A bacillus subtilis engineering bacterium for producing riboflavin and application thereof

Abstract

This invention discloses an engineered Bacillus subtilis strain for riboflavin production and its application, belonging to the field of biotechnology; the engineered Bacillus subtilis strain is named ZMBSB H-1, with accession number CCTCC NO: M 20252077; this invention utilizes Bacillus subtilis... Bacillus subtilis Knockout of the genome based on 168 spoOA Genes and ccpA Genes will ribBA‑ribD Mutant gene clusters integrated into spoOA In gene loci, targeted modification purF Mutant gene integration into ccpA In the gene locus, and expressed using the strong promoter P43. ribBA‑ribD Mutant gene clusters and purF The mutated gene was used to construct the Bacillus subtilis engineered strain; the Bacillus subtilis engineered strain can efficiently produce riboflavin using glucose, corn steep liquor powder and yeast extract as substrates.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered Bacillus subtilis strain for producing riboflavin and its applications. Background Technology

[0002] Riboflavin, also known as vitamin B2, is a water-soluble vitamin. While it doesn't directly participate in metabolic processes in living cells, it is a precursor to two key coenzymes: flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These two coenzymes play crucial roles in the catalytic processes of various metabolic enzymes, especially in redox reactions, where they are essential components of the electron transport chain. Therefore, riboflavin is vital for maintaining normal metabolism, participating in multiple physiological processes such as energy metabolism, fatty acid oxidation, amino acid breakdown, and cellular respiration. In nature, riboflavin is widely found in plants and microorganisms, where it can be produced autonomously through endogenous biosynthesis. However, humans and most animals lack this synthetic capacity and must obtain it through diet to meet their physiological needs.

[0003] Currently, riboflavin is mainly produced through chemical synthesis and microbial fermentation. Chemical synthesis uses chemical raw materials and involves multiple reactions to synthesize riboflavin, but it suffers from high production costs, particularly in raw material and energy consumption; potential for harmful byproducts that pollute the environment; and complex processes with long production cycles. Microbial fermentation, on the other hand, directly produces riboflavin by genetically modifying the metabolic pathways of microorganisms. It boasts advantages such as low production costs, short production cycles, environmental friendliness, and fewer byproducts, and is gradually becoming the mainstream production method. Compared to chemical synthesis, microbial fermentation not only reduces energy consumption and raw material costs but also minimizes the generation of harmful byproducts, aligning with the principles of green chemistry and sustainable development.

[0004] With the continuous advancement of synthetic biology and metabolic engineering technologies, the efficiency and yield of microbial riboflavin synthesis have been steadily improving. However, existing technologies still face numerous bottlenecks. For instance, the fermentation process is extremely sensitive to the genetic stability of the strain, the composition of the culture medium, and fermentation conditions, making process control difficult and susceptible to contamination by other microorganisms, resulting in insufficient production reproducibility and large-scale stability. Furthermore, traditional engineered strains still have limitations in carbon source utilization efficiency, precursor supply capacity, and metabolic flux regulation, restricting further increases in riboflavin yield. Therefore, developing novel industrial-scale production strains with high genetic stability, strong environmental adaptability, and efficient synthesis capabilities has become an important research direction in the field of riboflavin biomanufacturing.

[0005] For example, Chinese patent CN117568433A, filed on January 15, 2024, discloses a method for improving the fermentation production of riboflavin by Bacillus subtilis. The recombinant Bacillus subtilis possesses the MribO region, which is regulated by a strong promoter. This strong promoter is pJ23119, PtrnQ, or TP2. This invention eliminates feedback inhibition in the riboflavin synthesis process, increasing the riboflavin potency by 54.8% after 70 hours of fermentation. However, this invention only focuses on regulating gene expression levels and does not involve targeted evolution or modification of the key enzyme catalyzing riboflavin synthesis itself.

[0006] Chinese patent application dated June 30, 2022, publication number CN114958693A, discloses a strain of Bacillus subtilis, a recombinant Bacillus subtilis, and their applications. The described Bacillus subtilis RF1-6 uses the high-riboflavin-producing strain RF1 as the starting strain. Through genetic modification and mutagenesis, a mutant strain with the highest riboflavin yield was obtained and screened. Its accession number is CCTCCNO: M2022565. Compared to the high-riboflavin-producing strain RF1, the riboflavin yield can be increased by 22.8%. This invention uses traditional mutagenesis and partial genetic modification. Although the yield is improved, the method has high randomness and unclear genetic background.

[0007] Based on the above background, the focus is on Bacillus subtilis (B. subtilis) Bacillus subtilis This important industrial host, through research on metabolic pathway reconstruction and key enzyme molecule modification, aims to construct a high-yield riboflavin engineered bacterium that can efficiently utilize inexpensive carbon sources, possesses a stable genetic background, and exhibits superior fermentation performance. This will provide new technical solutions and strain resources for promoting the green biomanufacturing of riboflavin. Summary of the Invention

[0008] To address the problems existing in the prior art, the present invention provides an engineered Bacillus subtilis strain for producing riboflavin and its application. The engineered Bacillus subtilis strain can efficiently produce riboflavin using glucose, corn steep liquor powder and yeast extract as substrates.

