Riboflavin-producing strain, construction method and application thereof

Abstract

This invention provides a riboflavin-producing strain, its construction method, and its application. E. coli W3110 As a chassis strain, through the absence of purR FMN ribonucleoside switch gene, upregulated gmk, ndk, guaA, guaB Gene transcription level, heterologous expression originates from Bacillus subtilis 168 strains ribD , ribE , ribBA, ribH Genes and purine operon genes pur ( EKBCSQLFMNHD ), source Corynebacterium glutamicum K051 strains zwf Genes, dynamic regulation ribF The gene expression of the constructed strain has a clear genetic background, is plasmid-free, can be sustainably modified, and does not require the addition of resistance substances, effectively improving the efficiency of riboflavin synthesis and showing good prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and fermentation engineering technology, and in particular to a riboflavin-producing strain, its construction method, and its application. Background Technology

[0002] Riboflavin is an essential nutrient for living organisms. It can act as a coenzyme for FMN and FAD in redox reactions in the body, driving the energy metabolism of sugars, lipids, and proteins. It can also maintain the health of mucous membranes and epithelial tissues of animals, plants, and microorganisms, help synthesize visual photosensitive substances, and assist in the activation of other B vitamins. In addition, it can reduce oxidative damage to biological cells through its antioxidant function. It is a key factor in the growth and metabolism of microorganisms and a core additive in animal feed.

[0003] Currently, riboflavin (vitamin B2) is fully produced through microbial fermentation, with a global annual output exceeding 8,000 tons. The core mainstream strains for industrial production are *Ashurus hygroscopicus* (a riboflavin-producing yeast / filamentous fungus) and *Bacillus subtilis*, which together account for the vast majority of global production capacity. *Ashurus hygroscopicus* has a long growth cycle, low equipment turnover rate, and is prone to strain degradation. *Bacillus subtilis* often carries antibiotic resistance genes, posing a risk of drug resistance gene diffusion. *Escherichia coli*, with its mature genetic manipulation system, has become the core model strain for research. It has a rapid growth rate, extremely high theoretical production capacity, low culture cost, and wide adaptability, making it a promising candidate for constructing a riboflavin-producing strain with a clear genetic background and considerable yield. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a riboflavin-producing strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned riboflavin-producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned riboflavin-producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A riboflavin-producing strain, named strain RF-08, is produced using Escherichia coli. E.coli W3110, a chassis strain, was obtained through gene editing using metabolic engineering techniques: the purine repressor protein gene was knocked out. purR 3,4-Dihydroxy-2-butanone-4-phosphate synthase gene ribB The FMN riboswitch before the gene upregulates the guanylate kinase gene. gmk, Nucleoside diphosphate kinase gene ndk, Guanylate synthase gene guaA, Inosine nucleotide dehydrogenase gene guaBThe transcriptional level of heterologous expression of Bacillus subtilis Bacillus subtilis Riboflavin engineered strain B.subtilis 168 pyrimidine deaminase gene (ATCC 23857) ribD Riboflavin synthase gene ribE Guanosine triphosphate cyclase II gene ribBA Dioxane-tetrahydropurine synthase gene ribH and purine operon genes pur ( EKBCSQLFMNHD ), source Corynebacterium glutamicum K051 glucose-6 phosphate dehydrogenase gene of the strain zwf Dynamic regulation of the bifunctional riboflavin kinase gene ribF The expression.

[0008] Preferably, the above-mentioned riboflavin-producing strain, in the starting strain E.coli.W3110 Introducing heterogeneous sources B.subtilis 168 strain purine operon gene pur ( EKBCSQLFMNHD ), by P trc Promoter activation; knockout of purine repressor protein genes purR Overexpression originates from Bacillus subtilis 168 Riboflavin biosynthesis gene ribD , ribE , ribBA and ribH , by the same P T7 Promoter initiates transcription; overexpression of guanylate kinase gene gmk and nucleoside diphosphate kinase gene ndk , by P trc Promoter activation; overexpression of guanylate synthase gene guaA and hypoxanthine nucleotide dehydrogenase gene guaB , by P trc Promoter activation; knockout of the 3,4-dihydroxy-2-butanone-4-phosphate synthase gene ribB The FMN ribose switch before, by P trc Promoter activation; integration of the FMN riboswitch-coupled promoter into the bifunctional riboflavin kinase gene. ribF The preceding; overexpression originates from Corynebacterium glutamicum K051 glucose-6 phosphate dehydrogenase gene zwf , by P trc Startup sub-boot.

[0009] Preferably, the above-mentioned riboflavin-producing strain, *Escherichia coli* E.coli.W3110 Wild-type Escherichia coli E.coli W3110 ATCC 27325.

[0010] Preferably, the above-mentioned riboflavin-producing strain overexpresses the guanylate kinase gene. gmk and nucleoside diphosphate kinase gene ndk The specific method is as follows: Guanylate kinase gene gmk and nucleoside diphosphate kinase gene ndk They are arranged in tandem, with genes linked by RBS (ribosome binding sites), and transcription driven by the Ptrc promoter to form polycistronic expression units.

