CN114854780A - Method for efficiently synthesizing riboflavin based on balanced gene expression - Google Patents

Abstract

The invention discloses a method for efficiently synthesizing riboflavin based on balanced gene expression, belonging to the technical field of synthetic biology. The present invention modulates the expression of the mCherry and egfp genes within the operon by generating a library of tunable intergenic regions (TIGR) and confirms the relative expression of the two reporter genes. The TIGR library can coordinate the expression rate of the reporter gene in Escherichia coli to be more than 180 times, and the expression rate in Bacillus subtilis to be more than 70 times. TIGR libraries were used to modulate the expression of zwf, ribBA and ywlf genes to increase riboflavin biosynthesis. And (3) rapidly screening the optimal combination mutant by using a 96-well plate according to the fluorescence characteristic of the riboflavin. The best engineering bacteria are screened, the yield of the riboflavin is 2.7g/L, and the riboflavin is increased by 64.35 percent in a shake flask. Finally, in fed-batch fermentation, the riboflavin titer increased 59.27% to 11.77 g/L. It is helpful for the industrialized production of the bacillus subtilis riboflavin and related products.

Description

A method for efficient synthesis of riboflavin based on balanced gene expression

technical field

The invention relates to a method for efficiently synthesizing riboflavin based on balanced gene expression, in particular to a strategy for efficiently synthesizing riboflavin by balancing gene expression in an mRNA engineering operon, and belongs to the technical field of synthetic biology.

Background technique

Riboflavin (VB2) is an essential nutrient for humans and animals and is widely used in pharmaceuticals, food additives and cosmetics. Bacillus subtilis is a Gram-positive bacterium with excellent riboflavin production performance through a "classical" strain improvement strategy. Precursor supply is considered to be the major limiting factor for riboflavin biosynthesis. In Bacillus subtilis, glucose-6-phosphatase dehydrogenase (encoded by zwf) and 6-phosphogluconate dehydrogenase (encoded by gnd) convert glucose-6-phosphate to the nucleus via the pentose phosphate (PP) pathway Keto-5-phosphate. Riboflavin is synthesized from the immediate precursors ribose 5-phosphate and guanosine triphosphate (GTP) via the riboflavin synthesis pathway, catalyzed by a series of enzymes encoded by the rib operon. The gene ribBA encodes a bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase that catalyzes the conversion of ribulose-5-phosphate and 2,5-diamino- 6-ribosamino-4(3H)-pyrimidinone-50-phosphate (DARPP) was synthesized into 3,4-dihydroxy-2-butanone 4-phosphate (DHPB). Increasing the activity of the enzyme encoded by the gene ribBA increased the riboflavin titer by 25%. Phosphoribose pyrophosphate (PRPP) is a precursor of GTP formed in cells via the de novo purine pathway, first converted to inosine monophosphate (IMP), and then IMP is subsequently converted to GTP. Combined overexpression of the C. glutamicum mutant genes zwf243 and gnd361 increased riboflavin production by 39% in fed-batch fermentation. Riboflavin production was significantly increased by adding additional GTP to the medium and redirecting metabolism to the purine pathway through a metabolic engineering strategy. Therefore, GTP is the main limiting factor for riboflavin synthesis. Eliminating feedback inhibition by a metabolic engineering strategy increased riboflavin production 3-fold. Riboflavin production can be significantly enhanced by increasing the metabolic flux of the PP pathway and increasing the intracellular supply of the purine pathway precursor PRPP.

Gene deletion and overexpression have become the main strategies for traditional metabolic engineering and gene function studies. In E. coli, overexpression and knockout techniques are used to upregulate or downregulate gene expression to promote natural and non-natural products. When natural and non-natural product metabolic pathways are introduced into engineered strains to synthesize compounds, the simultaneous participation of multiple enzymes is often required. Fine-tune protein abundance in multi-enzyme-catalyzed reaction systems for optimal cell growth and production. Recent studies have used several strategies to control intracellular protein abundance, such as promoter libraries, RBS libraries, 5'-UTR libraries, mRNA stability, and the CRISPRi system. These strategies are used to fine-tune gene expression levels and control the coordinated expression of multiple genes to optimize metabolic flux. Promoters are key regulatory elements in synthetic biology that control the strength and timing of gene expression. Gene expression of this pathway is fine-tuned by promoter engineering to balance metabolic flux. To balance the parallelism of parallel polygene expression levels, a rapid and modular approach pairs genes with a tight set of ribosome-binding sites that can regulate protein abundance by orders of magnitude. EMOPEC (empirical model of protein expression changes and oligonucleotides) is a tool developed based on the contribution of SD (Shine Dalgarno) sequences to protein expression, which can modulate the expression level of any E. coli gene by changing a few bases. Furthermore, the expression of multiple genes in the operon is coordinated by generating tunable intergenic region libraries (TIGRs), recombining various post-transcriptional control elements, and screening for desired relative expression levels for the synthesis of non-natural products. This approach can effectively reduce the accumulation of toxic intermediate metabolites and the redundancy of protein expression through cooperative expression among genes.

In metabolic engineering practice, insufficient precursor supply is the main bottleneck limiting the yield of target products. High-level expression of rate-limiting enzymes to drive the metabolic flux of target product biosynthetic pathways is a rapid and straightforward strategy to increase precursor synthesis. Complex compounds, especially natural products, are often catalyzed by multiple precursors through a series of enzymatic reactions. For example, the biosynthetic pathways for the opioids thebaine and hydrocodone contain 21 and 23 enzymatic reactions from plants, mammals, yeast, and bacteria. The proportion of precursors in the cell directly affects the efficiency of compound synthesis. The lowest concentrations of precursors will be the limiting factor, while higher concentrations of precursors may be toxic to cells. During production, gene expression must be appropriate to avoid rate limiting and accumulation of toxic precursors. Various strategies based on synthetic biology have been developed to balance intracellular metabolism, including promoter engineering, RBS libraries, and dynamic control.

In the process of Bacillus subtilis synthesizing riboflavin, insufficient supply of precursors is the main factor that limits the further improvement of riboflavin production. At present, the synthesis of riboflavin can be promoted by simply expressing and knocking out key genes in the metabolic pathway. It is impossible to balance the metabolic flow in the cell, and the imbalance of the distribution of the metabolic flow in the cell leads to the increase of by-products, resulting in the phenomenon of waste of resources.