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

[0010] One of the objectives of this invention is to provide an engineered Bacillus subtilis strain for producing riboflavin, with accession number CCTCC NO: M 20252077 and named ZMBSB H-1.

[0011] Furthermore, the preservation information for Bacillus subtilis ZMBSB H-1 is as follows:

[0012] Bacterial strain name: Bacillus subtilis

[0013] Latin name:Bacillus subtilis

[0014] Strain number: ZMBSB H-1

[0015] Preservation Institution: China Center for Type Culture Collection

[0016] Abbreviation for depository institution: CCTCC

[0017] Address: Wuhan University, Wuhan, China

[0018] Date of preservation: September 22, 2025.

[0019] Furthermore, the recombinant engineered Bacillus subtilis strain is used to knock out... spoOA Genes and ccpA Bacillus subtilis gene Bacillus subtilis 168 is the host cell, in spoOA Gene locus integration ribBA-ribD Mutant gene clusters, in ccpA Gene locus integration purF Mutant gene.

[0020] Furthermore, the aforementioned ribBA-ribD The mutant gene cluster was obtained by tandem linking the ribBA mutant gene and the ribD mutant gene through the flexible linker (GGGGS)3.

[0021] Furthermore, the nucleotide sequence of the flexible linker (GGGGS)3 is shown in SEQ ID NO. 3.

[0022] Furthermore, the ribBA mutant gene originates from Bacillus subtilis. ribBA The ribBA mutant gene has a nucleotide sequence as shown in SEQ ID NO. 6 and an encoded amino acid sequence as shown in SEQ ID NO. 1. The ribBA mutant gene has a nucleotide sequence as shown in SEQ ID NO. 1.

[0023] Furthermore, the ribD mutant gene originates from Bacillus subtilis. ribD The ribD mutant gene has a mutation at amino acid position 152, Ala, which is changed to Phe, and an amino acid at position 209, Asn, which is changed to Arg. The nucleotide sequence of the mutant gene is shown in SEQ ID NO. 7, and the encoded amino acid sequence is shown in SEQ ID NO. 2.

[0024] Furthermore, the aforementioned purF The mutated gene originated from Bacillus subtilis. pur Gene F The 67th amino acid, Asn, is mutated to Ala, and the 295th amino acid, Ser, is mutated to Gln.purF The nucleotide sequence of the mutant gene is shown in SEQ ID NO. 8; the encoded amino acid sequence is shown in SEQ ID NO. 4.

[0025] Furthermore, the strong starter P43 is used to control the aforementioned ribBA - ribD Mutant gene clusters and purF The mutant gene was expressed, and the nucleotide sequence of the P43 promoter is shown in SEQ ID NO. 5.

[0026] Furthermore, the method for constructing the engineered Bacillus subtilis strain ZMBSB H-1 for producing riboflavin includes the following steps:

[0027] S1, with Bacillus subtilis Bacillus subtilis 168 is the host cell, and Bacillus thuringiensis is knocked out using a knockout plasmid via gene recombination. Bacillus subtilis On the 168 genome [[ID=2X]]spoOA Genes, to remove their influence on the expression of related metabolite genes;

[0028] S2, in spoOA Gene locus insertion ribBA-ribD Mutant gene clusters, co-overexpression of strong promoter P43 ribBA and ribD Riboflavin fermentation engineered bacteria Bacillus subtilis 168 spoOA :: ribBA-ribD ①③;

[0029] S3, engineered bacteria Bacillus subtilis 168 spoOA : :ribBA-ribD ① and ③ are chassis cells, and the strain was knocked out using gene recombination technology. ccpA Genes that alleviate the CCR effect caused by glucose and further increase the riboflavin production of the strain;

[0030] S4, gene encoding PRPP transaminases, a key enzyme in the purine synthesis pathway. purF Conduct targeted modifications, and in ccpA Gene loci expressed using the strong promoter P43 purF The mutant was ultimately used to construct the high-riboflavin-producing Bacillus subtilis engineered strain ZMBSB H-1.

[0031] The second objective of this invention is to provide an application of recombinant engineered Bacillus subtilis in the efficient production of riboflavin using glucose, corn steep liquor powder, and yeast extract as substrates.