[0011] Preferably, in the above-mentioned riboflavin-producing strain, the purine operon gene... pur ( EKBCSQLFMNHD The nucleotide sequence of the purine operon gene is shown in SEQ ID NO.1 of the sequence listing. pur ( EKBCSQLFMNHD The NCBI-GeneIDs of each gene in the dataset are as follows: purE : 939481; purK : 936039; purB : 936048; purC : 939389; purS : 936041; purQ : 938760; purL : 939233; purF : 936046; purM : 936044; purN : 936045; purH : 936053; purD :938741); Purine repressor protein gene purR The nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing; pyrimidine deaminase gene ribD The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing ( ribD (NCBI-GeneID: 936002); Riboflavin synthase gene ribE The nucleotide sequence is shown in SEQ ID NO.4 of the sequence listing ( ribE (NCBI-GeneID: 936004); Guanosyl triphosphate cyclase II gene ribBA The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing ( ribBA (NCBI-GeneID: 936003); dioxane synthase gene ribH The nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing ( ribH (NCBI-GeneID: 936005); guanylate kinase gene gmk The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing ( gmk(NCBI-GeneID: 946130); nucleoside diphosphate kinase gene ndk The nucleotide sequence is shown in SEQ ID NO. 8 ( ndk (NCBI-GeneID: 945611); guanylate synthase gene guaA The nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing (gua A (NCBI-GeneID: 945609); Inosine nucleotide dehydrogenase gene guaB The nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing ( guaB (NCBI-GeneID: 945608); The nucleotide sequence of the FMN riboswitch is shown in SEQ ID NO. 11 of the sequence listing; glucose-6 phosphate dehydrogenase gene zwf The nucleotide sequence is shown in SEQ ID NO. 13 (zwf; NCBI-GeneID: 12067307); 3,4-dihydroxy-2-butanone-4-phosphate synthase gene ribB The nucleotide sequence is shown in SEQ ID NO.16 of the sequence listing ( ribB (NCBI-GeneID: 946746); Bifunctional riboflavin kinase gene ribF The nucleotide sequence is shown in SEQ ID NO.17 of the sequence listing ( ribF (NCBI-GeneID: 944873); The nucleotide sequence of RBS is shown in SEQ ID NO.18 of the sequence listing.

[0012] Preferably, in the above-mentioned riboflavin-producing strain, the nucleotide sequence of the FMN riboswitch-coupled promoter is shown in SEQ ID NO. 12 of the sequence listing; P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.14 of the sequence listing; P T7 The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.15.

[0013] The above-mentioned method for constructing riboflavin-producing strains, starting with the strain E.coli Based on the W3110, targeted modifications were made, resulting in the following three modules: (1) Increase the production of precursors Ru5P and GTP and improve reducing power: Knock out the purine repressor protein gene purR ; Increase the purine operon gene pur ( EKBCSQLFMNHD The transcriptional level of guanylate kinase was increased; the gene encoding guanylate kinase was enhanced. gmk Transcription level; increase the transcription level of nucleoside diphosphate kinase-encoding genes ndk Transcription level; increase guanylate synthase geneguaA Transcription level; increase in hypoxanthine nucleotide dehydrogenase gene guaB Transcription level; increase glucose-6 phosphate dehydrogenase gene zwf transcriptional levels; (2) Increase riboflavin production: Introduce exogenous pyrimidine deaminase gene ribD, Riboflavin synthase gene ribE, Guanosine triphosphate cyclase II gene ribBA, Dioxatetrahydropurine synthase gene ribH Knockout of the 3,4-dihydroxy-2-butanone-4-phosphate synthase gene ribB The FMN ribose switch before; (3) Reduce riboflavin degradation: Replace the bifunctional riboflavin kinase gene with an FMN riboswitch-coupled promoter. ribF The original promoter, namely the promoter regulation that carries an FMN-responsive ribosomal switch. ribF Gene expression; the ribF The original promoter nucleotide sequence is shown in SEQ ID NO.19 of the sequence listing.

[0014] Preferably, the method for constructing the above-mentioned riboflavin-producing strain includes the following specific steps: (1) By E.coli W3110 genome yjiV Introducing heterologous purine operon genes at the site pur ( EKBCSQLFMNHD ), by P trc Promoter activation enhances the carbon metabolic flux in the purine metabolism pathway; (2) Knockout of purine repressor protein gene purR, Enhance purine metabolism; (3) Integration at the molR site in the genome originates from Bacillus subtilis 168 pyrimidine deaminase gene ribD Riboflavin synthase gene ribE Guanosine triphosphate cyclase II gene ribBA and dioxatetrahydropurine synthase gene ribH The genes are arranged in tandem according to the natural gene cluster sequence and are generated by the same P. T7 The promoter drives transcription to form polycistronic expression units, thereby enhancing the biosynthetic pathway of riboflavin; (4) Through the genome yeeL Site integration of guanylate kinase gene gmk and nucleoside diphosphate kinase gene ndk , by P trc Promoter activation enhances purine metabolism; (5) Through the genome ycjV Site integration of guanylate synthase geneguaA and hypoxanthine nucleotide dehydrogenase gene guaB The genes are arranged in tandem according to the natural gene cluster sequence, by P trc Promoter activation enhances purine metabolism; (6) Knockout 3,4-dihydroxy-2-butanone-4-phosphate synthase gene ribB The FMN ribose switch before, deactivate FMN to 3, 4-dihydroxy-2-butanone-4-phosphate synthase gene ribB Feedback inhibition; (7) Integrating the FMN riboswitch-coupled promoter into the bifunctional riboflavin kinase gene ribF Before replacing its original promoter, it achieves adaptive regulation of the riboflavin degradation pathway, reducing riboflavin degradation while maintaining intracellular FMN levels, thus avoiding its deficiency from affecting bacterial growth; (8) Through the genome yghE Site integration originates from Corynebacterium glutamicum K051 The glucose-6 phosphate dehydrogenase gene zwf, by P trc The promoter activates the pathway, causing more carbon metabolism to flow to the pentose phosphate pathway.