SUMMARY OF THE INVENTION

The invention provides a strategy that can efficiently and effectively regulate the metabolic balance in cells, and uses this strategy to construct an engineered strain with high riboflavin production, and uses the genetically engineered strain to produce riboflavin, so as to relieve the restriction of unbalanced metabolic flow in the fermentation process, Increase riboflavin production. A library of intergenic regulatable regions (TIGRs) was designed to regulate the expression of multiple genes within synthetic operons. Libraries of TIGRs consist of mRNA secondary structure, RNase cleavage sites, and RBS sequences, and are used to control mRNA processing and stabilization. The gene in the operon is transcribed into a complete mRNA chain, and the special site on the TIGR sequence is recognized by the intracellular RNase E (encoded from the E. coli gene rne), and the mRNA is divided into individual transcription units, and each transcription unit forms a unique The 5' and 3' structure of mRNA affects the protein level by affecting the stability of mRNA and the structure of RBS.

The present invention provides an element for regulating gene expression, the element is composed of mRNA secondary structure, RNase E cleavage site and RBS sequence, and the nucleotide sequence of the element is shown in SEQ ID NO.7.

The present invention provides a method for regulating gene expression, which utilizes the element to regulate the expression of genes, and the number of said genes is two or more;

The method is:

(1) utilize the element described in claim 1 to connect the gene, and integrate it into the expression vector to construct a recombinant plasmid;

(2) transfer the recombinant plasmid into Escherichia coli, and use the resistance gene on the expression vector to screen positive transformants;

(3) Extract the plasmid from the positive transformant, transfer it into the host cell integrating the RNase E gene to obtain recombinant cells, culture the recombinant cells and detect the fluorescence intensity, and screen the corresponding recombinant cells according to the positive correlation between the fluorescence intensity and the gene expression intensity .

The present invention provides a genetically engineered bacterium for producing riboflavin. The expression element is used to regulate the expression of zwf, ribBA and ywlf genes. The genome of the genetically engineered bacterium also integrates the RNase E gene derived from Escherichia coli.

In one embodiment, the nucleotide sequences of the genes zwf, ribBA and ywlf are shown in SEQ ID NO. 20-22, respectively

In one embodiment, the zwf gene and the ribBA gene are linked by SEQ ID NO.10, 12, 14, 16 or 18; the ribBA and ywlf genes are linked by SEQ ID NO.11, 13, 15 , 17 or 18 connections.

Preferably, the zwf gene and the ribBA gene, ribBA and ywlf gene are linked using SEQ ID NO. 14 and SEQ ID NO. 15, respectively.

In one embodiment, the plasmid pMA5-sat is used as the expression vector, and the plasmid pMA5-sat is described in the patent document with publication number CN104531745A.

In one embodiment, the genetically engineered bacteria use Bacillus subtilis RF1 as a host cell, and the Bacillus subtilis RF1 is disclosed in the patent document with publication number CN104531745A.

The invention provides a method for producing riboflavin, which is to use genetic engineering bacteria for producing riboflavin to ferment and produce riboflavin.

In one embodiment, the genetically engineered bacteria are cultured to an OD ₆₀₀ of 24-26 and added to a shake flask fermentation system in an amount of 3% (v/v) at 37° C. to 45° C. and 150 to 250 rpm. Fermentation production is not less than 24h.

In one embodiment, the shake flask fermentation system contains 15-25 g/L glucose, 15-25 g/L yeast powder, 2-5 g/L ammonium citrate, 0.5-2 g/LK ₂ HPO ₄ , 0.5-25 g/L ammonium citrate 2g/L KH ₂ PO ₄ , 1～5g/L MgSO ₄ ·7H ₂ O, 0.01～0.05g/L MnCl ₂ , 0.01～0.1g/L CaCl ₂ , 1～5g/L CuSO ₄ , pH 6.8.

In one embodiment, the genetically engineered bacteria are cultured to OD ₆₀₀ = 20-25 and added to a fed-batch fermentation system in an amount of 3% (v/v), and a feed medium is added to maintain The glucose concentration is not less than 5g/L, and the fermentation production is not less than 24h at 37℃～45℃ and 400～800rpm.

In one embodiment, the fed-batch fermentation system contains 15-25 g/L glucose, 15-25 g/L yeast powder, 2-9 g/L (NH ₄ ) ₂ HPO ₄ , and 3-8 g/LK ₂ HPO _4. 1～1.5g/L MgSO ₄ ·7H ₂ O, 0.01～0.05g/L ZnSO ₄ ·7H ₂ O, 0.01～0.05g/L MnCl ₂ , 0.01～0.02g/L FeSO ₄ ·7H ₂ O.

In one embodiment, the feed medium contains 500-600 g/L glucose, 5-10 g/L yeast powder, 4-6 g/L (NH ₄ ) ₂ HPO ₄ , 1-5 g/LK ₂ HPO ₄ , 0.1～1g/L MgSO ₄ ·7H ₂ O.

The present invention provides the application of the genetically engineered bacteria in the production of riboflavin and derivatives thereof.

Beneficial effects:

(1) Using the dual fluorescent reporter genes GFP and mCherry to characterize the regulatory ability of the TIGR sequence, in Bacillus subtilis, the expression of the second gene in the operon was significantly higher than that of the first gene, and the relative fluorescence ratio of red and green was More than 70-fold change (from 4:1 mCherry/EGFP to 18:1 EGFP/mCherry). In the TIGR library, the fluorescence range of mCherry was more than 80-fold and that of EGFP was more than 40-fold. A series of operons containing the zwf, ribBA, and ywlf genes were generated using the TIGR library, and the optimal operon combinations were screened. Five high-producing riboflavin-producing engineering bacteria were screened from 5000 polyclonal library.

(2) At the level of shake flask fermentation, the riboflavin titer of the screened engineered bacteria RF1-L3 reached 2.7 g/L, an increase of 64.35% compared with the parent.

(3) At the 5-L fermentation level, compared with the parental strain RF1, the riboflavin titer of the engineered strain RF1-L3 increased by 59.27% at 48 hours, reaching 11.77 g/L.

Description of drawings

Fig. 1 is a schematic diagram of the TIGR sequence structure;

Figure 2 is a schematic diagram of a TIGR reporter gene library;

Fig. 3 is the regulation ability analysis of TIGR library in Bacillus subtilis;

Fig. 4 is a shake flask fermentation analysis of the ability of high-producing riboflavin strain to synthesize riboflavin;

Figure 5 is the analysis of the riboflavin synthesis ability of RF1 and RF-L3 in a 5L fermenter.

Detailed ways

The media involved in the following examples are as follows:

Seed medium: 40 g/L glucose, 5 g/L yeast extract, 10 g/L peptone, 10 g/L NaCl and 10 μg/mL chloramphenicol.

Shake flask fermentation medium: 20g/L glucose, 20g/L yeast powder, 4g/L ammonium citrate, 1g/LK ₂ HPO ₄ , 1g/LKH ₂ PO ₄ , 2g/L MgSO ₄ ·7H ₂ O, 0.04g /L MnCl ₂ , 0.06 g/L CaCl ₂ , 2 g/L CuSO ₄ , pH 6.8.