[0032] Furthermore, the recombinant engineered Bacillus subtilis strain ZMBSB H-1 was cultured to obtain a seed culture, which was then inoculated into a fermentation medium. During the fermentation process, the pH was adjusted to 6.8-7.2, the fermentation temperature to 40±2℃, and the fermentation time to 36-40h. Riboflavin was produced by fed-feed fermentation using glucose, corn steep liquor powder, and yeast extract as substrates.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1. This invention discloses for the first time the engineered Bacillus subtilis strain ZMBSB H-1, which produces high levels of riboflavin, and its design concept. Firstly, CRISPR-Cas9 technology is used to precisely knock out the spores that control spore formation. spo0A First, by removing the influence of genes on the expression of the target metabolite gene, the metabolic flow is more focused on riboflavin synthesis. Second, targeting the riboflavin synthesis pathway itself, the ribBA and ribD genes in the riboflavin operon are mutated and fused in series using the flexible linker peptide (GGGGS)3 to form... ribBA-ribD Mutated gene clusters reduce steric hindrance and enhance the functional activity of the fusion protein, while the strong promoter P43 is used to integrate it as a whole into... spo0A Co-expression at the gene locus significantly improves the efficiency of riboflavin synthesis through constitutive high expression of the riboflavin operon; finally, to alleviate the glucose-induced CCR effect in cells, this invention simultaneously knocks out... ccpA The gene makes the central metabolism more favorable for riboflavin synthesis, synergistically increasing the riboflavin production of the strain. Furthermore, at this site, targeted modification was performed using P43 promoter overexpression. purF The mutant gene encodes a PRPP transaminases that optimizes the purine synthesis pathway in Bacillus subtilis, increases the supply of the riboflavin precursor GTP, and thus further promotes riboflavin production.

[0035] 2. The engineered Bacillus subtilis strain ZMBSB H-1 designed in this invention exhibits significant advantages in riboflavin fermentation production, mainly reflected in high yield, high efficiency, and high economic efficiency. See Example 6 and... Figure 3 As shown in the fermentation curve, this strain, using inexpensive glucose and corn steep liquor powder as the main substrates in a 100-liter scale-up fermenter and employing a fed-feedback process, achieved a riboflavin yield of up to 38 g / L in just 36 hours of fermentation. This yield level demonstrates its ideal synthetic capacity. Furthermore, compared to the traditional 70-hour fermentation cycle required by conventional processes, this strain can complete the process in 36-40 hours, significantly improving production efficiency and reducing energy consumption.

[0036] 3. The innovative Bacillus subtilis engineered strain ZMBSB H-1 of this invention provides simple application conditions for riboflavin production, with mild fermentation process control, easily achievable pH and temperature levels, and relatively relaxed dissolved oxygen requirements, which is beneficial for stable control during industrial scale-up. Precise detection of the fermentation broth using HPLC further verifies the effective synthesis of the product and the reliability of the method. The Bacillus subtilis engineered strain ZMBSB H-1 described in this invention has successfully achieved excellent production performance through systematic genetic modification, possessing advantages such as low production cost, environmental friendliness, and ease of large-scale control, and has broad application prospects in the feed, pharmaceutical, and food industries. Attached Figure Description

[0037] Figure 1 The recombinant plasmid pJOE8999 constructed in this invention - Schematic diagram of the structure of spoOA::ribBA-ribD;

[0038] Figure 2 The recombinant plasmid pJOE8999 constructed in this invention - Schematic diagram of the structure of ccpA::purF;

[0039] Figure 3 The fermentation curve of the engineered Bacillus subtilis strain ZMBSB H-1 described in this invention in a 100 L fermenter;

[0040] Figure 4 This is a liquid phase diagram showing the riboflavin content in the fermentation broth of the Bacillus subtilis engineered strain ZMBSB H-1 described in 100L of this invention. Detailed Implementation

[0041] The present invention will be further described below with reference to preferred embodiments. The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values. These ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed herein.

[0042] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] In the following examples, "codon optimization" refers to gene redesign that utilizes preferred codons and avoids codons with low utilization or rarity. Every organism exhibits some degree of codon utilization difference or preference, with the most frequently used codons being preferred codons.

[0045] Molecular biology experiments not specifically described in the following examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, which were mainly performed in accordance with *Molecular Cloning: A Laboratory Manual* (3rd edition). PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier.

[0046] The whole-genome synthesis, primer synthesis, and sequencing in the following examples were performed by Shanghai Sangon Biotech Co., Ltd. Host strain: Bacillus subtilis Bacillus subtilis 168 was purchased from Hangzhou Hongsai Biotechnology Co., Ltd.; the starting vector pJOE8999 in the following examples was purchased from Shanghai Haijihaoge Biotechnology Co., Ltd.

[0047] Table 1 Primer sequences in the following examples

[0048]

[0049] The culture medium formulations used in the following examples are as follows:

[0050] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride. For solid medium, add 20 g / L agar powder. Autoclave at 121°C for 20 min.

[0051] GM medium: LB + 0.5 mol / L sorbitol + 0.38 mol / L mannitol

[0052] RM resuscitation medium: LB + 0.5 mol / L sorbitol + 0.38 mol / L mannitol + 0.5 mol / L trehalose

[0053] ETM electroporation solution: LB + 0.5 mol / L sorbitol + 0.5 mol / L mannitol + 0.5 mol / L trehalose + 10% glycerol

[0054] Slant culture medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 5g / L, maltose 20g / L, agar powder 20g / L, pH 7.0, autoclaved at 118℃ for 15min.

[0055] Primary seed shake flask culture medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 5g / L, maltose 20g / L, agar powder 20g / L, pH 7.0, autoclaved at 118℃ for 15min.