[0015] Application of the above-mentioned riboflavin-producing strains in riboflavin production.

[0016] Preferably, the above-mentioned application involves producing riboflavin using a fermentation method in a shake flask or fermenter.

[0017] Preferably, in the above application, fermentation is carried out using a fermentation tank, and the specific steps are as follows: (1) Activation of strain: The strain was transferred from the glycerol tube to the slant medium and activated for 11-13 hours; the strain was transferred from the slant medium to the eggplant flask medium and activated and expanded for 10-12 hours. The culture temperature was 37℃. (2) Seed culture: During the culture process, the pH was maintained at 7.0±0.2, the temperature at 37±0.2℃, and the dissolved oxygen at 35%-50%, and the culture was carried out until the OD reached 70%. 600nm At 20:00, the cells were transferred to a fermenter for fermentation. (3) Fermentation culture: The seed liquid is inoculated into the fermentation medium for fermentation culture. During the fermentation culture, the pH is controlled at 7.0±0.2, the temperature is maintained at 37±0.2℃, and the dissolved oxygen is maintained at 30%-50%. During the culture, glucose solution is added to maintain the carbon source required by the cells during the fermentation process. During this period, ammonia water is added to adjust the pH to maintain it at 7.0±0.2.

[0018] Preferably, in the above application, the slant culture medium used is: glucose 2g / L, sodium chloride 2.5g / L, yeast extract 5g / L, peptone 10g / L, agar powder 25g / L, KH2PO4 1.5g / L, MgSO4·7H2O 0.2g / L, with the remainder being water.

[0019] Preferably, in the above application, the seed culture medium used is: glucose 30 g / L, yeast extract 8 g / L, peptone 2 g / L, citric acid 2 g / L, (NH4)2SO4 1 g / L, MgSO4·7H2O 1.5 g / L, KH2PO4 1.5 g / L, FeSO4·7H2O 10 mg / L, MnSO4 5 mg / L, vitamins B1, B3, B5, and C. 12 Each 1 mg / L, VH 1 mg / L, the remainder is water.

[0020] Preferably, in the above application, the fermentation medium used is: glucose 30 g / L, yeast extract 5 g / L, peptone 2 g / L, citric acid 2 g / L, (NH4)2SO4 2 g / L, MgSO4·7H2O 2 g / L, KH2PO4 4 g / L, FeSO4·7H2O 20 mg / L, MnSO4 10 mg / L, Vb1, Vb3, Vb5, Vb 12 Each 2 mg / L, VH 2 mg / L, the remainder is water.

[0021] All of the above-mentioned culture media can be prepared using standard methods.

[0022] Beneficial effects: The above-mentioned riboflavin-producing strains are wild-type. E.coli.W3110 For chassis microorganisms, heterologous purine operon genes were introduced. pur ( EKBCSQLFMNHD ), through genome integration and overexpression of key enzyme genes in the riboflavin biosynthesis pathway. ribD , ribE , ribBA, ribH, gmk, ndk, guaA, guaB, zwf Release 3,4-dihydroxy-2-butanone-4-phosphate synthase gene ribB Feedback inhibition was achieved, and a promoter carrying an FMN-responsive ribosomal switch was constructed to dynamically regulate the riboflavin kinase gene. ribFThis study demonstrates the ability to adaptively regulate the riboflavin degradation pathway, reducing riboflavin degradation while maintaining intracellular FMN levels to prevent its deficiency from negatively impacting cell growth. The constructed strain possesses a clear genetic background, is plasmid-free, and can be sustainably modified. Riboflavin production via fermentation using this strain requires no added antibiotics, exhibits excellent riboflavin synthesis capabilities, is simple to operate, and can synthesize riboflavin de novo using inexpensive carbon sources, effectively improving riboflavin synthesis efficiency, resulting in low production costs and stable performance. Using a fed-batch fermentation method in a 5L fermenter for 36 hours, a riboflavin yield of 15.6 g / L was achieved, significantly improving riboflavin production levels and demonstrating promising industrial application prospects. Specifically: (1) By knocking out the purine repressor protein gene purR, Introduction of purine operon gene pur ( EKBCSQLFMNHD ), increase the gene encoding guanylate kinase gmk, Nucleoside diphosphate kinase encoding gene ndk, Guanylate synthase gene guaA, Inosine nucleotide dehydrogenase gene guaB, glucose-6 phosphate dehydrogenase gene zwf The transcriptional level provides a large number of precursors and reducing power for riboflavin synthesis; (2) Introducing exogenous pyrimidine deaminase genes ribD, Riboflavin synthase gene ribE, Guanosine triphosphate cyclase II gene ribBA, Dioxatetrahydropurine synthase gene ribH, Significantly enhanced riboflavin synthesis capacity by knocking out the 3,4-dihydroxy-2-butanone-4-phosphate synthase gene. ribB The FMN riboswitch before the riboflavin decomposition product FMN is removed, thus eliminating the feedback inhibition effect of FMN on riboflavin synthesis and improving the efficiency of riboflavin synthesis. (3) In E.coli W3110 Bifunctional riboflavin kinase gene ribF It is primarily involved in the riboflavin degradation pathway by integrating the FMN riboswitch-coupled promoter into the bifunctional riboflavin kinase gene. ribF Before replacing its original promoter, it achieves adaptive regulation of the riboflavin degradation pathway, reducing riboflavin degradation while maintaining intracellular FMN levels, avoiding its deficiency from affecting bacterial growth, thereby increasing riboflavin production. Attached Figure Description

