High-riboflavin-producing Bacillus methanolic strain, its construction method and application

Abstract

This invention provides a high-riboflavin-producing Bacillus methanolicis engineered strain, its construction method, and its applications. By constructing a methanol-xylose co-metabolism platform, optimizing the RuMP cycle, reducing riboflavin kinase activity, and regulating purine metabolism, this invention alleviates formaldehyde toxicity in methanol metabolism, improves cell growth stability and riboflavin synthesis efficiency, and achieves efficient riboflavin production using methanol as a carbon source. Ultimately, a riboflavin yield of up to 2579 mg / L was obtained in a 5 L bioreactor. This invention demonstrates significant advantages in strain growth stability, riboflavin synthesis efficiency, and overall production cost, providing a new, efficient, and economical approach for the industrial production of riboflavin.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to an engineered Bacillus methylforminosus strain that produces high levels of riboflavin, its construction method, and its application. Background Technology

[0002] Riboflavin (vitamin B2) is an important water-soluble vitamin with wide applications in food, medicine, feed, and cosmetics. Currently, industrial riboflavin production mainly relies on microbial fermentation, in which… Bacillus subtilis and Ashbya gossypii These are the most commonly used production strains. These strains utilize sugars such as glucose as carbon sources, enhance riboflavin synthesis through metabolic engineering, and optimize fermentation processes to improve production efficiency. In recent years, to reduce raw material costs and achieve sustainable production, researchers have begun exploring the use of methanol as a low-cost, renewable single-carbon source for fermentation production. *Bacillus methanolii* (… Bacillus methanolicus It has advantages such as rapid growth and high temperature tolerance, and it naturally possesses a methanol metabolism pathway. However, traditional methanol strains have the following problems in riboflavin production: when using methanol as the only carbon source, the strain's growth stability is insufficient, and the toxic effects of formaldehyde during methanol metabolism are severe; there is carbon flow competition between methanol metabolism and the riboflavin biosynthesis pathway, resulting in low riboflavin synthesis efficiency.

[0003] The current riboflavin production process based on microbial fermentation has the following main drawbacks: 1. High carbon source cost of traditional strains: Fermentation strains that use glucose or other sugars as the main carbon source are expensive, affecting the economic feasibility of riboflavin.

[0004] 2. Limitations of using methanol as a single carbon source: When methanol is used as the only carbon source, the supply of Ru5P in the cell is insufficient, leading to the accumulation of formaldehyde intermediates and affecting cell growth and metabolic balance.

[0005] 3. Obvious metabolic bottleneck: During methanol metabolism, due to insufficient activity of key enzymes in the RuMP cycle, formaldehyde cannot be converted in time, resulting in toxic effects. At the same time, there is carbon flow competition between C1 metabolism and riboflavin synthesis.

[0006] 4. Insufficient regulation of riboflavin synthesis: The expression level of riboflavin synthesis gene clusters in natural strains is low, and riboflavin is easily converted into FMN / FAD, making it difficult to achieve high accumulation; in addition, the insufficient supply of GTP precursors in purine metabolism further limits the efficiency of riboflavin synthesis. Summary of the Invention

[0007] The purpose of this invention is to provide a high-riboflavin-producing engineered Bacillus methanolicus strain, its construction method, and its application.

[0008] The present invention is conceived as follows: a methanol-xylose co-metabolism strategy is adopted, and the RuMP cycle is optimized by introducing the xylose utilization pathway to assist methanol metabolism, thereby improving the supply of precursors. At the same time, genetic engineering technology is used to optimize the riboflavin biosynthesis pathway, so as to achieve efficient production of riboflavin in methanol culture medium.

[0009] To achieve the objectives of this invention, in a first aspect, this invention provides a method for constructing a high-riboflavin-producing Bacillus methanolicus engineered bacterium, comprising constructing Bacillus methanolicus heterologously expressing xylA and xylB genes (…). Bacillus methanolicus Engineered bacteria I; The xylA and xylB genes are derived from *Bacillus thermoglucosidase* (B. thermoglucosidase). Parageobacillus thermoglucosidasius The reference sequence numbers for xylA and xylB genes in NCBI are BCV53_11585 and BCV53_11590, respectively.

[0010] Furthermore, the xylA and xylB genes were introduced into Bacillus methanolica via plasmid and activated by a xylose-inducible promoter. The promoter drives the expression of xylA and xylB genes; The sequence is shown in SEQ ID NO:1.

[0011] Preferably, the plasmid is introduced into Bacillus methanolicus via electroporation under the following conditions: 2.5 kV / cm, 200 Ω, and 0.1 cm electroporation cup.

[0012] Furthermore, the riboflavin synthesis gene cluster derived from Bacillus pyrogallolase was heterologously expressed in engineered strain I to obtain engineered strain II; The riboflavin synthesis gene cluster derived from *Bacillus pyrogallolus* includes genes. ribD , ribE , ribBA and ribH Their reference sequence numbers in NCBI are: BCV53_16085, BCV53_16080, BCV53_16075 and BCV53_16070.