[0056] Secondary seed culture medium: 20 g / L corn steep liquor powder, 40 g / L white sugar, 5 g / L magnesium sulfate, 5 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 3 g / L dipotassium hydrogen phosphate, 5 g / L yeast extract, pH 7.0, autoclaved at 121℃ for 25 min.

[0057] Fermentation medium: 40 g / L corn steep liquor powder, 20 g / L glucose, 0.5 g / L magnesium sulfate, 1.7 g / L betaine, 0.73 g / L potassium dihydrogen phosphate, 2.5 g / L dipotassium hydrogen phosphate, 50 g / L yeast extract, 0.02 g / L zinc sulfate, pH 7.0, autoclaved at 121℃ for 25 min.

[0058] Method for detecting riboflavin in fermentation broth: Take fermentation broth and dilute it to an appropriate ratio with 0.01 mol / L NaOH. Stir with a magnetic stirrer. After the alkali dissolves and crystallizes, centrifuge at 12000 rpm for 2 min. Take the supernatant and filter it through a 0.22 μm filter membrane. Detect the riboflavin content by high performance liquid chromatography (HPLC). The HPLC parameters are as follows: mobile phase is 100% methanol and 0.12% acetic acid water in a ratio of 28:72, flow rate is 1 mL / min, UV detector, detection wavelength is 269 nm, chromatographic column is C18, column temperature is 30 degrees, and injection volume is 10 μL.

[0059] Example 1

[0060] This embodiment provides the recombinant plasmid pJOE8999- spoOA :: ribBA-ribD The method for constructing (original and mutant gene clusters) includes the following steps:

[0061] I. Mutant Gene Clusters ribBA - ribD Selection of mutation sites

[0062] (1) The information of ribBA and ribD proteins was submitted to the website for calculation using the HotSpot Wizard 3 web server. The top two optional mutation sites with higher scores were obtained from the results of functional hotspots represented by highly variable residues in catalytic pockets and / or channels and stability hotspots represented by flexible residues in protein engineering strategies. Among them, ribBA had Ser at position 60 mutated to Thr, Met at position 170 mutated to Ala, Val at position 252 mutated to Ile, and Pro at position 271 mutated to Glu; ribD had Gly at position 18 mutated to Gro, Ala at position 152 mutated to Phe, Asn at position 209 mutated to Arg, and Val at position 210 mutated to Ile.

[0063] (2) Constructing by combining functional mutations and stable mutation sites respectively. ribBA - ribD The mutated gene cluster consists of four genes synthesized by bioengineering. ribBA ① The Met at position 170 is mutated to Ala, and the Pro at position 271 is mutated to Glu (SEQ ID NO. 6 nucleotide sequence) ② The Ser at position 60 is mutated to Thr, and the Val at position 252 is mutated to Ile; ribD ③ The Ala at position 152 is mutated to Phe, and the Asn at position 209 is mutated to Arg (SEQ ID NO. 7 nucleotide sequence). ④ The Gly at position 18 is mutated to Gro, and the Val at position 210 is mutated to Ile. The mutant gene clusters are obtained by linking them together using a linker. ribBA-ribD ①③、 ribBA-ribD ①④、 ribBA-ribD ②③ and ribBA-ribD ②④;

[0064] II. The pJOE8999 plasmid backbone and spoOA -up-p43- ribBA-ribD - spoOA -do fragment concatenation conversion

[0065] (1) Design using the CHOPCHOP website (http: / / chopchop.cbu.uib.no / ) spoOA The gene's specific target is 20 bp. PCR amplification was performed using primers spoOA-sgRNA-F / R with pJOE8999 plasmid as a template. The amplified product was digested with the restriction endonuclease DpnI to eliminate the template. Homologous recombination was then performed according to the instructions of the seamless ligation kit (Novazan) to transform DH5α competent cells, constructing the pJOE8999- knockout plasmid. spoOA ;

[0066] (2) pJOE8999- spoOA Using plasmids as templates, homologous recombination linearized vector fragments were obtained by PCR amplification with primers JOE-F / R, and the corresponding vector backbone fragments were recovered using the Tiangen Gum Recovery Kit.

[0067] (3) Incubate in LB liquid medium at 37°C and 220 rpm. Bacillus subtilis 168, extracted using the Tiangen Bacterial Genome Extraction Kit Bacillus subtilis 168 genomes were amplified using primers spoOA-up-F / R and spoOA-dF / R, with the genome as a template. spoOAThe 500bp DNA fragments upstream and downstream of the gene were used to perform nucleic acid electrophoresis on the PCR product and the two DNA fragments spoOA-up and spoOA-d were recovered by gel extraction.

[0068] (4) with Bacillus subtilis The 168 genome and the mutant synthesized by Shanghai Sangon Biotech were respectively... ribBA and ribD Using the gene as a template, primers ribBA-F / ribBA-linker-R and ribD-linker-F / ribD-R were used to amplify and the ribBA-linker and ribD-linker fragments containing the linker and homologous recombination with the promoter P43 and spoOA-d DNA fragments were recovered using a kit.