Fed-batch fermentation medium: 20 g/L glucose, 20 g/L yeast powder, 6 g/L (NH ₄ ) ₂ HPO ₄ , 5 g/LK ₂ HPO ₄ , 1.5 g/L MgSO ₄ ·7H ₂ O, 0.03 g /L ZnSO ₄ ·7H ₂ O, 0.05 g/L MnCl ₂ , 0.02 g/L FeSO ₄ ·7H ₂ O.

Feed medium: 600 g/L glucose, 10 g/L yeast powder, 6 g/L (NH ₄ ) ₂ HPO ₄ , 5 g/LK ₂ HPO ₄ , 0.5 g/LMgSO ₄ ·7H ₂ O.

Minimum medium containing glucose: 20.0 g/L, (NH4) ₂ SO ₄ 2.0 g/L, KH ₂ PO ₄ 13.1 g/L, K ₂ HPO ₄ 6.0 g/L, NaC ₆ H ₅ O ₇ 2H ₂ O 1.2 g/L, MgSO ₄ ·7H ₂ O 0.05 g/L, and supplemented with tryptophan, phenylalanine and tyrosine (25 mg/L each).

The detection methods involved in the following examples are as follows:

Fluorescence values of EGFP and mCherry were measured to assess the expression range of the pMTG library. E. coli and B. subtilis containing the pMTG library were inoculated into sterile 96 black well plates (Corning 3603) containing 200 μl of LB medium and incubated at 37°C for 10 hours. In addition, EGFP fluorescence (excitation, 490 nm; emission, 530 nm), mCherry fluorescence (excitation, 588 nm; emission, 633 nm), optical density (absorbance at 600 nm) were measured at the end of the incubation with a microplate multimode reader (BIOTEK, Cytation 3) Determination. The relative fluorescence density was calculated using equation (1). FPbg represents the fluorescence value of the strain without fluorescent protein, and ODbg represents the absorbance of the medium.

Riboflavin emits yellow-green fluorescence under the irradiation of light with wavelengths of 440-500 nm, and its fluorescence intensity is proportional to its concentration in dilute solution. For the riboflavin standard curve, 10 mM riboflavin stock solutions were diluted to various concentrations including 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.313 mM and 0.1562 mM. Fluorescence values were measured at excitation wavelength 444 nm and absorption wavelength 500 nm, and a standard curve was drawn. Clones from the engineered strain library were inoculated into 96-well plates containing 200 μl of minimal medium and cultured at 37°C with shaking at 220 rpm for 24 hours. Cell cultures were diluted to appropriate concentrations using 0.01 M NaOH, and fluorescence intensity was measured by a multi-mode microplate reader (BIOTEK, Cytation 3). Cell growth at OD ₆₀₀ nm was monitored with a spectrophotometer.

The prepared fermentation broth was diluted with 0.01M NaOH, then centrifuged at 12,000 rpm for 2 min, and the supernatant was taken to measure the riboflavin concentration. Transfer the supernatant to a new EP tube and dilute to the appropriate concentration range (0.3-0.8) and measure the absorbance value at OD ₄₄₄ nm using a spectrophotometer. Riboflavin concentration was calculated from a standard curve of riboflavin concentration. According to the riboflavin standard curve, the calculation formula is: OD ₄₄₄ * dilution factor * 30/1000.

Glucose concentration was measured using Glucose analysis (Model-SBA40, Shandong, China).

The control strains RF1-aPaGaTgV and RF1 used in the following examples are disclosed in the patent document with publication number CN104531745A.

The primer sequences involved in the following examples are shown in Table 1:

Table 1 Primer sequences

Example 1: Heterologous expression of the gene rne in Bacillus subtilis

The RNase E gene rne from Escherichia coli was integrated into the gene amyE (Gene ID: 938356) in the genome of Bacillus subtilis 168 by means of homologous recombination.

Specific steps are as follows:

(1) Amplify the upstream homology arm (1000bp) and the downstream homology arm (1000bp) of the gene amyE according to the primer sequences in Table 1 to obtain the upstream and downstream homology arm fragments of amyE respectively; and separate the PCR products by agarose gel electrophoresis , and cut the gel to recover the target PCR product. Then, the upstream and downstream homology arms, the Pveg-rne fragment (the nucleotide sequence of the fragment is shown in SEQ ID NO.1) and the resistance Marker (bleomycin resistance gene, the nucleotide sequence is shown in SEQ ID NO.2) shown) fusions were performed by the strategy of fusion PCR.

First, the upstream and downstream homology arms, the Pveg-rne fragment and the resistance Marker fragment were mixed in a volume ratio of 1:1, and an equal volume of PCR enzyme was added to carry out a fusion PCR reaction (conditions were 98°C for 3 min, 98°C for 8s, 61°C for 5s, 72°C for 2min, amplification for 13 cycles), using the product after this step as a template, use primers amyE-veg-rne-F1 and amyE-veg-rne-R3 to amplify the fusion fragment (reaction conditions: 98°C for 3min , 98°C for 10s, 58°C for 15s, 72°C for 1min, and 34 cycles of amplification). The PCR product was purified and recovered for the knockout reaction. The fused fragment contained the bleomycin resistance gene and the lox66-lox71 recombination site, which facilitated the elimination of the later resistance Marker.

(2) The fusion PCR product obtained in step (1) was transformed into Bacillus subtilis168 competent cells using the transformation method, and spread on the LB plate containing bleomycin resistance, cultured at 37°C for 12 hours, and the resistance The colony PCR method was used to verify the successful integration of the strains grown on the plate. The primers used for verification were amyE-veg-rne-F1 and amyE-veg-rne-R3, and positive transformants with successful integration were obtained after PCR verification.

(3) Plasmidize pDG148 and transfer it to the correct positive transformants prepared in step (2), and shake at 180 rpm and 37 °C for 24 hours, then take part of the culture solution and spread it on LB plates, and culture at 37 °C for 12 hours to a long time. A single colony was obtained, and the single colony on the LB plate was one-to-one corresponding to another plate containing bleomycin with a sterile toothpick, and cultured at 37 °C for 12 hours. Colonies that failed to grow on the bleomycin-resistant plates were knockout strains that eliminated bleomycin resistance.

The resulting knockout strain was shaken at 180rpm and 42°C for 24h to eliminate the pDG148 plasmid. Part of the culture solution was spread on LB solid medium, and then cultured at 37°C for 12 hours. Use sterile toothpicks to point the colonies on the LB plate to another resistant plate containing ampicillin, and culture at 37°C. After culturing for 12 hours, the colonies could grow on LB plates, but the colonies that could not grow on bleomycin-resistant plates were knockout strains that eliminated the plasmid pDG148. The final strain obtained is the target strain BSF01 with no trace knockout.