[0013] Furthermore, a point mutation was introduced into the genome of engineered bacteria I or II, causing the 199th amino acid of the riboflavin kinase RibC encoded by it to be mutated from G to D, thus obtaining engineered bacteria III; The riboflavin kinase RibC has the reference sequence number BCV53_09330 in NCBI.

[0014] In one specific embodiment of the present invention, the method for constructing engineered bacteria III includes the following steps: 1) Construction of the thermosensitive suicide plasmid pBMe01: DNA fragment I containing sfgfp and colE ori was amplified from pUB-sfgfp plasmid using primers pBMe01-V1-F and pBMe01-V1-R; simultaneously, DNA fragment II containing RepB replicon and kanamycin resistance gene was amplified from pZL02 plasmid using primers pBMe01-V2-F and pBMe01-V2-R; after purification, DNA fragments I and II were recombined in vitro using recombinase to obtain plasmid pBMe01; The sequences of the primers pBMe01-V1-F and pBMe01-V1-R are as follows: pBMe01-V1-F: TGTGCTGCAAGGCGATTAA pBMe01-V1-R:CCATTTTGAACGATGACCTC The sequences of the primers pBMe01-V2-F and pBMe01-V2-R are as follows: pBMe01-V2-F:GAGGTCATCGTTCAAAATGGTATGCGTTTTGACACATCCACTAT pBMe01-V2-R:TTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAG; 2) Using Bacillus methanolicis genomic DNA as a template, site-directed mutagenesis primers were designed to introduce the G199D mutation, and the resulting amplified sample containing the site-directed mutation was obtained. ribC The gene fragment was ligated with the plasmid pBMe01 to obtain a recombinant vector. 3) The recombinant vector was introduced into engineered bacteria I or II via electroporation and cultured at 60 °C to obtain engineered bacteria III.

[0015] Furthermore, the key genes involved in GTP synthesis in the engineered bacteria I, II, or III are... purE The promoter was replaced with the strong constitutive promoter pH27 to obtain engineered bacteria IV; The gene purE The reference sequence number in NCBI is BCV53_04710; The sequence of the promoter pH27 is shown in SEQ ID NO:2.

[0016] Preferably, the starting strain used to construct the engineered bacteria is Bacillus methanolicus MGA3 (ATCC 53907).

[0017] Secondly, the present invention provides a high-riboflavin-producing Bacillus methylforminosus engineered strain prepared according to the method described above.

[0018] Thirdly, the present invention provides the application of the engineered bacteria in the production of riboflavin through fermentation or in increasing the yield of riboflavin through fermentation.

[0019] The application includes: fermenting the engineered bacteria and collecting riboflavin from the fermentation products.

[0020] Preferably, the fermentation medium is MVcMY medium containing 150 mM methanol (the medium formula can be found in the literature Brautaset T, Jakobsen MO, Flickinger MC, et al. Journal of Bacteriology, 2004, 186(5): 1229–1238).

[0021] Preferably, the fermentation conditions are: 50°C, stirring speed 300-800 rpm (preferably 500 rpm), aeration rate set at about 2.5 L / min, pH 7.0, and the methanol concentration maintained at 150 mM during fermentation.

[0022] Preferably, in the later stage of fermentation (OD) 600 (Approximately 30%) Xylose is added to the fermentation system (as an auxiliary carbon source) to maintain the xylose concentration at 0.5-10 g / L. The fermentation cycle is 72 hours.

[0023] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention provides a highly efficient riboflavin production method based on Bacillus methanolicus. This method constructs a methanol-xylose co-metabolism platform, optimizes the RuMP cycle, reduces riboflavin kinase activity, and regulates purine metabolism, thereby alleviating formaldehyde toxicity during methanol metabolism, improving cell growth stability and riboflavin synthesis efficiency, and achieving highly efficient riboflavin production. Ultimately, a riboflavin yield of up to 2579 mg / L was obtained in a 5 L bioreactor, significantly reducing production costs. Specifically, (i) Introducing the xylose co-metabolism pathway to optimize the RuMP cycle: Heterologous expression of xylA and xylB genes allows xylose to enter the cell as an auxiliary carbon source, enhancing the supply of Ru5P in the RuMP cycle, thereby alleviating the toxic effects of formaldehyde in methanol metabolism and increasing cell growth rate.

[0024] (ii) Reduce RibC activity to enhance riboflavin accumulation: Using site-directed mutagenesis, the activity of riboflavin kinase RibC is reduced (by introducing the G199D mutation), thereby reducing the conversion of riboflavin to FMN / FAD and thus increasing the accumulation of riboflavin in cells.

[0025] (III) Promoter engineering to optimize riboflavin biosynthesis gene expression: using synthetic promoters Replacing the natural promoter significantly increases the transcriptional levels of riboflavin biosynthesis-related genes (ribD, ribE, ribBA, ribH), thereby increasing riboflavin production.

