R (SEQ ID NO.24), as primers, PCR fusion was performed to obtain UGT1 (SEQ ID NO.16), with a size of 5459 bp; using two fragments, T2 (SEQ ID NO.14) and yajI-L (SEQ ID NO.12), as templates, and tet-F (SEQ ID NO.25) and yajI LR (SEQ ID NO.22), as primers, PCR fusion was performed to obtain T2L (SEQ ID NO.17), with a size of 1238 bp; using two fragments, UGT1 (SEQ ID NO.16) and T2L (SEQ ID NO.17), as templates, and yajI UF (SEQ ID NO.39) and yajI LR (SEQ ID NO.13 ... and yajI UF (SEQ ID NO.39) and yajI LR (SEQ ID NO.13), as primers, PCR fusion was performed to obtain T2L (SEQ ID NO.17), with a size of 1238 bp; using two fragments, UGT1 (SEQ ID NO Using primer NO.22, PCR fusion yielded the integrated fragment RF-yajI-tet (SEQ ID NO.18) of the artificial riboflavin operon, with a size of 6553 bp.

[0026] Step 2: The plasmid pTKRED was electroporated into strain SN02 and electroporation competent cells were prepared. The fragment RF-yajI-tet (SEQ ID NO.18) obtained in the above step was electroporated into the electroporation competent cells obtained in this step. Using tet-F (SEQ ID NO.25) and yajI LR (SEQ ID NO.22) as primers, colony PCR was performed to verify the recombination of RF-yajI-tet (SEQ ID NO.18), and the correct band size was 1238 bp. Using yajI UF (SEQ ID NO.39) and yajI LR (SEQ ID NO.22) as primers, colony PCR was performed to verify the ejection of the tetracycline resistance gene, and the correct band size was 5240 bp. The strain that successfully integrated the artificial riboflavin operon was named RSN01.

[0027] Example 2: Screening of key amino acids for Escherichia coli 3,4-dihydroxy-2-butanone-4-phosphate synthase, including the following steps: The amino acid sequence of DHBPs derived from Escherichia coli in this embodiment is shown in SEQ ID NO.1.

[0028] (1) Substrate docking was performed on 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPs) (SEQ ID NO.1) to obtain a simulated crystal structure; (2) Based on the simulated crystal structure obtained in step (1), the coding gene for the mutant was constructed by scanning 16 amino acid residues within 5 Å of the substrate using the alanine scanning method and by selecting seven other riboflavin-producing strains and Escherichia coli DHBPs for multiple sequence alignment. The obtained gene was overexpressed in plasmid pZY48 to obtain a series of plasmids carrying the mutant gene. (3) The series of plasmids in step (2) were introduced into strain RSN01 to obtain the strain and fermented in a shake flask; the key amino acid residues of the enzyme were determined to be P46, G151 and T58. (3) Construct a saturated mutant library of the three key amino acid residues in step (3) and a multi-site combination effect; overexpress the obtained gene in plasmid pZY48 to obtain a series of plasmids carrying mutant genes and introduce them into strain RSN01 respectively, obtain the strain and ferment it in a shake flask, and select the enzyme mutant after the mutation combination based on the fermentation data.

[0029] DHBPs mutant is DHBPs G151N DHBPs P46I DHBPs T58E or DHBPs T58E-G151N DHBPs G151N The amino acid sequence is shown in SEQ ID NO.2, DHBPs P46I The amino acid sequence is shown in SEQ ID NO.3, DHBPs T58E The amino acid sequence is shown in SEQ ID NO.4, DHBPs T58E-G151N The amino acid sequence is shown in SEQ ID NO.5.

[0030] Encoding DHBPs G151N The nucleotide sequence of the gene, as shown in SEQ ID NO.6, encodes DHBPs. P46I The nucleotide sequence of the gene is shown in SEQ ID NO.7, encoding DHBPs. T58E The nucleotide sequence of the gene, as shown in SEQ ID NO.8, encodes DHBPs. T58E-G151N The nucleotide sequence of the gene is shown in SEQ ID NO.9.

