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

Method for efficiently synthesizing 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 gene expression in an mRNA engineering balanced operon, belonging to the technical field of synthetic biology.

Background

Riboflavin (VB2) is a nutrient essential to humans and animals and is widely used in pharmaceuticals, food additives and cosmetics. Bacillus subtilis is a gram-positive bacterium with excellent riboflavin-producing properties by a "classical" strain improvement strategy. The precursor supply is considered to be a major limiting factor in riboflavin biosynthesis. In Bacillus subtilis, glucose-6-phosphatase dehydrogenase (encoded by zwf) and 6-phosphogluconate dehydrogenase (encoded by gnd) convert glucose-6-phosphate to ribulose-5-phosphate via the Pentose Phosphate (PP) pathway. Riboflavin is synthesized enzymatically from the direct precursors ribose-5-phosphate and Guanosine Triphosphate (GTP) via the riboflavin synthesis pathway, 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 which catalyzes the synthesis of 3, 4-dihydroxy-2-butanone 4-phosphate (DHPB) from ribulose-5-phosphate and 2, 5-diamino-6-ribosyl-4 (3H) -pyrimidinone-50-phosphate (DARPP). Increasing the activity of the enzyme encoded by the gene ribBA increased the riboflavin titer by 25%. Phosphoribosyl Pyrophosphate (PRPP) is a GTP precursor formed in cells by the pathway from the headpurine, first converted to Inosine Monophosphate (IMP), which is then subsequently converted to GTP. The combined overexpression of the mutant genes zwf243 and gnd361 of C.glutamicum increased the riboflavin production in fed-batch fermentations by 39%. By adding additional GTP in the culture medium and redirecting metabolism to the purine pathway through metabolic engineering strategies, riboflavin production is significantly increased. GTP is therefore the major limiting factor in riboflavin synthesis. By eliminating feedback inhibition through metabolic engineering strategies, riboflavin production was increased by 3-fold. By increasing the metabolic flux of the PP pathway and increasing the supply of PRPP, a purine pathway precursor in cells, the yield of riboflavin can be remarkably improved.

Gene deletion and overexpression have become the main strategies for traditional metabolic engineering and gene function studies. In E.coli, overexpression and knock-out techniques are used to up-or down-regulate gene expression to promote both natural and non-natural products. When natural and non-natural product metabolic pathways are introduced into engineered strains to synthesize compounds, multiple enzymes are often required to participate simultaneously. The protein abundance in the multi-enzyme catalytic reaction system is finely adjusted, so that the cells reach the optimal growth and production state. Recent studies have used several strategies to control intracellular protein abundance, such as promoter libraries, RBS libraries, 5' -UTR libraries, mRNA stability, and CRISPRi systems. 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, controlling the strength and timing of gene expression. Gene expression in this pathway is fine-tuned by promoter engineering to balance metabolic flux. To balance the parallelism of parallel multi-gene expression levels, a rapid and modular approach pairs genes with a tight set of ribosome binding sites that can modulate protein abundance by orders of magnitude. EMOPEC (empirical model and oligonucleotides of protein expression changes) is a tool developed based on the contribution of the SD (shine Dalgarno) sequence to protein expression, and can modulate the expression level of any E.coli gene by altering some bases. In addition, the expression of multiple genes in an operon is coordinated by generating a library of regulatable intergenic regions (TIGR), recombining various post-transcriptional control elements and screening for the relative expression levels required for the synthesis of non-natural products. The method can effectively reduce the accumulation of toxic intermediate metabolites and the redundancy of protein expression through the synergistic expression among genes.

In metabolic engineering practice, insufficient supply of precursors is a major bottleneck limiting the yield of the target product. High levels of expression rate-limiting enzymes to drive the metabolic flux of the biosynthetic pathway of the target product are a fast and direct strategy to increase the synthesis of precursors. Complex compounds, especially natural products, are often catalyzed by a series of enzymatic reactions from a variety of precursors. For example, the biosynthetic pathway of thebaine and hydrocodone opioids comprises 21 and 23 enzymatic reactions from plants, mammals, yeast and bacteria. The ratio of precursors in the cell directly affects the efficiency of synthesis of the compound. The lowest concentration of precursor will be the limiting factor, while higher concentrations of precursor may be toxic to the 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 synthesizing riboflavin by using bacillus subtilis, insufficient supply of precursors is a main factor for limiting further improvement of riboflavin yield, and currently, the balance of intracellular equilibrium metabolic flow cannot be realized by simply expressing and knocking out key genes of metabolic pathways, but the distribution imbalance of the intracellular metabolic flow causes increase of byproducts, so that the phenomenon of resource waste is caused.