[0069] (5) Using primers P43-BAD-F / R, PCR amplification was performed using the laboratory-preserved pP43NMK vector as a template to obtain a sample containing pP43NMK. spoOA DNA fragment of the P43 promoter arm of the gene upstream homologous arm;

[0070] (6) The pJOE8999 backbone fragment was combined with spoOA-up, P43, ribBA-linker, ribD-linker and spoOA-d fragments according to the instructions of the seamless ligation kit (Novazan); the recombinant product was added to 100µL of E. coli DH5α competent cells, the tube was gently tapped to mix, placed on ice for 30min, heat-shocked in a 42℃ water bath for 45s, and immediately placed on ice for 2min. 500µL of liquid culture medium was added, and the cells were shaken and cultured at 37℃ for 1h; after the culture was completed, 100µL of the culture was spread evenly on a Kan resistance plate, and incubated upside down in a 37℃ incubator for 12h. Single colonies were picked for colony PCR verification and then sent to Sangon Biotech for sequencing; the correctly sequenced bacteria were cultured overnight in liquid shaker, and the plasmid was extracted using a plasmid extraction kit (Tiangen) to obtain the recombinant plasmid pJOE8999- spoOA :: ribBA-ribD A total of 5 plasmids (original and mutant gene clusters).

[0071] Example 2

[0072] This embodiment provides Bacillus subtilis. Bacillus subtilis 168 genome spoOA Gene knockout and ribBA-ribD The method for co-expressing (original and mutant gene clusters) includes the following steps:

[0073] one, Bacillus subtilis Preparation and plasmid transformation of 168 competent cells

[0074] (1) Activate the strain stored at -80℃ by streaking on LB plates and incubating at 37℃;

[0075] (2) Pick a single colony from the LB plate and inoculate it into a 5 mL LB tube. Incubate overnight at 37°C and 220 rpm.

[0076] (3) Inoculate 5% of the culture medium into 50 mL of GM medium and culture until the logarithmic phase;

[0077] (4) Incubate the bacterial culture on ice for 30 minutes, and collect the bacterial cells using a refrigerated centrifuge at 4°C, 4000 rpm for 10 minutes;

[0078] (5) Wash the bacterial cells twice with pre-cooled deionized water and ETM respectively;

[0079] (6) Resuspend the bacterial cells in 500 μL of ETM and dispense 50 μL into each tube;

[0080] (7) Add 1ug pJOE8999 to 50uL of competent cells. [[ID=XX]]spoOA :: ribBA-ribD The plasmids (original and mutant gene clusters) were gently mixed and transferred to pre-cooled 2mm-spaced electrode cups and incubated on ice for 5 minutes.

[0081] (8) Wipe the surface of the electric shock cup to remove moisture, and set the electric shock parameters as follows: 2.5kV, 200Ω, 25uF, electric shock 5ms;

[0082] (9) Immediately after the electric shock, add 1 mL of RM medium, incubate at 37°C and 150 rpm for 3 h, then spread the bacterial solution onto LB plates containing 0.2% mannose and 5 μg / mL kanamycin, and incubate upside down in a 30°C incubator for about 2 days.

[0083] two, spoOA Gene knockout and ribBA-ribD Co-expression verification of mutant gene clusters

[0084] (1) Pick a single colony from the LB plate and put it into 10 uL of deionized water and lyse it at 95℃ for 5-10 min;

[0085] (2) Using cross primers spoOA-up-F / spoOA-dR; P43-BAD-F / ribD-R, take 1 μL of the above bacterial lysate as a template. Bacillus subtilis DNA amplification was performed by colony PCR using the 168 genome as a control template.

[0086] (3) The PCR products were initially verified by electrophoresis, and single colonies with the correct band size were inoculated into LB tubes for overnight culture.

[0087] (4) Take an appropriate amount of bacterial culture and extract its genome according to the instructions of the bacterial genome extraction kit. Use the genome as a template and verify it again using cross primers.

[0088] (5) After electrophoresis verification, the PCR products are sent to Sangon Biotech for sequencing. The correctly sequenced strain is the... [[ID=XX]]spoOA The gene has been knocked out and ribBA-ribD Gene clusters (original and mutant gene clusters) co-express engineered Bacillus subtilis Bacillus subtilis 168 spoOA ::ribBA - ribD, Bacillus subtilis 168 spoOA :: ribBA-ribD ①③、 Bacillus subtilis 168 spoOA :: ribBA-ribD ①④、 Bacillus subtilis 168 spoOA :: ribBA-ribD ②③ and 168 Bacillus subtilis :: spoOA ribBA- ②④;

[0089] III. Bacillus subtilis ribD 168 Bacillus subtilis :: spoOA Elimination of plasmids in strains containing both original and mutated gene clusters

[0090] (1) pJOE8999 contains a temperature-sensitive replication origin from pE194ts, and its replication ability is significantly reduced at higher temperatures, resulting in the plasmid being unable to maintain stability.

[0091] (2) ribBA-ribD 168 Bacillus subtilis :: spoOA , ribBA-ribD 168 Bacillus subtilis :: spoOA ①③、 ribBA-ribD 168 Bacillus subtilis :: spoOA ①④、 ribBA-ribD 168 Bacillus subtilis :: spoOA ②③ and ribBA-ribD 168 Bacillus subtilis :: spoOA Strains ② and ④ were streaked onto LB plates without antibiotics and incubated overnight at 50°C.