[0026] (iv) Regulating purine metabolism to enhance precursor supply: By increasing the expression of key genes in purine metabolism through promoter substitution strategies, the production of GTP is increased, providing sufficient precursors for riboflavin biosynthesis.

[0027] (v) System optimization of fermentation process parameters: Determine the optimal fermentation conditions such as 50°C, pH 7.0, aeration rate of about 2.5 L / min and stirring to ensure that the strain maintains high activity and achieves high-density growth and high product accumulation. Attached Figure Description

[0028] Figure 1 In a preferred embodiment of the present invention B. methanolicus Enhanced formaldehyde detoxification and growth during methanol-xylose co-metabolism. (a) B. methanolicus Schematic diagram of formaldehyde detoxification. Increasing the effectiveness of Ru5P through xylose metabolism can enhance formaldehyde detoxification. (b) Promoter optimization of xylose utilization genes (xylA and xylB) in *Methanolacobacterium*. We tested promoters with progressively increasing expression intensity (pL6, pL14, pL20, ...). pH 7, pH 27), select Optimize xylose metabolism. (c) OD600 values ​​under different promoters after 24 hours of growth with xylose as the sole carbon source. (d) B. methanolicus Growth curves on methanol (5 g / L), xylose (5 g / L), and a methanol-xylose mixture (2.5 g / L each). (e) Simultaneous consumption of methanol and xylose during co-metabolism. (f) Molar consumption ratio of methanol to xylose during co-metabolism ranging from 4:1 to 8:1.

[0029] Figure 2 The constitutive promoter in a preferred embodiment of the present invention Sequencing validation at pH 7 and pH 27. (ac) Sequencing results of the pH7 and pH27 promoters confirmed the presence of mutations in the -10 region.

[0030] Figure 3 The preferred embodiment of the present invention is expressed from B. methanolicus (Bmerib) P. thermoglucosidasius (Pthrib) and B. subtilisRiboflavin production of the test strain of (Bsurib) riboflavin biosynthetic cluster. xylAB(+) and xylAB(−) represent the presence and absence of the xylose utilization pathway, respectively.

[0031] Figure 4 This invention provides an analysis of riboflavin production changes in the Pthrib strain, a preferred embodiment of the present invention. Sequencing analysis revealed the deletion of riboflavin biosynthesis gene clusters in low-yielding strains; these included genes ribD, ribE, ribBA, and ribH. The starter is under the control of the starter.

[0032] Figure 5 This is a growth curve of an engineered strain induced by xylose in a preferred embodiment of the present invention. Labeled as pxylA-xylAB-pH27i- The strain indicated co-expression of the xylose metabolism pathway and the riboflavin biosynthesis pathway, while the strain labeled pH27i- The strains shown represent isolated expression of the riboflavin biosynthesis pathway. Pink arrows indicate xylose addition time points (xylose addition). Error bars represent the standard deviation of biological replicates.

[0033] Figure 6 In a preferred embodiment of the present invention, plasmid pBMe03 is used in a strictly regulated promoter. The diagram below shows the gene cluster that co-expresses the xylose metabolic pathway (xylAB) and riboflavin biosynthesis.

[0034] Figure 7 This invention relates to a preferred embodiment of the metabolic pathway alteration in the production of riboflavin from B. methanolicus.

[0035] Figure 8 This invention provides a preferred embodiment for sequencing verification of the RibC G199D mutation.

[0036] Figure 9 The riboflavin yield of strains BmeRF-1 and BmeRF-2 in a preferred embodiment of the present invention is shown. Both carry plasmid pBMe03 and co-express xylAB and the riboflavin biosynthesis gene cluster.

[0037] Figure 10 The successful construction of the BmeRF-2 strain was verified by sequencing in a preferred embodiment of the present invention. The natural promoter of the purine operon was replaced with the constitutive promoter pH27.

[0038] Figure 11 The fermentation results of producing riboflavin in a 5-L bioreactor using methanol and xylose as co-substrate are shown in the preferred embodiment of the present invention. Detailed Implementation

[0039] This invention aims to solve the problems of high carbon source cost, low fermentation stability and metabolic bottleneck of traditional strains, and provides a high-efficiency riboflavin production method based on Bacillus methanolicus.

[0040] The present invention adopts the following technical solution: 1. Constructing a methanol-xylose co-metabolism platform: From Parageobacillus thermoglucosidasius The xylose isomerase (xylA) and xylose kinase (xylB) genes were amplified to construct the recombinant vector pBMe02-xylAB. The recombinant vector was then introduced into the target cell using an optimized electroporation method. B. methanolicus In MGA3, positive clones were screened using a suitable selective medium (containing Kanamycin) and cultured in MVcMY medium containing 150 mM methanol and 50 mM xylose.