[0023] Figure 1 A diagram illustrating the construction method of the riboflavin-engineered strain RF-08. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0025] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0026] The starting strain used in the examples was wild-type Escherichia coli. E.coli W3110 ATCC 27325 (commercially available), the corresponding promoter and gene are shown in the sequence listing. Primers used in the construction of the involved strains are shown in Table 1.

[0027] Table 1 Primers used in strain construction

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] The gene editing methods used in the gene manipulations in this embodiment refer to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). Unless otherwise specified, all technical terms used in this invention are explained in this article. In this invention, "knockout" refers to the inactivation of the target gene, and "introduction" refers to the insertion of a foreign gene into the engineered bacterial genome after linking it with a promoter and terminator.

[0035] Example 1 like Figure 1 As shown, the riboflavin-producing strain RF-08 was constructed using wild-type... E.coli.W3110 As chassis microorganisms, the strains were modified according to the metabolic pathway of the entire strain modification process. The modification process mainly includes the following three modules: (1) Increase the production of precursors Ru5P and GTP and improve reducing power: including knocking out purine repressor protein genes. purR ; Increase the purine operon gene pur ( EKBCSQLFMNHD The transcriptional level of guanylate kinase was increased; the gene encoding guanylate kinase was enhanced. gmk Transcription level; increase the transcription level of nucleoside diphosphate kinase-encoding genes ndk Transcription level; increase guanylate synthase gene guaA Transcription level; increase in hypoxanthine nucleotide dehydrogenase gene guaB Transcription level; increase glucose-6 phosphate dehydrogenase gene zwf transcriptional levels; (2) Increase riboflavin production: including the introduction of exogenous pyrimidine deaminase genes ribD, Riboflavin synthase gene ribE, Guanosine triphosphate cyclase II gene ribBA, Dioxatetrahydropurine synthase gene ribH Knockout of the 3,4-dihydroxy-2-butanone-4-phosphate synthase gene ribB The FMN ribose switch before; (3) Reduce riboflavin degradation: Integrate the FMN riboswitch-coupled promoter into the bifunctional riboflavin kinase gene. ribF Before that, replace its original promoter, that is, construct a promoter regulation that carries an FMN-responsive ribosomal switch. ribF Gene expression. The specific methods are as follows: 1.1 Purine nucleoside operon gene pur ( EKBCSQLFMNHD Integration because B.subtilis The 168-purine nucleoside operon gene is 12805 bp in length. It was integrated into the genome sequentially using a segmented integration method (the method is based on the segmented integration of the Ptrc-carAB segment gene in CN114774341A specification

[0096] ). yjiV The five segments are: purEKB, purCSQ, purL, purFMN, purHD .

[0036] 1.1.1 Genes purEKB integration pGRB- yjiV -s、pGRB- yjiV -a is a primer, used to construct pGRB- yjiV Plasmids. yjiV -ups, yjiV -up- purEKB -a、 purEKB -s、 purEKB -a、 yjiV -dn- purEKB -s、yjiV -dn-a as primer, and then respectively with B.subtilis 168 and E.coli W3110 Using the genome as a template, upstream homologous arms were obtained. yjiV -up, downstream homologous arm yjiV -dn, target gene purEKB Then obtain the fusion fragment. yjiV :: purEKB Finally, pGRB- yjiV Plasmids and overlapping fragments yjiV :: purEKB Electroporation to the starting strain E.coliW3110 In electrocompetent cells; then with yjiV -ups, yjiV -dn-a is the identification primer; positive transformants are screened to obtain strain RF-01- purEKB .