[0092] (3) Spot the single colonies on the plate onto non-resistant LB plates again and incubate overnight at 42°C in an incubator;

[0093] (4) Pick a single colony from the plate and streak it parallel to the Kan plate and the non-resistant plate. The strain that grows on the non-resistant plate and does not grow on the Kan plate is the one whose plasmid has been eliminated.

[0094] Example 3

[0095] This embodiment provides recombinant riboflavin-producing Bacillus subtilis. ribBA-ribD Bacillus subtilis It should be noted that there seems to be a duplicate "spoOA" tag with ID "20" and ID "21" and some other potential issues in the original text which might need further clarification for a more accurate translation. Also, there are some "XX" in the translation which are placeholders for the original text where the ID might be incorrect or there are some unclear elements. 168 spoOA::ribBA - The screening method for ribD (original and mutant gene clusters) engineered strains includes the following steps:

[0096] (1) Bacillus subtilis 168 spoOA :: ribBA-ribD , Bacillus subtilis 168 spoOA :: ribBA-ribD ①③、 Bacillus subtilis 168 spoOA :: ribBA-ribD ①④、 Bacillus subtilis 168 spoOA :: ribBA-ribD ②③ and Bacillus subtilis 168 spoOA :: ribBA-ribD ②④ Five recombinant Bacillus subtilis strains were inoculated on slant agar and cultured overnight at 37°C. The bacterial cells were resuspended in 7 mL of sterile water and then inoculated into 50 mL of fermentation shake flask medium at a 2% inoculation rate. The culture was carried out at 37°C and 180 rpm for 16 h.

[0097] (2) Take 1 mL of fermentation broth and dilute it to an appropriate ratio with 0.01 mol / L NaOH. Stir with a magnetic stirrer. After the alkali dissolves and crystallizes, centrifuge at 12000 rpm for 2 min. Take the supernatant and filter it through a 0.22 μm filter membrane. Detect the riboflavin content by high performance liquid chromatography (HPLC). The HPLC parameters are as follows: mobile phase is 100% methanol and 0.12% acetic acid water in a ratio of 28:72, flow rate is 1 mL / min, UV detector, detection wavelength is 269 nm, chromatographic column is C18, column temperature is 30 degrees, injection volume is 10 μL. The results of shaking flask are shown in Table 2.

[0098] Table 2. Recombinant riboflavin-producing Bacillus subtilis 168 Riboflavin content of spoOA::ribBA-ribD (original and mutant gene cluster) engineered strain after 16 h of shake-flask fermentation

[0099]

[0100] Example 4

[0101] This embodiment provides recombinant Bacillus subtilis. Bacillus subtilis 168 spoOA : :ribBA-ribD ①③ ccpA :: purF The method for constructing (original and mutant genes) includes the following steps:

[0102] one, purF Prediction of mutant gene sites

[0103] (1) The purF protein information was submitted to the website for calculation using the HotSpot Wizard 3 web server. The top two optional mutation sites with higher scores were obtained from the results of the functional hotspots represented by highly variable residues in the catalytic pocket and / or channels in the protein engineering strategy and the stability hotspots represented by flexible residues. The purF protein was mutated from Gly at position 30 to Ser, from Asn at position 67 to Ala, from Ser at position 295 to Gln, and from Ser at position 385 to Ala.

[0104] (2) Starting from functional and stability mutations, two genes were synthesized by bioengineering. purF ⑤ The Asn at position 67 is mutated to Ala, and the Ser at position 295 is mutated to Gln (nucleotide sequence as shown in SEQ ID NO. 8). ⑥ The Gly at position 30 is mutated to Ser, and the Ser at position 385 is mutated to Ala.

[0105] two, Bacillus subtilis 168 spoOA :: ribBA-ribD ①③Preparation of competent cells and plasmid transformation

[0106] (1) Construct the recombinant plasmid pJOE8999- using the corresponding primers according to the method in Example 1. ccpA :: purF (Original and mutated genes);

[0107] (2) Activate the strain stored at -80℃ by streaking on LB plates and incubating at 37℃;

[0108] (3) Pick a single colony from the LB plate and inoculate it into a 5 mL LB tube. Incubate overnight at 37°C and 220 rpm.

[0109] (4) Inoculate 5% of the culture medium into 50 mL of GM medium and culture until the logarithmic phase;

[0110] (5) Incubate the bacterial culture on ice for 30 minutes, and collect the bacterial cells using a refrigerated centrifuge at 4°C, 4000 rpm for 10 minutes;

[0111] (6) Wash the bacterial cells twice with pre-cooled deionized water and ETM respectively;

[0112] (7) Resuspend the bacterial cells in 500 μL of ETM and dispense 50 μL into each tube;

[0113] (8) Add 1ug pJOE8999- to 50uL of competent cells ccpA :: purF Gently mix the plasmids and transfer them to pre-cooled electrode cups with a 2 mm spacing. Incubate in an ice bath for 5 minutes.