[0041] 2. Enhancing the riboflavin synthesis pathway: A site-directed mutagenesis strategy was employed to reduce the activity of riboflavin kinase RibC. Site-directed mutagenesis primers were designed to change glycine (G) to aspartic acid (D) at position 199 of the ribC gene. The mutated fragment was constructed into the temperature-sensitive suicide plasmid pBMe01, and an engineered strain containing the RibC G199D mutation was obtained through double crossover recombination. Simultaneously, the riboflavin biosynthesis gene cluster was optimized through promoter engineering. ribD , ribE , ribBA and ribH The expression of ) is achieved using a synthetic promoter. This drives the efficient expression of these genes.

[0042] 3. Regulating purine metabolism to increase GTP supply: For key genes involved in GTP synthesis, the promoter regions were amplified using high-fidelity PCR, replacing the original promoters with the strong promoter pH27 during amplification. After double enzyme digestion and ligation, a recombinant vector was constructed and transformed into the aforementioned RibC G199D strain, thereby enhancing the expression levels of key purine metabolism genes and increasing the supply of riboflavin precursor (GTP).

[0043] 4. Fermentation process parameter optimization: Large-scale fermentation was carried out in a 5 L bioreactor. The temperature was set at 50 ℃, the pH was controlled at 7.0 (adjusted by automatic addition of 8 N ammonia), the aeration rate was set at 2.5 L / min, and the stirring rate was set at 300-900 rpm, with stirring based on dissolved oxygen at 30%. The fermentation medium was MVcMY methanol medium, supplemented with 150 mM methanol (and xylose as an auxiliary carbon source if necessary), and cultured for 72 hours.

[0044] After adopting the technical solution of this invention, the experimental results show that: 1. Enhanced formaldehyde detoxification ability: By introducing a heteroxylose utilization pathway, the intracellular Ru5P pool was enhanced, thereby effectively promoting the conversion of formaldehyde into the non-toxic intermediate H6P, significantly reducing the impact of formaldehyde toxicity on strain growth, and improving... B. methanolicus Robustness.

[0045] 2. Optimization of the xylose co-metabolism system: The optimized xylose metabolic pathway enabled the strain to utilize the carbon source more efficiently under methanol-xylose co-metabolism conditions. Experimental results showed that this strategy accelerated the growth rate during methanol-xylose co-metabolism, significantly improving the growth rate compared to using methanol alone.

[0046] 3. Increased Riboflavin Production: Co-expression of the riboflavin synthesis gene cluster with the xylose metabolism pathway significantly increased intracellular Ru5P supply, promoting riboflavin synthesis. Compared with strains expressing the riboflavin gene cluster alone, the co-expressed strains exhibited higher riboflavin production (up to 264 mg / L), and their growth rate under xylose induction was not significantly affected.

[0047] 4. Optimization effect of RibC mutation: By introducing the RibC G199D site-directed mutation, the activity of RibC enzyme was reduced, thereby reducing the process of riboflavin being converted into FMN and FAD, increasing the accumulation of riboflavin, and further improving the production capacity of riboflavin.

[0048] 5. Purine metabolism optimization: Replacing the natural promoter of the purine operon with the strong promoter pH27 further enhanced the precursor supply for riboflavin synthesis, thereby improving the efficiency of riboflavin synthesis and increasing riboflavin production by approximately 38.7%.

[0049] 6. Industrial Application Potential: Fermentation experiments in a 5 L bioreactor showed that, through a methanol-xylose co-metabolism strategy, the final riboflavin yield reached 2579 mg / L, which is... B. methanolicus This lays the foundation for the industrial production of riboflavin and demonstrates its great potential as a methanol-based microbial cell factory. At the same time, using methanol as a carbon source reduces dependence on sugar-based carbon sources and improves the economic feasibility of the process.

[0050] In summary, this invention demonstrates significant advantages in terms of strain growth stability, riboflavin synthesis efficiency, and overall production cost, providing a new, efficient, and economical approach for the industrial production of riboflavin.

[0051] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0052] The following examples use P. thermoglucosidasius NCIMB 11955 (CGMCC 1.3473).

[0053] Example 1: Construction and optimization of a xylose co-metabolism platform A major challenge for methyltrophic strains is formaldehyde toxicity, which severely impairs growth during high-concentration methanol or low-concentration pulsed methanol additions. These conditions lead to formaldehyde accumulation, reducing strain robustness. B. methanolicus In this process, formaldehyde detoxification is mainly promoted by Hps enzyme. Hps enzyme catalyzes the reaction of formaldehyde with Ru5P to generate the non-toxic intermediate H6P. Figure 1 a). Thermodynamic analysis based on Gibbs free energy change (ΔG) indicates that increasing the intracellular Ru5P pool can accelerate the conversion of formaldehyde to H6P, thereby reducing formaldehyde toxicity and enhancing robustness. Therefore, we investigated this in xylose, which cannot be naturally metabolized. B. methanolicus Introducing heteroxylose utilization pathway enhances the Ru5P library and promotes formaldehyde detoxification. Figure 1 a).