Example 2: Construction of TIGR library containing dual fluorescent reporter genes

Libraries of TIGRs include mRNA secondary structures, RNase E cleavage sites, and RBS sequence control elements. Libraries of TIGRs utilize mRNA secondary structure, RNase E enzymatic cleavage sites, and RBS sequences to control mRNA processing and stabilization, thereby affecting gene expression on operons (Figure 1).

In order to further amplify the synthetic biology tools suitable for B. subtilis, an M-TIGR-G library was constructed based on TIGR (Fig. 2), and the TIGRs library was introduced into strain BSF01, which overexpressed the gene rne derived from E. coli at the amyE site . The fluorescence values of the two reporter genes were determined to assess the regulatory capacity of the TIGR library in B. subtilis.

Specific steps are as follows:

1. Preparation of TIGR library

(1) Use PCR to synthesize TIGR, and assemble the oligonucleotide sequences TIGR-A, TIGR-B, TIGR-C and TIGR-D (the nucleotide sequences are shown in SEQ ID NO. 3 to 6, respectively) into a chimera DNA sequence (nucleotide sequence is shown in SEQ ID NO. 7). The five nucleotides in each oligonucleotide sequence were replaced with random nucleotides "N" to synthesize diverse sequences. The fusion fragment M-TIGRs-G was constructed by integrating mCherry gene, TIGR sequence (chimeric DNA sequence) and GFP gene by fusion PCR. Then, the fusion PCR fragment M-TIGRs-G was inserted into the plasmid pP43NMK by Gibson assembly to construct the reporter plasmid pMTG for screening TIGR, so that the transcription of the reporter operon was controlled by the strong promoter P43. Plasmid pMTG was transformed into viable E. coli JM109 cells and transformants were selected with ampicillin. E. coli library colonies were scraped and plasmids were extracted, collected from three separate plates, and transformed into BSF01.

(2) Inoculate the polyclonal library obtained in step (1) into a 96-well plate, measure the fluorescence values of GFP and mCherry, and construct a TIGR library.

The results showed that the expression of the second gene in the operon was significantly higher than that of the first gene, and the relative fluorescence ratio of red and green changed more than 70-fold depending on the intergenic region (from mCherry/EGFP=4:1 to EGFP/mCherry=18:1; Figure 3).

Example 3: Construction of TIGR library to screen high-yielding riboflavin engineered strains

According to the method of Example 1, the RNase E gene rne derived from Escherichia coli was integrated into the gene amyE of the riboflavin-producing strain RF1, and the strain RF1-R was constructed.

The TIGR library was used to generate a series of synthetic operons containing the zwf, ribBA and ywlf genes, which were then screened for the optimal combination to increase riboflavin production. Using the macroprimer PCR method, the TIGR library was introduced simultaneously between the first and second genes and the second and third genes of the operon. The gene zwf was used as the first gene of the operon, and ribBA and ywf were used as the second and third genes of the operon.

Specific steps are as follows:

(1) Construct gene fragments containing TIGR library by fusion PCR. First, the primers ZRY-F2/ZRY-R2 and ZRY-F4/ZRY-R4 were designed, and the TIGR library in Example 2 (the chimeric DNA sequence whose nucleotide sequence was shown in SEQ ID NO. 7) was used as a template, Amplify the TIGR library between the genes zwf, ribBA and ywlf. The PCR fragment zwf and TIGR library were subjected to fusion PCR to obtain zwf-TIGR (the nucleotide sequence is shown in SEQ ID NO. 8). The PCR fragment of ribBA-TIGR was obtained according to the above method (the nucleotide sequence is shown in SEQ ID NO. 9). Plasmid pMA5-sat was treated with restriction enzymes EcoRI and KpnI at 37°C for 30 min and purified and recovered. Then the purified PCR fragments zwf-TIGR, ribBA-TIGR and ywlf were ligated into a PCR fragment by fusion PCR method and inserted into The plasmid pMA5-sat was assembled using Gibson to obtain the zwf-TIGR-ribBA-TIGR-ywlf library, which was transformed into E. coli DH5α competent cells, cultured at 37°C for 16 hours, and the correct transformants were screened by colony PCR. The resulting transformants were mixed and extracted with plasmids to obtain a library and transformed into RF1-R to construct an engineering strain library. The clones from the engineering strain library were inoculated into a 96-well plate containing 200 μl of minimal medium at 37°C at a speed of 220 rpm. Shake culture for 24 hours. The cell culture was diluted to the appropriate concentration with 0.01M NaOH, and the fluorescence intensity was measured by a multi-mode microplate reader (BIOTEK, Cytation 3), and the mutant with the highest fluorescence intensity was screened.

Example 4: Fermentation production of riboflavin using genetically engineered strains in shake flask stage

The mutant strain with the highest fluorescence intensity screened in Example 3 was re-screened using shake flask fermentation.

A single colony was inoculated in 10 ml-LB medium and incubated at 37°C with shaking at 180 rpm. After 16 hours, the culture was transferred to a 250ml block containing 50ml LBG medium (40g/L glucose, 5g/L yeast extract, 10g/L peptone and 10g/L NaCl) at 1% (v/v) inoculum size. Plate in shake flasks and shake at 220 rpm for 24 hours at 41 °C to obtain seed cultures. The seed culture ( _OD600 = 25.21) was transferred to a 500 ml baffled shake flask containing 50 ml of fermentation medium (3% (v/v) inoculum) and incubated at 41°C with shaking at 220 rpm for 48 hours. During shake flask fermentation, 1 ml of cell suspension was sampled every 12 hours to measure _OD600 and riboflavin concentration.

The results showed that the riboflavin titers of the five engineered strains were significantly increased, and the titer advantage of the engineered strain RF1-L3 was the most obvious. The riboflavin titer of the engineered bacteria RF1-L3 reached 2.7 g/L, which was 64.35% higher than that of the parental 1.67 g/L (Figure 4 shows the riboflavin yield of each strain after 48 hours of fermentation). The engineered bacteria RF1-L1, RF1 The riboflavin titers of -L2, RF1-L4 and RF1-L5 were 1.94g/L, 2.01g/L, 2.16g/L and 2.24g/L, respectively.