[0037] 1.1.2 Genes purCSQ integration The pGRB-1 plasmid was constructed using pGRB-1-s and pGRB-1-a as primers. The plasmid was then constructed using 1-1-up-s, 1-1-up-a, and... purCSQ -s、 purCSQ -a、 yjiV -dn- purCSQ -s、 yjiV -dn-a as primer, and then respectively with B. subtilis 168 and E.coliW3110 Using the genome as a template, upstream homologous arm 1-1-up and downstream homologous arm 1-1-DN were obtained, along with the target gene. purCSQ Then obtain the fusion fragment 1::purEKB. Finally, combine the pGRB-1 plasmid with the overlapping fragment 1:: purEKB Electrical transfer to RF-01- purEKB In electrocompetent cells; then with 1-1-up-s, yjiV -dn-a was used as the identification primer to screen for positive transformants and obtain strain RF-01. -purEKBCSQ .

[0038] 1.1.3 Genes purL integration The pGRB-4 plasmid was constructed using pGRB-4-s and pGRB-4-a as primers. The plasmid was then constructed using 4-1-up-s, 4-1-up-a, and... purL -s、 purL -a、 yjiV -dn- purL -s、 yjiV Using -dn-a as primers, respectively with B. subtilis168 and E.coliW3110Using the genome as a template, upstream homologous arm 4-1-up and downstream homologous arm 4-1-dn were obtained, along with the target gene. purL Then obtain fusion fragment 4:: purL Finally, pGRB- yjiV Plasmid and overlapping fragment 4:: purL Electrical transfer to RF-01- purEKBCSQ In electrocompetent cells; then with 4-1-up-s, yjiV -dn-a is the identification primer; positive transformants are screened to obtain strain RF-01- purEKBCSQL .

[0039] 1.1.4 Genes purFMN integration The pGRB-5 plasmid was constructed using pGRB-5-s and pGRB-5-a as primers. The plasmid was then constructed using 5-1-up-s, 5-1-up-a, and... purFMN -s、 purFMN -a、 yjiV -dn- purFMN -s、 yjiV Using -dn-a as primers, respectively with B. subtilis168 and E.coliW3110 Using the genome as a template, upstream homologous arm 5-1-up and downstream homologous arm 5-1-dn were obtained, along with the target gene. purFMN Then, the fusion fragment 5::purEKB was obtained. Finally, the pGRB-5 plasmid was combined with the overlapping fragment 5:: purFMN Electrical transfer to RF-01- purEKBCSQL Electrocompetent cells; then with 5-1-up-s, yjiV -dn-a is the identification primer; positive transformants are screened to obtain strain RF-01- purEKBCSQLFMN .

[0040] 1.1.5 Genes purHD integration The pGRB-4 plasmid was constructed using pGRB-4-s and pGRB-4-a as primers. Then, using 4-2-up-s, 4-2-up-a, and... purHD -s、 purHD -a、 yjiV -dn- purL -s、 yjiV Using -dn-a as primers, respectively with B. subtilis168 and E.coliW3110 Using the genome as a template, upstream homologous arm 4-2-up and downstream homologous arm 4-2-dn were obtained, along with the target gene. purHD Then obtain fusion fragment 4:: purHD Finally, the pGRB-4 plasmid was combined with the overlapping fragment 4:: purHDElectrical transfer to RF-01- purEKBCSQLFMN Electrocompetent cells; then with 4-2-up-s, yjiV -dn-a is the identification primer; positive transformants are screened to obtain strain RF-01- pur(EKBCSQLFMNHD), That is, RF-01.

[0041] 1.2 Genes purR Knockout pGRB- purR -s、pGRB- purR -a is a primer, used to construct pGRB- purR Plasmid. Then with purR -ups, purR -up-a、 purR -dn-s、 purR -dn-a is used as a primer, with E.coliW3110 Using the genome as a template, upstream homologous arms were obtained. purR -up, downstream homologous arm purR -dn; then obtain the fusion fragment. purR Finally, pGRB- purR Plasmids and overlapping fragments purR Electroporation into RF-01 electrocompetent cells; then... purR -ups, purR -dn-a was used as the identification primer to screen for positive transformants and obtain strain RF-02.

[0042] 1.3 Genes ribD, ribE, ribBA, ribH integration pGRB- molR -s、pGRB- molR -a is a primer, used to construct pGRB- molR Plasmid. Then with molR -ups, molR -up-a、 ribD -s、 ribH -a、 molR -dn-s、 molR Using -dn-a as primers, respectively with B. subtilis 168 and E.coli W3110 Using the genome as a template, upstream homologous arms were obtained. molR -up, downstream homologous arm molR -dn, target gene rib(DEBAH) Then obtain the fusion fragment. molR :: rib(DEBAH) Finally, pGRB- molR Plasmids and overlapping fragments molR :: rib(DEBAH)Electroporation into RF-02 electrocompetent cells; then... molR -ups, molR -dn-a was used as the identification primer to screen for positive transformants and obtain strain RF-03.

[0043] 1.4 Genes gmk and ndk integration pGRB- yeeL -s、pGRB- yeeL -a is a primer, used to construct pGRB- yeeL Plasmid. Then with yeeL -ups, yeeL -up-a、 yeeL -dn-s、 yeeL -dn-a、 gmk -s、 gmk -a、 ndk -s、 ndk -a is a primer, with E.coliW3110 Using the genome as a template, upstream homologous arms were obtained. yeeL -up, downstream homologous arm yeeL -dn, target segment gmk and ndk Then obtain the fusion fragment. yeeL :: gmk+ndk Finally, pGRB- yeeL Plasmids and overlapping fragments yeeL :: gmk+ndk Electroporation into RF-03 electrocompetent cells; then... yeeL -ups, yeeL -dn-a was used as the identification primer to screen for positive transformants and obtain strain RF-04.