[0114] (9) Wipe the surface of the electric shock cup to remove moisture, and set the electric shock parameters as follows: 2.5kV, 200Ω, 25uF, electric shock 5ms;

[0115] (10) Immediately after the electric shock, add 1 mL of RM medium, incubate at 37℃ and 150 rpm for 3 h, take the bacterial solution and spread it on 0.2% mannose, 5 ug / mL Kan resistance LB plate, and incubate upside down in a 30℃ incubator;

[0116] three, ccpA Gene knockout and purF Site-specific insertion of (original and mutated) genes;

[0117] (1) Pick a single colony from the LB plate and put it into 10 uL of deionized water and lyse it at 95℃ for 5-10 min;

[0118] (2) Use cross primers ccpA -up-F / ccpA -dR; P43-purF-F / purF-R, use 1 μL of the above bacterial lysate as a template. Bacillus subtilis 168 (重复部分直接保留原文,下同) :: spoOA ①③Using the genome as a control template, colony PCR was performed to amplify DNA;

[0119] (3) The PCR products were initially verified by electrophoresis, and single colonies with the correct band size were inoculated into LB tubes for overnight culture.

[0120] (4) Take an appropriate amount of bacterial culture and extract its genome according to the instructions of the bacterial genome extraction kit. Use the genome as a template and verify it again using cross primers.

[0121] (5) After electrophoresis verification, the PCR products are sent to Sangon Biotech for sequencing. The correctly sequenced strain is the... ribBA-ribD The gene has been knocked out and ccpA Bacillus subtilis engineered bacteria with mutated gene overexpression purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA , purF 168 Bacillus subtilis :: spoOA ribBA- ①③ ribD :: ccpA ⑤ and purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA ⑥;

[0122] IV. Recombinant Bacillus subtilis purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA Elimination of plasmids in engineered strains (both original and mutated genes)

[0123] (1) purF 168 Bacillus subtilis ::spoOA ①③ ribBA-ribD :: ccpA , purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA ⑤ and purF Bacillus 168 subtilis :: spoOA ①③ ribBA-ribD :: ccpA ⑥ The strain was streaked onto LB plates without antibiotics and incubated overnight at 50°C.

[0124] (2) Spot the single colonies on the plate onto non-resistant LB plates again and incubate overnight at 42°C in an incubator;

[0125] (3) Pick a single colony from the plate and streak it parallel to the Kan plate and the non-resistant plate. The strains that grow on the non-resistant plate but do not grow on the Kan plate are the strains whose plasmids have been eliminated.

[0126] Example 5

[0127] This embodiment provides recombinant Bacillus subtilis with high riboflavin production. purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA , purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA ⑤ and purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA ⑥ The strain screening method includes the following steps:

[0128] According to Example 3 purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA , purF168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA ⑤ and purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA ⑥ Three recombinant Bacillus subtilis strains were inoculated on slant culture medium and cultured overnight at 37°C. The bacterial cells were resuspended in 7 mL of sterile water and then inoculated into 50 mL of fermentation shake flask culture medium at a 2% inoculation rate. The culture was carried out at 37°C and 180 rpm for 16 h. The results of the shake flask culture are shown in Table 3.

[0129] Table 3 purF 168 Bacillus subtilis : spoOA ①③ :ribBA-ribD :: ccpA Riboflavin content after 16 h of shake-flask fermentation (original and mutant genes)

[0130]

[0131] The selected Bacillus subtilis purF 168 Bacillus subtilis :: spoOA ①③ ribBA-ribD :: ccpA ⑤ It was named ZMBSB H-1.

[0132] ZMBSB H-1 was deposited at the China Center for Type Culture Collection (CCTCC) on September 22, 2025, with accession number CCTCC NO: M 20252077. The deposit address is Wuhan University, Wuhan, China, 430072, China.

[0133] Example 6

[0134] This embodiment provides a method for producing riboflavin by fermentation of Bacillus subtilis ZMBSB H-1 in a 100 L fermenter, including the following steps:

[0135] I. Bacillus subtilis ZMBSB H-1 slant and primary seed shake flask culture

[0136] Bacillus subtilis ZMBSB H-1 monoclonal inoculation was carried out on slant medium and cultured overnight at 37°C. The bacterial cells were resuspended in 7 mL of sterile water and then inoculated into seed shake flasks containing seed shake flask medium at a 2% inoculation rate. The culture was carried out at 37°C and 180 rpm for 7-16 h to obtain the primary seed culture.

[0137] II. Secondary seed culture of Bacillus subtilis ZMBSB H-1

[0138] The primary seed culture was inoculated into a 3L fermenter containing secondary seed culture medium at a transfer rate of 8%. The fermenter pressure was controlled at 0.05MPa, dissolved oxygen (DO) ≥20%, and cultured at 37±0.5℃ for 13h. The riboflavin titer was 800~1200, thus obtaining the secondary seed culture.