[0054] The construction process of the xylose metabolism pathway plasmid pBMe02 is as follows: First, the primer pair xylAB-F / R (ACGAGGCCCTTTCGTCGAGCTCCATAAACTTTGTTTGTACACTAGACAAAC / CTTTCGTTTTATTTGATGCCTGCTAGCTCCGAAATGCTTTTAATTTTC) was used to... P. thermoglucosidasius The xylAB gene was amplified from the genomic DNA of NCIMB 11955. Simultaneously, the xylAB gene was amplified from the pZL02 plasmid (provided by Wang Weishan's laboratory at the Institute of Microbiology, Chinese Academy of Sciences). Synth Syst Biotechnol 2024, 9(4), 658-666) used primer pairs pZL02-V1-F / R (CAGGCATCAAATAAAACGAAAGGC / ACGCCAGAGCTGTACAAGTGAGTCGACTCTAG) and pZL02-V2-F / R (GTCGACTCACTTGTACAGCTCTGGCGTAATCATGGTCATAGCTG / GAGCTCGACGAAAGGGCCTCGTG) to amplify two fragments of the plasmid backbone. PCR amplification conditions were: 98°C pre-denaturation for 2 min; 30 cycles (98°C 10 s, 58°C 30 s, 72°C 45 s); final extension at 72°C for 5 min. After purification by agarose gel electroporation, the amplified product was recombined in vitro using NovoRec Plus recombinase and transformed into *E. coli* JM 109 (ATCC 53323) to obtain the pBMe02 plasmid. The plasmid was then introduced into the *E. coli* JM 109 (ATCC 53323) according to the optimized electroporation protocol. B. methanolicus MGA3 was electroporated under the following conditions: 2.5 kV / cm, 200 Ω, and a 0.1 cm electroporation cuvette. After electroporation, 1 mL of preheated TGP medium was added, and the cells were incubated at 50°C for 2 hours before being inoculated onto TSA plates containing 12.5 ng / μL of Kanamycin for selection.

[0055] To optimize the xylose pathway, we first enhanced xylAB expression. We tested a series of constitutive promoters with progressively increasing strength (…). Figure 1 b): pL6: 5′-TACACTAGACACCCACCGTACGCTTTCCTATAATTTAGTTG-3′ pL14: 5′-TACACTAGACAAACAAATTTAACCGCATTATAATTGAGGAC-3′ pL20: 5′-TACACTAGACACAAGTAGTTATGAAGATTATAATTTAGTTG-3′ :5′-TACACTAGACATCCTCGTGTCGGTATTGTATAATTTAGTTG-3′ pH7: 5′-TACACTAGACAGGCATTGTTCTTCACGATATAATTTAGTTG-3′ pH27: 5′-TACACTAGACAAACAAATTTAACCGCATTATAATGAAGATA-3′ Plasmids containing weaker promoters (such as pL6, pL14, and pL20) were successfully constructed in E. coli, but stronger promoters failed due to toxicity associated with xylAB overexpression, as evidenced by mutations in the -10 region of the plasmids during assembly. Figure 2 Of the six selected promoters, pL20, with moderate strength, showed the best performance on xylose-containing medium. B. methanolicus The growth of [the organism] was well supported, indicating that a promoter stronger than pL20 can promote [the growth of the organism]. B. methanolicus growth ( Figure 1 c). To avoid the toxicity problem of E. coli, and at the same time... B. methanolicus Using a stronger promoter, we employed a tightly regulated xylose-inducible promoter. (SEQ ID NO:1) is used to drive xylAB expression. The results indicate that this promoter supports... B. methanolicus Growth using xylose as the sole carbon source ( Figure 1 c).

[0056] We hypothesized that increased Ru5P availability in xylose metabolism would accelerate the RuMP pathway in the methanol-xylose co-metabolism process, leading to faster growth than with methanol alone. Growth experiments confirmed this hypothesis, as the strain grew faster on the methanol-xylose mixture than on methanol alone. Figure 1 d). To confirm that the enhanced growth was due to the co-utilization of methanol and xylose, we measured carbon source consumption. The results showed that growth cessation after 22 hours was attributed to complete carbon source depletion, with simultaneous consumption of methanol and xylose during the co-metabolism. Figure 1 d and Figure 1 e). Furthermore, analysis of the molar consumption ratio of methanol to xylose in the mixed substrates showed a range of 4:1 to 8:1. Figure 1 f). These results indicate that xylose primarily provides Ru5P for formaldehyde detoxification, while methanol remains the main carbon source for growth. The above results demonstrate that the optimized xylose utilization pathway... Driven by the promoter, the detoxification effect of formaldehyde was enhanced, and the growth in the methanol-xylose co-metabolism process was promoted.