[0044] 1.5 genes guaA and guaB integration pGRB- ycjV -s、pGRB- ycjV -a is a primer, used to construct pGRB- ycjV Plasmid. Then with ycjV -ups, ycjV -up-a、 ycjV -dn-s、 ycjV -dn-a、 ycjV - guaA -s、 ycjV - guaB -a is a primer, with E.coliW3110 Using the genome as a template, upstream homologous arms were obtained. ycjV -up, downstream homologous arm ycjV -dn, target segment guaA+ guaB Then obtain the fusion fragment. ycjV :: guaA + guaB Finally, pGRB- ycjV Plasmids and overlapping fragments ycjV :: guaA + guaB Electroporation into RF-04 electrocompetent cells; then... ycjV -ups, ycjV -dn-a was used as the identification primer to screen for positive transformants and obtain strain RF-05.

[0045] 1.6 Genes ribB Knockout of the FMN riboswitch pGRB -ribB -s、pGRB- ribB -a is a primer, used to construct pGRB- ribB Plasmid. Then with ribB -ups, ribB -up-a、 ribB -dn-s、 ribB -dn-a is used as a primer, with E.coliW3110 Using the genome as a template, upstream homologous arms were obtained. ribB -up, downstream homologous arm ribB -dn; then obtain the fusion fragment. ribB FMN. Finally, pGRB- ribB Plasmids and overlapping fragments ribB FMN was electroporated into RF-05 electrocompetent cells; then... ribB -ups, ribB -dn-a was used as the identification primer to screen for positive transformants and obtain strain RF-06.

[0046] 1.7 Integration of FMN Ribose Switch Coupled Promoters pGRB- ribF -s、pGRB- ribF -a is a primer, used to construct pGRB- ribF Plasmid. Then with ribF -ups, ribF -up-a、 ribF -dn-s、 ribF Using -dn-a, FMN riboswitch-s, and FMN riboswitch-a as primers, with E.coliW3110 Using the genome as a template, upstream homologous arms were obtained. ribF -up, downstream homologous arm ribF-dn, the target fragment FMNriboswitch; then obtain the fusion fragment ribF::FMN riboswitch. Finally, pGRB -ribF Plasmids and overlapping fragments ribF ::FMN riboswitch was electroporated into RF-06 electroporation competent cells; then... ribF -ups, ribF -dn-a was used as the identification primer to screen for positive transformants and obtain strain RF-07.

[0047] 1.8 genes zwf integration pGRB- yghE -s、pGRB- yghE -a is a primer, used to construct pGRB- yghE Plasmid. Then with yghE -ups, yghE -up-a、 yghE -dn-s、 yghE -dn-a、 yghE - zwf -s、 yghE-zwf -a is a primer, with E.coliW3110 Using the genome as a template, upstream homologous arms were obtained. yghE -up, downstream homologous arm yghE -dn, target segment zwf Then obtain the fusion fragment. yghE :: zwf Finally, pGRB- yghE Plasmids and overlapping fragments yghE :: zwf Electroporation into RF-07 electrocompetent cells; then... yghE -ups, yghE -dn-a was used as the identification primer to screen for positive transformants and obtain strain RF-08.

[0048] Example 2 The strains RF-03, RF-04, RF-05, RF-06, RF-07, and RF-08 obtained in Example 1 were used for fermentation verification. Riboflavin was produced by fermentation in a 5L fermenter. The specific steps are as follows: (1) Activation of the strain: The strain was transferred from the glycerol tube to the slant medium for activation and culture for 12 h. The slant medium used was: glucose 2 g / L, sodium chloride 2.5 g / L, yeast extract 5 g / L, peptone 10 g / L, agar powder 25 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, and the remainder was water. The strain was then transferred from the slant medium to the flask medium for further activation and expansion culture for 12 h. The culture temperature was 37℃.

[0049] (2) Seed culture: The seed culture medium used was glucose 30 g / L, yeast extract 8 g / L, peptone 2 g / L, citric acid 2 g / L, (NH4)2SO4 1 g / L, MgSO4·7H2O 1.5 g / L, KH2PO4 1.5 g / L, FeSO4·7H2O 10 mg / L, MnSO4 5 mg / L, Vb1, Vb3, Vb5, Vb 12 Each 1 mg / L, VH 1 mg / L, the remainder water; during cultivation, pH was maintained at 7.0 ± 0.2, temperature at 37℃ ± 0.2, and dissolved oxygen at 35%-50%, and cultivation continued until OD reached... 600nm At 20:00, the cells were transferred to a fermenter for fermentation culture.