[0139] III. Production of Riboflavin from Bacillus subtilis ZMBSB H-1 Fermentation in a 100 L Fermenter

[0140] The secondary seed culture was inoculated at a rate of 10% into a 100 L (30 L) fermenter containing fermentation medium. The fermentation was carried out at 40℃±2℃ and a pressure of 0.05~0.07 MPa. DO ≥ 20% was maintained by adjusting the turbine rotation speed and aeration rate. OD was continuously monitored. 600 To control residual sugar content, a glucose solution of 600 g / L is automatically added to the fermenter, maintaining the glucose content of the fermentation system between 5 g / L and 12 g / L. The pH during fermentation is controlled between 6.8 and 7.2. The fermentation process curve is shown below. purF As shown;

[0141] IV. Detection of Riboflavin Content in Bacillus subtilis ZMBSB H-1 Produced in a 100 L Fermenter

[0142] Every 4 hours, samples were taken from the fermenter. The fermentation broth was diluted to an appropriate concentration with 0.01 mol / L NaOH, stirred with a magnetic stirrer, and centrifuged at 12000 rpm for 2 minutes after the alkali dissolved and crystallized. The supernatant was then filtered through a 0.22 μm filter membrane, and the riboflavin content in the diluted fermentation broth was determined according to the riboflavin liquid chromatography method. The HPLC results are as follows: Figure 3 Figure 4 As shown, Bacillus subtilis ZMBSB H-1 can reach 38 g / L after fermentation in a 100 L fermenter for 36 h.

[0143] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention's specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A recombinant engineering bacterium of Bacillus subtilis (B. subtilis) for producing riboflavin, characterized in that, Bacillus subtilis ) the bacterium comprises a nucleotide sequence of SEQ ID NO: 1, and The preservation number is CCTCC NO: M 20252077, and the name is ZMBSB H-1.

2. The recombinant engineering bacteria of Bacillus subtilis for riboflavin production according to claim 1, characterized in that, The Bacillus subtilis recombinant engineering bacteria are constructed by knocking out spoOA the genes of ccpA Bacillus subtilis Bacillus subtilis 168 as a host cell, in spoOA the gene site integration ribBA-ribD a mutant gene cluster, in ccpA the gene site integration purF a mutant gene.

3. The recombinant engineering bacteria of Bacillus subtilis for riboflavin production according to claim 2, characterized in that, The ribBA-ribD The mutant gene cluster is obtained by concatenation of the ribBA mutant gene and the ribD mutant gene via a flexible linker (GGGGS)3.

4. The recombinant engineering bacteria of Bacillus subtilis for producing riboflavin according to claim 3, characterized in that, The nucleotide sequence of the flexible linker (GGGGS)3 is shown in SEQ ID NO.

3.

5. The recombinant engineering bacteria of Bacillus subtilis for riboflavin production according to claim 3, characterized in that, The ribBA mutant gene is derived from Bacillus subtilis ribBA The 170th amino acid Met in the gene is mutated into Ala, and the 271st amino acid Pro is mutated into Glu; the nucleotide sequence of the ribBA mutant gene is shown as SEQ ID NO. 6; and the encoded amino acid sequence is shown as SEQ ID NO.

1.

6. The recombinant engineering bacteria of Bacillus subtilis for riboflavin production according to claim 3, characterized in that, The ribD mutant gene is derived from a B. subtilis ribD The 152th amino acid Ala of the gene is mutated into Phe, and the 209th amino acid Asn is mutated into Arg; the nucleotide sequence of the ribD mutant gene is shown as SEQ ID NO. 7; and the encoded amino acid sequence is shown as SEQ ID NO.

2.

7. The recombinant engineering bacteria of Bacillus subtilis for riboflavin production according to claim 2, characterized in that, The purF The mutant gene is derived from the pur The gene F The 67th amino acid Asn is mutated into Ala, and the 295th amino acid Ser is mutated into Gin, and the purF The nucleotide sequence of the mutant gene is shown as SEQ ID NO. 8; the encoded amino acid sequence is shown as SEQ ID NO.

4.

8. The recombinant engineering bacteria of Bacillus subtilis for riboflavin production according to claim 2, characterized in that, Controlled by strong starter P43 ribBA - ribD Mutant gene clusters and purF The mutant gene was expressed, and the nucleotide sequence of the P43 promoter is shown in SEQ ID NO.

5.

9. The application of the recombinant engineering bacteria of Bacillus subtilis for producing riboflavin according to any one of claims 1 to 8 in the production of riboflavin with glucose, corn syrup dry powder and yeast extract as substrates.

10. The use of the recombinant engineering bacteria of Bacillus subtilis for producing riboflavin according to claim 9, characterized in that, The seed liquid is obtained by culturing the recombinant engineering bacteria of Bacillus subtilis ZMBSB H-1, the seed liquid is inoculated into the fermentation medium, the pH in the fermentation process is controlled to be 6.8-7.2, the fermentation temperature is 40±2℃, the fermentation time is 36-40 h, and riboflavin is obtained by feedback fed-batch fermentation production with glucose, corn syrup dry powder and yeast extract as substrates.

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