Table 2 TIGR sequences of strains RF1-L1~RF1-L5

Example 5: Fermentation production of riboflavin using genetically engineered strains at the fermenter level

Specific steps are as follows:

(1) Bacillus subtilis strains RF1-aPaGaTgV and RF1 cultured in 10 mL LB medium for 24 h were inoculated into 100 mL of seed medium at an inoculum of 3% (v/v) by volume (the seed medium included 20 g/v/v). L glucose, 20g/L yeast powder, 4g/L ammonium citrate, 1g/LK ₂ HPO ₄ , 1g/L KH ₂ PO ₄ , 2g/L MgSO ₄ ·7H ₂ O, 0.04g/LMnCl ₂ , 0.06g /L CaCl ₂ , 2g/L CuSO ₄ ), the temperature was 41°C, the rotation speed was 180rpm, and after culturing for 16h, the seed solution (OD ₆₀₀ =23.5) was prepared;

(2) All 100 mL of the prepared seed solution was inoculated into a 5 L fermentor containing 1900 mL of fermentation medium, and fed-batch fermentation was performed.

The residual glucose concentration in the fermentation broth was maintained at not less than 5 g/L by controlling the flow rate of the feed medium. During the fermentation, the pH of the fermentation broth was maintained at 6.8 using 1M _H2SO4 and 50% ammonia _. The rotation speed was maintained at 400 rpm before starting the batch feed, and then gradually increased to 900 rpm until the end of the fermentation, and the temperature was maintained at 41°C throughout.

The results showed that fed-batch fermentation was carried out in a 5L bioreactor to verify the large-scale fermentation performance of engineered bacteria Bacillus subtilis RF1-L3 in riboflavin production. In fed-batch fermentation, there was no significant difference in growth between the engineered and parental strains. Compared with the parental strains RF1 (7.39g/L) and RF1-aPaGaTgV (10.71g/L), the riboflavin titer of the engineered strain RF1-L3 increased by 59.27% at 48h, reaching 11.77g/L (Fig. 5A). and Figure 5B).

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> BAA220441A

<130> A method for efficient synthesis of riboflavin based on balanced gene expression

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 3263

<212> DNA

<213> Artificial sequences

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accagcgtgc ggtagaaaac cgtctgcgtg aagcggtgcg tcaggaccgt gcgcgtattc 1200

aaatcagcca tatttctcgc tttggcctgc tggaaatgtc ccgtcagcgc ctgagcccat 1260

cactgggtga atccagtcat cacgtttgtc cgcgttgttc tggtactggc accgtgcgtg 1320

acaacgaatc gctgtcgctc tctattctgc gtctgatcga agaagaagcg ctgaaagaga 1380

acacccagga agttcacgcc attgttcctg tgccaatcgc ttcttacctg ctgaatgaaa 1440

aacgttctgc ggtaaatgcc attgaaactc gtcaggacgg tgtgcgctgt gtaattgtgc 1500

caaacgatca gatggaaacc ccgcactacc acgtgctgcg cgtgcgtaaa ggggaagaaa 1560

ccccaacctt aagctacatg ctgccgaagc tgcatgaaga agcgatggcg ctgccgtctg 1620

aagaagagtt cgctgaacgt aagcgtccgg aacaacctgc gctggcaacc tttgccatgc 1680

cggatgtgcc gcctgcgcca acgccagctg aacctgccgc gcctgttgta gctccagcac 1740

cgaaagctgc accggcaaca ccagcagctc ctgcacaacc tgggctgttg agccgcttct 1800

tcggcgcact gaaagcgctg ttcagcggtg gtgaagaaac caaaccgacc gagcaaccag 1860

caccgaaagc agaagcgaaa ccggaacgtc aacaggatcg tcgcaagcct cgtcagaaca 1920

accgccgtga ccgtaatgag cgccgcgaca cccgtagtga acgtactgaa ggcagcgata 1980

atcgcgaaga aaaccgtcgt aatcgtcgcc aggcacagca gcagactgcc gagacgcgtg 2040

agagccgtca gcaggctgag gtaacggaaa aagcgcgtac cgccgacgag cagcaagcgc 2100

cgcgtcgtga acgtagccgc cgccgtaatg atgataaacg tcaggcgcaa caagaagcga 2160

aggcgctgaa tgttgaagag caatctgttc aggaaaccga acaggaagaa cgtgtacgtc 2220

cggttcagcc gcgtcgtaaa cagcgtcagc tcaatcagaa agtgcgttac gagcaaagcg 2280

tagccgaaga agcggtagtc gcaccggtgg ttgaagaaac tgtcgctgcc gaaccaattg 2340

ttcaggaagc gccagctcca cgcacagaac tggtgaaagt cccgctgcca gtcgtagcgc 2400

aaactgcacc agaacagcaa gaagagaaca atgctgataa ccgtgacaac ggtggcatgc 2460

cgcgtcgttc tcgccgctcg cctcgtcacc tgcgcgtaag tggtcagcgt cgtcgtcgct 2520

atcgtgacga gcgttatcca acccagtcgc caatgccgtt gaccgtagcg tgcgcgtctc 2580

cggaactggc ctctggcaaa gtctggatcc gctatccaat tgtacgtccg caagatgtac 2640

aggttgaaga gcagcgcgaa caggaagaag tacatgtgca gccgatggtg actgaggtcc 2700

ctgtcgccgc cgctatcgaa ccggttgtta gcgcgccagt tgttgaagaa gtggccggtg 2760

tcgtagaagc ccccgttcag gttgccgaac cgcaaccgga agtggttgaa acgacgcatc 2820

ctgaagtgat cgctgccgcg gtaactgaac agccgcaggt gattaccgag tctgatgttg 2880

ccgtagccca ggaagttgca gaacaagcag aaccggtggt tgaaccgcag gaagagacgg 2940

cagatattga agaagttgtc gaaactgctg aggttgtagt tgctgaacct gaagttgttg 3000

ctcaacctgc cgcgccagta gtcgctgaag tcgcagcaga agttgaaacg gtagctgcgg 3060

tcgaacctga ggtcaccgtt gagcataacc acgctaccgc gccaatgacg cgcgctccag 3120

caccggaata tgttccggag gcaccgcgtc acagtgactg gcagcgccct acttttgcct 3180

tcgaaggtaa aggtgccgca ggtggtcata cggcaacaca tcatgcctct gccgctcctg 3240

cgcgtccgca acctgttgag taa 3263

<210> 2

<211> 497

<212> DNA

<213> Artificial sequences

<400> 2

taccgttcgt atagcataca ttatacgaag ttatcttgat atggcttttt atatgtgtta 60

ctctacatac agaaaggagg aactaaatat ggccaagttg accagtgccg ttccggtgct 120

caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg 180

ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag 240

cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct 300

ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg 360

gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc 420

ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgaataactt cgtatagcat 480

acattatacg aacggta 497

<210> 3

<211> 70

<212> DNA

<213> Artificial sequences

<220>

<221> misc_feature

<222> (32)..(36)