[0050] (3) Fermentation culture: The seed culture was inoculated into the fermentation medium at an inoculation rate of 20% for fermentation culture. The fermentation medium used was glucose 30 g / L, yeast extract 5 g / L, peptone 2 g / L, citric acid 2 g / L, (NH4)2SO4 2 g / L, MgSO4·7H2O 2 g / L, KH2PO4 4 g / L, FeSO4·7H2O 20 mg / L, MnSO4 10 mg / L, Vb1, Vb3, Vb5, Vb 12 Each component was 2 mg / L, VH 2 mg / L, and the remainder was water. During fermentation, the pH was controlled at 7.0 ± 0.2, the temperature at 37 ± 0.2℃, and dissolved oxygen at 30%-50%. During cultivation, 80% (m / v) glucose solution was added to maintain the carbon source required by the fermentation cells. 25% ammonia was added to adjust the pH, maintaining it at 7.0 ± 0.2. The fermentation time was 36 hours. The fermentation results in a 5L fermenter are shown in Table 2 below.

[0051] Table 2

[0052] Example 3 Based on strain RF-06 in Example 1, the bifunctional riboflavin kinase gene was further modified and expressed using different attenuation methods. ribF : Use P respectively J23115 promoters and P J23114 Promoter replacement ribF The original promoter corresponds to strain P. J23115 - ribF and P J23114 - ribF The P J23115 promoters and P J23114 See https: / / registry.igem.org / parts / bba-b0015 for the promoter.

[0053] For strain P J23115 - ribF P J23114 - ribF Fermentation verification was performed with RF-07, and the fermentation method was the same as in Example 2. The fermentation results of the 5L fermenter are shown in Table 3 below.

[0054] Table 3

[0055] The fermentation results of Examples 2 and 3 above show that the riboflavin production of strain RF-07 is significantly higher than that of strain RF-06, and compared with the strain using the weak promoter P J23115 And weaker P J23114 Startup Sub-boot ribF Genes using FMN riboswitch-coupled promoters exhibit higher bacterial biomass and riboflavin production, indicating that integrating the FMN riboswitch into... ribF Before the original gene promoter, a promoter regulator carrying an FMN-responsive ribosomal switch is constructed for regulation. ribF Gene expression was used to adaptively regulate the riboflavin degradation pathway, reducing riboflavin degradation while maintaining intracellular FMN levels, thus preventing its deficiency from affecting cell growth and effectively increasing riboflavin production. The engineered strain RF-08 was fermented in a 5L fermenter for 36 hours using a fed-batch fermentation method, achieving a riboflavin yield of 15.6 g / L.

[0056] It is evident that the genetically engineered bacteria for producing riboflavin described in this invention can synthesize higher-value riboflavin de novo using inexpensive glucose as a substrate. It has advantages such as stable genetic performance, clear genetic background, no defect type, and high production efficiency, and has good prospects for industrialization.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A riboflavin-producing strain, characterized in that: E. coli E. coli W3110, a chassis strain, was obtained through gene editing using metabolic engineering techniques: the purine repressor protein gene was knocked out. purR Knockout of the 3,4-dihydroxy-2-butanone-4-phosphate synthase gene ribB The FMN ribose switch before, by P trc Promoter activation upregulated the guanylate kinase gene. gmk、 Nucleoside diphosphate kinase gene ndk、 Guanylate synthase gene guaA and hypoxanthine nucleotide dehydrogenase gene guaB The transcriptional level, by P trc Promoter-initiated; heterologous expression derived from Bacillus subtilis Bacillus subtilis pyrimidine deaminase gene ribD Riboflavin synthase gene ribE Guanosine triphosphate cyclase II gene ribBA and dioxatetrahydropurine synthase gene ribH , by the same P T7 The promoter initiates transcription; heterologous expression of Bacillus subtilis Bacillus subtilis purine operon gene pur ( EKBCSQLFMNHD ), by P trc Starter-based startup; heterogeneous expression originating from Corynebacterium glutamicum glucose-6 phosphate dehydrogenase gene zwf , by P trc Promoter activation; integration of the FMN riboswitch-coupled promoter into the bifunctional riboflavin kinase gene. ribF Previously, the bifunctional riboflavin kinase gene was dynamically regulated. ribF The expression.

2. The riboflavin-producing strain according to claim 1, characterized in that: The E. coli E. coli W3110 Wild-type Escherichia coli E. coli W3110 ATCC 27325.

3. The riboflavin-producing strain according to claim 1, characterized in that: Overexpression of guanylate kinase gene gmk and nucleoside diphosphate kinase gene ndk The specific method is as follows: Guanylate kinase gene gmk and nucleoside diphosphate kinase gene ndk Arranged in tandem, genes are linked by RBS and connected by P. trc The promoter drives transcription to form a polycistronic expression unit, and the nucleotide sequence of the RBS is shown in SEQ ID NO.

18.