[0057] Example 2: Heterologous expression assay of riboflavin synthesis gene clusters from different sources In order to B. methanolicus We modified the strain to produce riboflavin from... P. thermoglucosidasius NCIMB 11955 Bacillus subtilis 168 (ATCC 23857) and B. methanolicus The cloning riboflavin biosynthesis gene cluster in MGA3 ( P. thermoglucosidasius The NCIMB 11955 riboflavin synthesis gene cluster includes genes ribD, ribE, ribBA and ribH, which are numbered BCV53_16085, BCV53_16080, BCV53_16075 and BCV53_16070, respectively. Bacillus subtilis The 168 riboflavin biosynthesis gene cluster includes genes ribD, ribE, ribBA, and ribH, with sequence numbers BSU_23280, BSU_23270, BSU_23260, and BSU_23250, respectively. B. methanolicus The riboflavin biosynthesis gene cluster includes genes ribD, ribE, ribBA, and ribH (sequence numbers BMMGA3_09905, BMMGA3_09900, BMMGA3_09895, and BMMGA3_09890, respectively), and places them in a strictly controlled environment. Expression was performed under a xylose-inducible promoter. These plasmids were then transformed into wild-type strains to construct three test strains: Pthrib, Bsurib, and Bmerib.

[0058] In shake-flask experiments, gene cluster expression during the exponential growth phase induced by 60 mM xylose was used to assess riboflavin production. Among the three tested strains, Bsurib showed the highest production, reaching 175 mg / L. However, the Pthrib strain exhibited significant yield fluctuations across three biological replicates, with riboflavin production ranging from 60 mg / L to 202 mg / L. Figure 3 Sequencing analysis indicated that this difference was due to the loss of the riboflavin biosynthesis gene cluster. Figure 4 Furthermore, the growth rate of the high-yielding Pthrib strains was significantly reduced under xylose-induced conditions. Figure 5 These results indicate that P. thermoglucosidasius The riboflavin biosynthesis gene cluster from this source imposed a high metabolic burden on the host strain, possibly due to a decrease in intracellular Ru5P (ribulose-5-phosphate) levels caused by increased metabolic flux. Ru5P is both a key precursor in riboflavin biosynthesis and an important intermediate metabolite in the RuMP pathway for formaldehyde detoxification; its depletion may trigger metabolic stress.

[0059] Example 3 Co-expression of xylose metabolic pathway and riboflavin gene cluster To alleviate the metabolic burden caused by Ru5P depletion, we co-expressed the xylose metabolism pathway and riboflavin synthesis gene cluster on the same plasmid, constructing the pBMe03 plasmid to enhance the intracellular Ru5P pool. Figure 6 To construct plasmid pBMe03, from... P. thermoglucosidasiusThe riboflavin biosynthesis gene cluster (pthrib) was amplified from NCIMB 11955 using primer pairs. -pthrib-F / R (ACCGCATTATAATGAAGATATAAGCTTAGTTTGTATACCAAACTAAACTAGAAGGAGGAAAAAAGTGCG / TTGATGCCTGGTTTCAATTCTTATTCAGCAAACGCGCGC). Use primers The plasmid backbone was amplified from pBme02 using the pthrib-VF / R (TGAATAAGAATTGAAACCAGGCATCAAATAAAACG / CTTATATCTTCATTATAATGCGGTTAAATTTGTTTGTCTAGTGTACAAACAAAGTTTATGCTAGCTCCGAAATGCTTTTAATTTTCTCG) fragment. These fragments were then assembled into plasmid pBMe03 and transformed into... B. methanolicus Pthrib-xyl strain was obtained from MGA3. Bsurib-xyl and Bmerib-xyl strains were obtained using a similar method.

[0060] Shake-flask experiments showed that the modified Pthrib-xyl strain produced the highest riboflavin yield, reaching 264 mg / L, with good reproducibility. Figure 3 Furthermore, co-expression of the xylose metabolic pathway did not significantly affect the growth rate of the strain under xylose supplementation conditions. Figure 5 These results indicate that co-expression of the xylose metabolic pathway not only increases the supply of Ru5P for formaldehyde detoxification but also provides a sufficient direct precursor for riboflavin biosynthesis. Simultaneously, Ru5P can indirectly promote GTP production, which is another important precursor for riboflavin synthesis. Figure 7 ).

[0061] Example 4: RibC Site-Directed Mutagenesis and Riboflavin Production Test RibC is a bifunctional enzyme encoding riboflavin kinase and FMN adenylate transferase, which converts riboflavin to FMN and FAD, the latter being crucial for cell growth. To promote riboflavin accumulation, we attenuated RibC activity by introducing a G199D mutation. B. methanolicus Using MGA3 genomic DNA as a template, site-directed mutagenesis primers were designed to introduce the G199D mutation, resulting in a site-directed mutagenesis of the ribC gene. The mutated fragment was ligated into the thermosensitive suicide plasmid pBMe01 (containing a 6 kb homologous arm and an sfGFP anti-selection marker) to construct an editing vector. The vector was then electroporated into... B. methanolicusSubsequently, a single-crossover integration strain was obtained by culturing at 50°C with antibiotics, followed by double-crossover recombination at 60°C without antibiotics. Finally, the RibC G199D mutation was successfully introduced into the Pthrib-xyl strain, resulting in the BmeRF-1 strain. Figure 8 BmeRF-1 increased riboflavin production to 301 mg / L during shake-flask fermentation. Figure 9 ).