<223> n is a, c, g, or t

<400> 3

gcctagcaag atctcctgat cagagggaca annnnnaagg tcattgcagc tcagacatct 60

gcatagtctg 70

<210> 4

<211> 66

<212> DNA

<213> Artificial sequences

<220>

<221> misc_feature

<222> (31)..(35)

<223> n is a, c, g, or t

<400> 4

aaatactgta aattcaaggc agaaggtcaa nnnnnaaggc cactgcagct cagactatgc 60

agatgt 66

<210> 5

<211> 65

<212> DNA

<213> Artificial sequences

<220>

<221> misc_feature

<222> (31)..(35)

<223> n is a, c, g, or t

<400> 5

gccttgaatt tacagtattt tagtggcctt nnnnnnatact attcggtcac cttatccgct 60

caaga 65

<210> 6

<211> 71

<212> DNA

<213> Artificial sequences

<220>

<221> misc_feature

<222> (39)..(42)

<223> n is a, c, g, or t

<400> 6

ggatacagta tctgcggtac cctagattta gtggccttnn nnatactact cggtctcttg 60

agcggataag g 71

<210> 7

<211> 272

<212> DNA

<213> Artificial sequences

<220>

<221> misc_feature

<222> (32)..(36)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (101)..(105)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (167)..(171)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (240)..(243)

<223> n is a, c, g, or t

<400> 7

gcctagcaag atctcctgat cagagggaca annnnnaagg tcattgcagc tcagacatct 60

gcatagtctg aaatactgta aattcaaggc agaaggtcaa nnnnnaaggc cactgcagct 120

cagactatgc agatgtgcct tgaatttaca gtattttagt ggccttnnnn natactattc 180

ggtcacctta tccgctcaag aggatacagt atctgcggta ccctagattt agtggccttn 240

nnnatactac tcggtctctt gagcggataa gg 272

<210> 8

<211> 1762

<212> DNA

<213> Artificial sequences

<220>

<221> misc_feature

<222> (1528)..(1532)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1582)..(1586)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1627)..(1631)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1675)..(1678)

<223> n is a, c, g, or t

<400> 8

gcatcgcgcg cggggaattc tgataggtga aaacaaacca acaaccaaaa gcagtaattg 60

tcatattcgg tgcaactgga gatttagcaa aacgaaaatt gtatccgtct attcaccgtt 120

tatatcaaaa cggacaaatc ggagaagagt ttgcagtggt aggagttgga agaagacctt 180

ggtctaatga ggatcttcgc caaactgtta aaacatccat ttcctcatct gcagataagc 240

atatagatga tttcacgtct catttttact atcacccgtt tgacgtgaca aaccctggtt 300

cttatcaaga gctaaacgta ttgcttaacc agctggaaga tacatatcaa attcctaaca 360

acagaatgtt ctacttggca atggctcctg aattcttcgg aacgattgca aaaacattaa 420

aatcagaggg tgtaacagct acaaccggct ggtcccgcct tgtcatcgaa aaaccgttcg 480

gccatgatct gccaagcgca caggcattga ataaagaaat ccgcgaagca tttacggaag 540

atcaaattta cagaatcgac cattatctag gcaaacaaat ggttcagaac attgaagtga 600

ttcgatttgc caatgcgatt ttcgaaccgc tttggacaaa ccgctacatt tcaaacattc 660

aaatcacatc tagcgaatca ctaggcgttg aagacgcgc aagatattac gaaaaatcag 720

gcgcccttcg cgacatggtg caaaaccata ttatgcagat ggttgccctt cttgcaatgg 780

agccgcctat caaattgaac acagaagaaa tccgcagcga gaaagtgaag gtgctgagag 840

cactgcgtcc tattgcaaaa gacgaagtgg atgaatactt tgtgcgcgga caatatcatg 900

ctggtgaaat tgacggtgta ccggttcctg cttatacaga tgaagataat gtcgctcctg 960

actccaatac agaaaccttt gttgccggca agctcttgat cgacaacttc agatgggctg 1020

gtgttccatt ctacatcaga accggaaaac gaatgaaaga aaagtccaca aaaattgtcg 1080

ttcaatttaa ggacattccg atgaacctgt actacggtaa tgaaaacaac atgaatccga 1140

acttgcttgt cattcatatt cagcctgacg aaggcattac gctttactta aatgctaaaa 1200

agcttggcgg agcagcacac gcacagccaa tcaaactcga ttattgcagc aattgcaatg 1260

acgagttgaa cacccctgaa gcatatgaaa aactaattca cgactgtctt cttggcgatg 1320

caacaaactt tgcacactgg gatgaagttg ccctttcttg gagctttgtc gactctattt 1380

ctgaaacatg ggcagcaaac aaaaccttat ctcctaacta cgaatcaggc tcaatgggac 1440

cgaaagaatc tgatgatctt ttggtgaaag acggcttaca ctggtggaac atataagcct 1500

agcaagatct cctgatcaga gggacaannn nnaaggtcat tgcagctcag acatctgcat 1560

agtctgagct gcagtggcct tnnnnnttga ccttctgcct tgaatttaca gtattttagt 1620

ggccttnnnn natactattc ggtcacctta tccgctcaag agaccgagta gtatnnnnaa 1680

ggccactaaa tctagggtac cgcagatact gtatccgtaa gagaggaatg tacacatgtt 1740

tcatccgata gaagaagcac tg 1762

<210> 9

<211> 1463

<212> DNA

<213> Artificial sequences

<220>

<221> misc_feature

<222> (1229)..(1233)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1283)..(1287)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1328)..(1332)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1376)..(1379)