4. The riboflavin-producing strain according to claim 1, characterized in that: The purine operon gene pur ( EKBCSQLFMNHD The nucleotide sequence of the purine repressor protein gene is shown in SEQ ID NO.1 of the sequence listing. purR The nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing; pyrimidine deaminase gene ribD The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing; riboflavin synthase gene ribE The nucleotide sequence is shown in SEQ ID NO.4 of the sequence listing; guanosine triphosphate cyclase II gene ribBA The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing; dioxetine synthase gene ribH The nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing; guanylate kinase gene gmk The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing; nucleoside diphosphate kinase gene. ndk The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing; guanylate synthase gene guaA The nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing; inosine nucleotide dehydrogenase gene guaB The nucleotide sequence of the gene is shown in SEQ ID NO. 10 of the sequence listing; the nucleotide sequence of the FMN riboswitch is shown in SEQ ID NO. 11 of the sequence listing; glucose-6 phosphate dehydrogenase gene. zwf The nucleotide sequence is shown in SEQ ID NO.13 of the sequence listing; bifunctional riboflavin kinase gene. ribF The nucleotide sequence is shown in SEQ ID NO.17 of the sequence listing.

5. The riboflavin-producing strain according to claim 1, characterized in that: The nucleotide sequence of the FMN riboswitch-coupled promoter is shown in SEQ ID NO. 12 of the sequence listing; P trc The nucleotide sequence of the promoter is shown in SEQ ID NO. 14 of the sequence listing; P T7 The nucleotide sequence of the promoter is shown in SEQ ID NO.15 of the sequence listing; the 3,4-dihydroxy-2-butanone-4-phosphate synthase gene ribB The nucleotide sequence is shown in the sequence listing SEQ ID NO.

16.

6. A method for constructing the riboflavin-producing strain according to any one of claims 1-5, characterized in that: In the originating strain E. coli Based on the W3110, targeted modifications were made, resulting in the following three modules: (1) Knockout of purine repressor protein gene purR ; Increase the purine operon gene pur ( EKBCSQLFMNHD The transcriptional level of guanylate kinase was increased; the gene encoding guanylate kinase was enhanced. gmk Transcription level; increase the transcription level of nucleoside diphosphate kinase-encoding genes ndk Transcription level; increase guanylate synthase gene guaA Transcription level; increase in hypoxanthine nucleotide dehydrogenase gene guaB Transcription level; increase glucose-6 phosphate dehydrogenase gene zwf transcriptional levels; (2) Introducing exogenous pyrimidine deaminase genes ribD, Riboflavin synthase gene ribE, Guanosine triphosphate cyclase II gene ribBA and dioxatetrahydropurine synthase gene ribH Knock out the FMN ribonucleoside switch; (3) Replace the bifunctional riboflavin kinase gene with an FMN riboswitch-coupled promoter. ribF The original promoter.

7. The method for constructing the riboflavin-producing strain according to claim 6, characterized in that: The specific steps are as follows: (1) In E. coli W3110 genome yjiV Introducing heterologous purine operon genes at the site pur ( EKBCSQLFMNHD ), by P trc Startup sub-boot; (2) Knockout of purine repressor protein gene purR ; (3) Integration at the molR site in the genome originates from Bacillus subtilis pyrimidine deaminase gene ribD Riboflavin synthase gene ribE Guanosine triphosphate cyclase II gene ribBA and dioxatetrahydropurine synthase gene ribH The genes are arranged in tandem according to the natural gene cluster sequence and are generated by the same P. T7 Promoters drive transcription to form polycistronic expression units; (4) In the genome yeeL Site integration of guanylate kinase gene gmk and nucleoside diphosphate kinase gene ndk , by P trc Startup sub-boot; (5) In the genome ycjV Site integration of guanylate synthase gene guaA and hypoxanthine nucleotide dehydrogenase gene guaB The genes are arranged in tandem according to the natural gene cluster sequence, by P trc Startup sub-boot; (6) Knockout of 3,4-dihydroxy-2-butanone-4-phosphate synthase gene ribB The FMN ribose switch before; (7) Integrating the FMN riboswitch-coupled promoter into the bifunctional riboflavin kinase gene ribF Before that, replace its original promoter; (8) In the genome yghE Site integration originates from Corynebacterium glutamicum The glucose-6 phosphate dehydrogenase gene zwf, by P trc Startup sub-boot.

8. The use of the riboflavin-producing strain according to any one of claims 1-5 in the production of riboflavin.

9. The application according to claim 8, characterized in that: Riboflavin is produced by fermentation in shake flasks or fermenters.

10. The application according to claim 8 or 9, characterized in that: Fermentation is carried out using a fermentation tank. The specific steps are as follows: (1) Activation of strain: The strain was transferred from the glycerol tube to the slant medium and activated for 11-13 hours; the strain was transferred from the slant medium to the eggplant flask medium and activated and expanded for 10-12 hours. The culture temperature was 37℃. (2) Seed culture: During the culture process, the pH was maintained at 7.0±0.2, the temperature at 37±0.2℃, and the dissolved oxygen at 35%-50%, and the culture was carried out until the OD reached 70%. 600nm At 20:00, the cells were transferred to a fermenter for fermentation. (3) Fermentation culture: The seed liquid is inoculated into the fermentation medium for fermentation culture. During the fermentation culture, the pH is controlled at 7.0±0.2, the temperature is maintained at 37±0.2℃, and the dissolved oxygen is maintained at 30%-50%. During the culture, glucose solution is added to maintain the carbon source required by the cells during the fermentation process. During this period, ammonia water is added to adjust the pH so that the pH is maintained at 7.0±0.2.

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