[0062] The construction process of the thermosensitive suicide plasmid pBMe01 is as follows: First, using primers pBMe01-V1-F / R (TGTGCTGCAAGGCGATTAA / CCATTTTGAACGATGACCTC), the plasmid pUB-sfgfp (provided by Wang Weishan's laboratory at the Institute of Microbiology, Chinese Academy of Sciences) was constructed. Microbial Biotechnology DNA fragments containing the sfgfp and colE ori regions were amplified from pZL02 plasmid (provided by Wang Weishan's laboratory at the Institute of Microbiology, Chinese Academy of Sciences) using primers pBMe01-V2-F / R (GAGGTCATCGTTCAAAATGGTATGCGTTTTGACACATCCACTAT / TTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAG). Synth Syst Biotechnol A DNA fragment containing the RepB replicon and kanamycin resistance gene was amplified in (2024, 9 (4), 658-666). KOD One was used to amplify the fragment. TM Amplification was performed using PCR Master Mix, following the instructions provided with the polymerase. The extension time per kb of DNA was set to 1 min. Specific thermal cycling conditions were as follows: initial denaturation phase at 98 °C for 3 min; denaturation at 98 °C for 10 seconds per cycle, annealing at 60 °C for 5 seconds per cycle, extension at 68 °C for 1 min per kb, and a final extension phase at 68 °C for 3 min. After purification, these two DNA fragments were recombined in vitro using NovoRec Plus recombinase to construct the temperature-sensitive suicide plasmid pBMe01 (sequence shown in SEQ ID NO:3).

[0063] Example 5: Purine metabolism optimization to enhance riboflavin precursor supply To further enhance the precursor supply for riboflavin biosynthesis, we replaced the natural promoter of the purine operon in strain BmeRF-1 with the strong constitutive promoter pH27. High-fidelity PCR was used to amplify the promoter region, and during amplification, designed primers were used to replace the natural promoter with the strong promoter pH27. Figure 10 The sequence is shown in SEQ ID NO:2. The modified strain BmeRF-2 was inoculated into 50 mL of MVcMY medium (supplemented with 150 mM methanol) and cultured in a shake flask at 50°C and 220 rpm for 72 hours. HPLC analysis showed that after promoter replacement, riboflavin production increased from 369 mg / L to 512 mg / L, an increase of approximately 38.7%. Figure 9 This indicates that enhanced purine metabolism significantly improves the supply of riboflavin precursors, thereby further enhancing riboflavin synthesis efficiency.

[0064] Example 6: Production of Riboflavin in a 5-L Bioreactor To evaluate the industrial application potential of this riboflavin production cell factory, we conducted methanol-xylose co-substrate fermentation of strain BmeRF-2 in a 5 L bioreactor. Riboflavin fermentation was carried out in a 5 L fermenter with an initial working volume of 2 L, using MVcMY medium supplemented with 150 mM methanol. The pre-culture process began with 5–10 single colonies inoculated into 50 mL of SOBsuc medium and cultured at 37°C and 250 rpm for 12 hours. Subsequently, the culture was transferred to 50 mL of MVcMY methanol medium at a 10% inoculation ratio and cultured at 50°C for 6 hours. This was then scaled up to four 500 mL Erlenmeyer flasks (50 mL of medium per flask) and cultured for another 6 hours before finally inoculating the fermenter at a 10% inoculation ratio.

[0065] The SOBsuc culture medium was prepared as follows: 20 g / L tryptone, 5 g / L yeast extract, 0.186 g / L potassium chloride, 0.5 g / L sodium chloride and 0.95 g / L magnesium chloride, adjusted to pH 7.0, and then 8.4 g / L sucrose was added after sterilization.

[0066] During fermentation, the aeration rate was set to 2.5 L / min, and the pH was maintained at 7.0 by automatically adding 8 N ammonia. To supplement phosphate and calcium, 1 M KH₂PO₄ and 0.01 M CaCl₂ solutions were used, with the addition rate matched to the ammonia replenishment rate. The fermentation temperature was maintained at 50°C, and the stirring speed was adjusted between 300-900 rpm to ensure the dissolved oxygen concentration remained above 30%. Foam levels were monitored by a level controller and suppressed by automatically adding Antifoam 204 defoamer. Trace metal elements were pre-added to the supplemental methanol solution. Applied and environmental microbiology 1990, 56 (4), 963-970). Methanol concentration was monitored in real time using an online monitoring system and maintained at 150 mM to ensure a continuous supply. Furthermore, during the later stages of fermentation (OD... 600 = 30) Supplement xylose at a constant rate, maintaining a xylose concentration of 0.5-10 g / L, to further support cell growth and riboflavin production.