[0031] Example 3: Construction of a recombinant expression plasmid containing the above-mentioned encoding gene, comprising the following steps: (1) Using plasmid pLS01 as a template, PCR amplification was performed using BF (SEQ ID NO.29) and BR (SEQ ID NO.30) as primers to obtain the DHBPs gene fragment (including the RBS region), which is 682 bp in size; using plasmid pZY48 as a template, PCR amplification was performed using pzy-F (SEQ ID NO.31) and pzy-R (SEQ ID NO.32) as primers to obtain the pZY48 vector fragment (SEQ ID NO.19), which is 3623 bp in size; the DHBPs gene fragment and the pZY48 vector fragment (SEQ ID NO.19) were combined to construct plasmid pZY48-ribB using a seamless cloning method; (2) Using plasmid pZY48-ribB as a template, G151N-F (SEQ ID NO.33) and G151N-R (SEQ ID NO.34) as primers, PCR amplification was performed to obtain the linear fragment of G151N, and plasmid pB-G151N was constructed by seamless cloning method; (3) Using plasmid pZY48-ribB as a template, P46I-F (SEQ ID NO.35) and P46I-R (SEQ ID NO.36) as primers, PCR amplification was performed to obtain the linear fragment of P46I, and plasmid pB-P46I was constructed by seamless cloning method; (4) Using plasmid pZY48-ribB as a template, T58E-F (SEQ ID NO.37) and T58E-R (SEQ ID NO.38) as primers, PCR amplification was performed to obtain the T58E linear fragment, and plasmid pB-T58E was constructed by seamless cloning method; (5) Using plasmid pB-T58E as a template and G151N-F (SEQ ID NO.33) and G151N-R (SEQ ID NO.34) as primers, PCR amplification was performed to obtain the linear fragment T58E-G151N. Plasmid pB-T58E-G151N was constructed using a seamless cloning method, see [link to relevant documentation]. Figure 1 .

[0032] Example 4: Genetically engineered bacteria containing the above recombinant expression plasmid, comprising the following steps Plasmid pZY48-ribB and mutant plasmids pB-G151N, pB-P46I, pB-T58E, and pB-T58E-G151N were electroporated into competent cells of strain RSN01 to construct strains RFB01, RFB02, RFB03, RFB04, and RFB05.

[0033] Example 5: Preparation of fermentation medium, including the following steps Take 1 mL of the mixture of component I, 1 mL of component II, 1 mL of component III and IV, add glucose to make a final concentration of 10 g / L, add yeast extract to make a final concentration of 5 g / L, and add distilled water to 50 mL; thus obtaining the fermentation medium. Component I is: take 10g (NH4)2SO4 and 2g MgSO4, make up to 200mL with distilled water, and sterilize at 121℃ for 20min. Component II is: Take 38.3g Na2HPO4 and 15g KH2PO4, dilute to 200mL with distilled water, and sterilize at 121℃ for 20min; Component III is: Take 5g of ferric ammonium citrate, 2g of CaCl2·2H2O, and 41.7mL of HCl aqueous solution with a concentration of 12mol / L, and dilute to 1000mL with distilled water. The component IV is as follows: Take 1g ZnSO4·7H2O, 0.3g MnCl2·4H2O, 3g H3BO3, 2g CoCl2·6H2O, 0.1g CuSO4·5H2O, 0.2g NiCl2·6H2O, and 0.3g NaMoO4·2H2O, and dilute to 1000mL with distilled water; The mixture of components III and IV is prepared by taking 100 ml of component III and 1 ml of component IV, making up to 200 mL with distilled water, adjusting the pH to 4.5-5.5 with 5 M NaOH aqueous solution, and sterilizing at 121℃ for 20 min.