<223> n is a, c, g, or t

<400> 9

atgtttcatc cgatagaaga agcactggac gctttaaaaa aaggcgaagt catcatcgtt 60

gtagatgatg aagacagaga aaatgaagga gactttgtgg ctcttgccga gcatgcaacg 120

ccggaagtca ttaactttat ggcgacacat gggagaggac tgatctgcac gccgctcagt 180

gaggaaatcg cagacaggct tgatcttcac cctatggttg agcataatac agactctcac 240

cacactgcat ttaccgtaag catagaccat cgtgaaacga agacaggtat cagcgctcaa 300

gaaagatctt ttaccgttca agcattgctg gacagcaaat ccgtgccatc tgattttcag 360

cgtccggggc acatttttcc actgattgcg aaaaaaggag gtgtcctgaa aagagcgggc 420

catacagaag ctgctgttga tcttgctgaa gcttgcggat ctccaggagc cggcgtcatt 480

tgtgaaatta tgaatgaaga cggaacgatg gcgagagtgc ctgagctcat tgaaattgcg 540

aaaaagcatc aattaaaaat gatcaccatt aaggatttga ttcaataccg ttacaatctg 600

acaacacttg tcgagcgtga agttgacatt acgctgccta ctgattttgg gacatttaag 660

gtttatggat acacaaatga ggtagatgga aaagagcatg tcgcatttgt gatgggagat 720

gtgccgttcg gagaagaacc ggtattggtc cgggtgcatt cagaatgtct cacaggtgac 780

gtgtttggct ctcatcgctg tgattgcgga ccgcagctgc acgccgcgct gaaccaaatt 840

gccgcagaag gccgtggagt gctcctgtac ttgcgccaag aaggacgagg catcggttta 900

atcaataaat taaaagctta taagcttcag gaacaaggct atgacaccgt agaagccaat 960

gaggcgcttg gattcttgcc ggatcttcgc aactatggca tcggagcaca aattttacgc 1020

gacctcggtg tccggaatat gaagcttttg acgaataatc cgcgaaaaat cgcaggcctt 1080

gaaggctacg gactcagtat ttcagaaaga gtgccgcttc aaatggaggc gaaagaacac 1140

aataaaaaat atttgcaaac caaaatgaac aagctaggtc atttacttca tttctaagcc 1200

tagcaagatc tcctgatcag agggacaann nnnnaaggtca ttgcagctca gacatctgca 1260

tagtctgagc tgcagtggcc ttnnnnnttg accttctgcc ttgaatttac agtattttag 1320

tggccttnnn nnatactatt cggtcacctt atccgctcaa gagaccgagt agtatnnnna 1380

aggccactaa atctagggta ccgcagatac tgtatccgta agagaggaat gtacacatga 1440

aagtagccat tgcatcggat cat 1463

<210> 10

<211> 239

<212> DNA

<213> Artificial sequences

<220>

<221> misc_feature

<222> (131)..(131)

<223> n is a, c, g, or t

<400> 10

gcctagcaag atctcctgat cagagggaca aaatcaaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gccttccgac ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt nacagatact attcggtcac cttatccgct caagagaccg agtagtatga 180

ccaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 11

<211> 239

<212> DNA

<213> Artificial sequences

<400> 11

gcctagcaag atctcctgat cagagggaca attcgcaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gccttgatta ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt gtcctatact attcggtcac cttatccgct caagagaccg agtagtatca 180

aaaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 12

<211> 239

<212> DNA

<213> Artificial sequences

<400> 12

gcctagcaag atctcctgat cagagggaca aaacgcaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gccttctaat ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt gaaccatact attcggtcac cttatccgct caagagaccg agtagtatga 180

ccaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 13

<211> 239

<212> DNA

<213> Artificial sequences

<400> 13

gcctagcaag atctcctgat cagagggaca ataaacaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gccttgctta ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt cgaatatact attcggtcac cttatccgct caagagaccg agtagtattc 180

caaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 14

<211> 239

<212> DNA

<213> Artificial sequences

<400> 14

gcctagcaag atctcctgat cagagggaca accattaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gccttgttat ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt gcccaatact attcggtcac cttatccgct caagagaccg agtagtattt 180

aaaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 15

<211> 239

<212> DNA

<213> Artificial sequences

<400> 15

gcctagcaag atctcctgat cagagggaca aaatgaaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gcctttacgc ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt ttagaatact attcggtcac cttatccgct caagagaccg agtagtattt 180

aaaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 16

<211> 239

<212> DNA

<213> Artificial sequences

<400> 16

gcctagcaag atctcctgat cagagggaca agccgaaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gccttgtaag ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt gattaatact attcggtcac cttatccgct caagagaccg agtagtatca 180

ccaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 17

<211> 239

<212> DNA

<213> Artificial sequences

<400> 17

gcctagcaag atctcctgat cagagggaca attagaaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gccttatccg ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt aagatatact attcggtcac cttatccgct caagagaccg agtagtatgg 180

aaaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 18

<211> 239

<212> DNA

<213> Artificial sequences

<400> 18

gcctagcaag atctcctgat cagagggaca acgagaaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gccttgagac ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt attgaatact attcggtcac cttatccgct caagagaccg agtagtatgg 180

aaaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 19

<211> 239

<212> DNA

<213> Artificial sequences

<400> 19

gcctagcaag atctcctgat cagagggaca acaagaaagg tcattgcagc tcagacatct 60

gcatagtctg agctgcagtg gccttttacc ttgaccttct gccttgaatt tacagtattt 120

tagtggcctt aagaaatact attcggtcac cttatccgct caagagaccg agtagtatga 180

ctaaggccac taaatctagg gtaccgcaga tactgtatcc gtaagagagg aatgtacac 239

<210> 20

<211> 1470

<212> DNA

<213> Artificial sequences

<400> 20

gtgaaaacaa accaacaacc aaaagcagta attgtcatat tcggtgcaac tggagattta 60

gcaaaacgaa aattgtatcc gtctattcac cgtttatatc aaaacggaca aatcggagaa 120

gagtttgcag tggtaggagt tggaagaaga ccttggtcta atgaggatct tcgccaaact 180

gttaaaacat ccatttcctc atctgcagat aagcatatag atgatttcac gtctcatttt 240

tactatcacc cgtttgacgt gacaaaccct ggttcttatc aagagctaaa cgtattgctt 300

aaccagctgg aagatacata tcaaattcct aacaacagaa tgttctactt ggcaatggct 360

cctgaattct tcggaacgat tgcaaaaaca ttaaaatcag agggtgtaac agctacaacc 420

ggctggtccc gccttgtcat cgaaaaaccg ttcggccatg atctgccaag cgcacaggca 480

ttgaataaag aaatccgcga agcatttacg gaagatcaaa tttacagaat cgaccattat 540

ctaggcaaac aaatggttca gaacattgaa gtgattcgat ttgccaatgc gattttcgaa 600

ccgctttgga caaaccgcta catttcaaac attcaaatca catctagcga atcactaggc 660

gttgaagacc gcgcaagata ttacgaaaaa tcaggcgccc ttcgcgacat ggtgcaaaac 720

catattatgc agatggttgc ccttcttgca atggagccgc ctatcaaatt gaacacagaa 780

gaaatccgca gcgagaaagt gaaggtgctg agagcactgc gtcctattgc aaaagacgaa 840

gtggatgaat actttgtgcg cggacaatat catgctggtg aaattgacgg tgtaccggtt 900

cctgcttata cagatgaaga taatgtcgct cctgactcca atacagaaac ctttgttgcc 960

ggcaagctct tgatcgacaa cttcagatgg gctggtgttc cattctacat cagaaccgga 1020

aaacgaatga aagaaaagtc cacaaaaatt gtcgttcaat ttaaggacat tccgatgaac 1080

ctgtactacg gtaatgaaaa caacatgaat ccgaacttgc ttgtcattca tattcagcct 1140

gacgaaggca ttacgcttta cttaaatgct aaaaagcttg gcggagcagc acacgcacag 1200

ccaatcaaac tcgattattg cagcaattgc aatgacgagt tgaacacccc tgaagcatat 1260

gaaaaactaa ttcacgactg tcttcttggc gatgcaacaa actttgcaca ctgggatgaa 1320

gttgcccttt cttggagctt tgtcgactct atttctgaaa catgggcagc aaacaaaacc 1380

ttatctccta actacgaatc aggctcaatg ggaccgaaag aatctgatga tcttttggtg 1440

aaagacggct tacactggtg gaacatataa 1470

<210> 21

<211> 1197

<212> DNA

<213> Artificial sequences

<400> 21

atgtttcatc cgatagaaga agcactggac gctttaaaaa aaggcgaagt catcatcgtt 60

gtagatgatg aagacagaga aaatgaagga gactttgtgg ctcttgccga gcatgcaacg 120

ccggaagtca ttaactttat ggcgacacat gggagaggac tgatctgcac gccgctcagt 180

gaggaaatcg cagacaggct tgatcttcac cctatggttg agcataatac agactctcac 240

cacactgcat ttaccgtaag catagaccat cgtgaaacga agacaggtat cagcgctcaa 300

gaaagatctt ttaccgttca agcattgctg gacagcaaat ccgtgccatc tgattttcag 360

cgtccggggc acatttttcc actgattgcg aaaaaaggag gtgtcctgaa aagagcgggc 420

catacagaag ctgctgttga tcttgctgaa gcttgcggat ctccaggagc cggcgtcatt 480

tgtgaaatta tgaatgaaga cggaacgatg gcgagagtgc ctgagctcat tgaaattgcg 540

aaaaagcatc aattaaaaat gatcaccatt aaggatttga ttcaataccg ttacaatctg 600

acaacacttg tcgagcgtga agttgacatt acgctgccta ctgattttgg gacatttaag 660

gtttatggat acacaaatga ggtagatgga aaagagcatg tcgcatttgt gatgggagat 720

gtgccgttcg gagaagaacc ggtattggtc cgggtgcatt cagaatgtct cacaggtgac 780

gtgtttggct ctcatcgctg tgattgcgga ccgcagctgc acgccgcgct gaaccaaatt 840

gccgcagaag gccgtggagt gctcctgtac ttgcgccaag aaggacgagg catcggttta 900

atcaataaat taaaagctta taagcttcag gaacaaggct atgacaccgt agaagccaat 960

gaggcgcttg gattcttgcc ggatcttcgc aactatggca tcggagcaca aattttacgc 1020

gacctcggtg tccggaatat gaagcttttg acgaataatc cgcgaaaaat cgcaggcctt 1080

gaaggctacg gactcagtat ttcagaaaga gtgccgcttc aaatggaggc gaaagaacac 1140

aataaaaaat atttgcaaac caaaatgaac aagctaggtc atttacttca tttctaa 1197

<210> 22

<211> 450

<212> DNA

<213> Artificial sequences

<400> 22

atgaaagtag ccattgcatc ggatcatggc ggcgttcaca ttcgaaatga aatcaaagag 60

ttaatggacg aattgcaaat tgaatatatt gatatgggct gtgactgcgg cagcggctct 120

gtcgattatc cggattatgc ttttccggtg gccgaaaaag tggttagcgg cgaagttgac 180

agaggcattt taatttgcgg gacaggcatc ggcatgagca tttccgctaa taaagtaaaa 240

gggattcgct gcgcgctggc gcacgatacc ttcagcgcga aggcgacgag ggagcataat 300

gacacaaaca tccttgcgat gggtgaacgg gtgatcggac ctggtttggc tcgggaaatc 360

gcaaaaatct ggctgactac tgagtttacc gggggaagac accaaacgcg tattggaaaa 420

atctccgatt atgaagagaa aaacctgtag 450

Claims (10)

1. An element for regulating gene expression level, which is characterized by consisting of mRNA secondary structure, RNase E cleavage site and RBS sequence, wherein the nucleotide sequence of the element is shown as SEQ ID NO. 7.

2. A method for regulating the expression of genes, wherein the expression of two or more genes is regulated by using the element of claim 1;

the method comprises the following steps:

(1) connecting genes by using the element of claim 1, and integrating the genes on an expression vector to construct a recombinant plasmid;

(2) transferring the recombinant plasmid into a microbial cell integrated with an RNase E gene, and screening a positive transformant by using a resistance gene on an expression vector;

(3) extracting plasmid from the positive transformant, transferring the plasmid into an expression host cell to obtain a recombinant cell, culturing the recombinant cell, detecting fluorescence intensity, and screening the corresponding recombinant cell according to the positive correlation between the fluorescence intensity and the gene expression intensity.

3. The genetic engineering bacteria for producing the riboflavin is characterized in that the expression of zwf, ribBA and ywlf genes is regulated by utilizing the expression element, and the genome of the genetic engineering bacteria is also integrated with an RNase E gene derived from escherichia coli.

4. The genetically engineered bacterium of claim 3, wherein the zwf gene and the ribBA gene are linked by SEQ ID No.10, 12, 14, 16 or 18; the ribBA and ywlf genes are linked by SEQ ID NO.11, 13, 15, 17 or 18.

5. The genetically engineered bacterium of claim 3, wherein the genetically engineered bacterium uses plasmid pMA5-sat as an expression vector.

6. The genetically engineered bacterium of claim 3, wherein the genetically engineered bacterium uses Bacillus subtilis RF1 as a host cell.

7. A method for producing riboflavin by fermentation using the genetically engineered bacterium according to any one of claims 3 to 6.

8. The method of claim 7, wherein the method is performed byCulturing the genetically engineered bacteria to OD ₆₀₀ Adding the 24-26 of the strain into a shake flask fermentation system in an amount of 1-5% (v/v), and fermenting at 37-45 ℃ and 150-250 rpm for not less than 24 h.

9. The method of claim 7, wherein the genetically engineered bacteria are cultured to OD ₆₀₀ Adding the medium into a fed-batch fermentation system in an amount of 1-5% (v/v) at 20-25, adding a fed-batch culture medium, keeping the glucose concentration not lower than 5g/L, and fermenting at 37-45 ℃ and 400-800 rpm for not less than 24 h.

10. Use of the genetically engineered bacterium of any one of claims 3 to 6 for the production of riboflavin or derivatives thereof.

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