[0067] Riboflavin concentration was determined using HPLC (YMC Polymer C18 column, mobile phase 60% H2O, 10% methanol, 20% acetonitrile, 10% phosphoric acid, flow rate 1 mL / min, detection wavelength 370 nm). After a final fermentation period of 46 h, the OD... 600 The riboflavin production reached 2579 mg / L, reaching 63. Figure 11 ), is currently B. methanolicus The highest production record for riboflavin.

[0068] The above experimental results demonstrate that the methanol-xylose co-metabolism strategy established in this invention, along with the modification of strain targets and the selection of gene clusters, effectively achieves [the desired results]. B. methanolicus It has significant value in engineering applications, laying the foundation for its broad prospects as a methanol-based microbial cell factory in industrial production.

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for constructing a high-riboflavin-producing engineered Bacillus methanolicus strain, characterized in that, Constructing Bacillus methylformate expressing xylA and xylB genes heterologously ( Bacillus methanolicus Engineered bacteria I; The xylA and xylB genes are derived from *Bacillus thermoglucosidase* (B. thermoglucosidase). Parageobacillus thermoglucosidasius The reference sequence numbers for xylA and xylB genes in NCBI are BCV53_11585 and BCV53_11590, respectively.

2. The method according to claim 1, characterized in that, The xylA and xylB genes were introduced into Bacillus methanolicus via plasmid and were activated by a xylose-inducible promoter. The promoter drives the expression of xylA and xylB genes; The sequence is shown in SEQ ID NO:

1.

3. The method according to claim 2, characterized in that, The plasmid was introduced into Bacillus methylmercurate by electroporation under the following conditions: 2.5 kV / cm, 200 Ω, and 0.1 cm electroporation cup.

4. The method according to any one of claims 1-3, characterized in that, In engineered strain I, a riboflavin synthesis gene cluster derived from thermoglucosidase Bacillus was heterologously expressed to obtain engineered strain II; The riboflavin synthesis gene cluster derived from *Bacillus pyrogallolus* includes genes. ribD , ribE , ribBA and ribH Their reference sequence numbers in NCBI are: BCV53_16085, BCV53_16080, BCV53_16075 and BCV53_16070.

5. The method according to any one of claims 1-4, characterized in that, A point mutation was introduced into the genome of engineered bacteria I or II, causing the 199th amino acid of the encoded riboflavin kinase RibC to be mutated from G to D, thus obtaining engineered bacteria III; The riboflavin kinase RibC has the reference sequence number BCV53_09330 in NCBI.

6. The method according to claim 5, characterized in that, The method includes the following steps: 1) Construction of the thermosensitive suicide plasmid pBMe01: DNA fragment I containing sfgfp and colE ori was amplified from pUB-sfgfp plasmid using primers pBMe01-V1-F and pBMe01-V1-R; simultaneously, DNA fragment II containing RepB replicon and kanamycin resistance gene was amplified from pZL02 plasmid using primers pBMe01-V2-F and pBMe01-V2-R; after purification, DNA fragments I and II were recombined in vitro using recombinase to obtain plasmid pBMe01; The sequences of the primers pBMe01-V1-F and pBMe01-V1-R are as follows: pBMe01-V1-F: TGTGCTGCAAGGCGATTAA pBMe01-V1-R:CCATTTTGAACGATGACCTC The sequences of the primers pBMe01-V2-F and pBMe01-V2-R are as follows: pBMe01-V2-F:GAGGTCATCGTTCAAAATGGTATGCGTTTTGACACATCCACTAT pBMe01-V2-R:TTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAG; 2) Using Bacillus methanolicis genomic DNA as a template, site-directed mutagenesis primers were designed to introduce the G199D mutation, and the resulting amplified sample containing the site-directed mutation was obtained. ribC The gene fragment was ligated with the plasmid pBMe01 to obtain a recombinant vector. 3) The recombinant vector was introduced into engineered bacteria I or II via electroporation and cultured at 60 °C to obtain engineered bacteria III.

7. The method according to any one of claims 1-6, characterized in that, The key genes involved in GTP synthesis in the engineered bacteria I, II, or III purE The promoter was replaced with the strong constitutive promoter pH27 to obtain engineered bacteria IV; The gene purE The reference sequence number in NCBI is BCV53_04710; The sequence of the promoter pH27 is shown in SEQ ID NO:

2.

8. A high-riboflavin-producing Bacillus methanolic strain prepared according to the method of any one of claims 1-7.

9. The application of the engineered bacteria described in claim 8 in the production of riboflavin by fermentation or in increasing the yield of riboflavin by fermentation.

10. The application according to claim 9, characterized in that, The engineered bacteria were fermented and cultured, and riboflavin was collected from the fermentation products; The fermentation medium used was MVcMY medium containing 150 mM methanol; Fermentation conditions were: 50-52°C, stirring speed 300-900 rpm, aeration rate set at 2.5 L / min, pH 6.5-7.2, and methanol concentration maintained at 150 mM during fermentation. Preferably, xylose is added to the fermentation system in the later stage of fermentation to maintain the xylose concentration at 0.5-10 g / L until the fermentation is completed.

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