[0034] Example 6: Shake-flask fermentation of the strain, comprising the following steps: (1) Activation of strains: The strains obtained in Example 4 were streaked on LB solid medium and cultured at 37°C for 12-16 h to rejuvenate the strains; (2) Seed culture: The single colony obtained in step (1) was inoculated into LB liquid medium and cultured at 37℃ and 220rpm for 12-16 h. (3) Shake flask fermentation: The seed liquid obtained in step (2) is fermented according to the initial OD. 600 An inoculum of 0.025 μL was added to the fermentation medium, and the volume of the liquid in the shake flask was 50 mL / 500 mL. Chloramphenicol was added to bring the final concentration to 10 mg / L, and the mixture was incubated at 37°C and 220 rpm for 36 h. The riboflavin yields of strains RFB02, RFB03, RFB04, and RFB05 were 419.18 mg / L, 430.01 mg / L, 471.01 mg / L, and 490.78 mg / L, respectively, which were 37.81%, 41.37%, 54.85%, and 61.35% higher than that of the control strain RFB01 (304.17 mg / L).

[0035] The above embodiments only describe a portion of the specific implementation methods of the present invention in detail, and are not limited to the embodiments disclosed herein. Furthermore, the substantive content protected by the present invention is not limited thereto. Any other modifications, equivalent substitutions, improvements, etc., made based on the principles and techniques of the present invention without departing from its design scope are all within the protection scope of the present invention.

[0036] References [1] S. Liu, N. Diao, Z. Wang, W. Lu, YJ Tang, T. Chen, ModularEngineering of the Flavin Pathway in Escherichia coli for Improved FlavinMononucleotide and Flavin Adenine Dinucleotide Production, J Agric Food Chem, 67 (2019) 6532-6540. [2] J. You, [3] A. Rana, NK Taneja, N. Singh, AK Puniya, MetabolicEngineering in Microbial System for Riboflavin Production: A ComprehensiveReview, Indian Journal of Microbiology, DOI 10.1007 / s12088-025-01531-7(2025). [4] E. Zhang, Y. Cai, Y. Luo, Z. Piao, Riboflavin-shuttledextracellular electron transfer from Enterococcus faecalis to electrodes inmicrobial fuel cells, Can J Microbiol, 60 (2014) 753-759. [5] S. Wang, Q. Zhu, C. Liu, H. Dong, M. Xia, Z. Jin, D. Zhang,Engineering riboflavin-overproducing Bacillus subtilis via pathway geneoverexpression, Synthetic and Systems Biotechnology, 11 (2026) 91-101. [6] R. Ledesma-Amaro, C. Serrano-Amatriain, A. Jiménez, J.L.Revuelta, Metabolic engineering of riboflavin production in Ashbya gossypiithrough pathway optimization, Microb Cell Fact, 14 (2015) 163. [7] M. Lehmann, S. Degen, H.P. Hohmann, M. Wyss, A. Bacher, N.Schramek, Biosynthesis of riboflavin. Screening for an improved GTPcyclohydrolase II mutant, FEBS J, 276 (2009) 4119-4129。

Claims

1. A mutant of 3,4-dihydroxy-2-butanone-4-phosphate synthase characterized in that, The mutants are DHBPs G151N , DHBPs P46I , DHBPs T58E , or DHBPs T58E-G151N ; the amino acid sequence of DHBPs G151N is shown as SEQ ID NO. 2, the amino acid sequence of DHBPs P46I is shown as SEQ ID NO. 3, the amino acid sequence of DHBPs T58E is shown as SEQ ID NO. 4, and the amino acid sequence of DHBPs T58E-G151N is shown as SEQ ID NO.

5.

2. A gene encoding the 3,4-dihydroxy-2-butanone-4-phosphate synthase mutant of claim 1, characterized in that, Encoding DHBPs G151N The nucleotide sequence of the gene, as shown in SEQ ID NO.6, encodes DHBPs. P46I The nucleotide sequence of the gene is shown in SEQ ID NO.7, encoding DHBPs. T58E The nucleotide sequence of the gene, as shown in SEQ ID NO.8, encodes DHBPs. T58E-G151N The nucleotide sequence of the gene is shown in SEQ ID NO.

9.

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

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

5. Use of the genetically engineered bacterium of claim 4 in the fermentation preparation of riboflavin.

Images