Disclosure of Invention

The invention provides a strategy capable of efficiently and effectively regulating intracellular metabolic balance, and an engineering strain with high riboflavin production is constructed by using the strategy, and the genetic engineering strain is adopted to produce riboflavin, so that the limitation of metabolic flow imbalance in the fermentation process is removed, and the riboflavin yield is improved. A pool of intergenic regulatory regions (TIGERs) was designed to regulate the expression of multiple genes within a synthetic operon. TIGRs libraries consist of secondary mRNA structures, rnase cleavage sites, and RBS sequences and are used to control mRNA processing and stabilization. The genes in the operon are transcribed into a complete mRNA chain, the cellular RNase E (from gene rne of e.coli) recognizes a specific site on the TIGR sequence, the mRNA is split into individual transcription units, and each transcription unit forms a unique 5 'and 3' structure, which in turn affects the protein level by affecting the stability of the mRNA and the structure of the RBS.

The invention provides an element for regulating gene expression quantity, which consists of an mRNA secondary structure, an RNase E cutting site and an RBS sequence, wherein the nucleotide sequence of the element is shown as SEQ ID NO. 7.

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

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 escherichia coli, 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 a host cell integrated with the RNase E gene to obtain a recombinant cell, culturing the recombinant cell, detecting the fluorescence intensity, and screening the corresponding recombinant cell according to the positive correlation between the fluorescence intensity and the gene expression intensity.

The invention provides a genetic engineering bacterium for producing riboflavin, which utilizes the expression element to regulate and control the expression of zwf, ribBA and ywlf genes, and the genome of the genetic engineering bacterium is also integrated with an RNase E gene from escherichia coli.

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

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.

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, plasmid pMA5-sat is used as an expression vector, and the plasmid pMA5-sat is described in patent document CN 104531745A.

In one embodiment, the genetically engineered bacterium has bacillus subtilis RF1 as a host cell, and bacillus subtilis RF1 is disclosed in patent document CN 104531745A.

The invention provides a method for producing riboflavin by fermentation of genetically engineered bacteria for producing riboflavin.

In one embodiment, the genetically engineered bacteria are cultured to OD ₆₀₀ Adding the powder 24-26 in an amount of 3% (v/v) into a shake flask fermentation system, and fermenting at 37-45 ℃ and 150-250 rpm for not less than 24 h.

In one embodiment, the shake flask fermentation system comprises 15-25 g/L glucose, 15-25 g/L yeast powder, 2-5 g/L ammonium citrate, and 0.5-2 g/L K ₂ HPO ₄ ，0.5～2g/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 Adding the mixture into a fed-batch fermentation system in an amount of 3% (v/v) at 20-25, adding a fed-batch culture medium to keep the glucose concentration not lower than 5g/L, and fermenting at 37-45 ℃ and 400-800 rpm for not less than 24 h.

In one embodiment, the fed-batch fermentation system comprises 15-25 g/L glucose, 15-25 g/L yeast powder, and 2-9 g/L (NH) ₄ ) ₂ HPO ₄ ，3～8g/L K 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, and 4-6 g/L (NH) ₄ ) ₂ HPO ₄ ，1～5g/L K ₂ HPO ₄ ，0.1～1g/L MgSO ₄ ·7H ₂ O。

The invention provides application of the genetic engineering bacteria in the production of riboflavin and derivatives thereof.

Has the advantages that:

(1) the regulatory capacity of the TIGR sequence was characterized using the dual fluorescent reporter genes GFP and mCherry, and in Bacillus subtilis the expression of the second gene in the operon was significantly higher than the expression of the first gene, with a relative fluorescence ratio of red and green that varied by more than 70-fold (from 4:1mCherry/EGFP to 18:1 EGFP/mCherry). In the TIGR library, the fluorescence change range of mCherry is over 80 times, and the fluorescence change range of EGFP is over 40 times. A series of operons containing zwf, ribBA and ywlf genes were generated using a TIGR library and the optimal operon combinations were screened. 5 high riboflavin-producing engineering bacteria are screened from 5000 polyclonal libraries.

(2) At the shake flask fermentation level, the riboflavin titer of the screened engineering bacteria RF1-L3 reaches 2.7g/L, and is increased by 64.35 percent compared with that of the parent strain.

(3) At the 5-L fermentation level, the riboflavin titer of the engineered strain RF1-L3 increased 59.27% at 48 hours, reaching 11.77g/L, compared to the parent strain RF 1.

Drawings

FIG. 1 is a schematic diagram of a TIGR sequence structure;

FIG. 2 is a schematic representation of a TIGR reporter library;

FIG. 3 is a regulatory ability analysis of TIGR library in Bacillus subtilis;

FIG. 4 is a shake flask fermentation analysis of the riboflavin synthesizing ability of a high riboflavin-producing strain;

FIG. 5 is a 5L fermenter which was analyzed for the ability of RF1 and RF-L3 to synthesize riboflavin.

Detailed Description

The media involved in the following examples are as follows:

seed culture medium: 40g/L glucose, 5g/L yeast extract, 10g/L peptone, 10g/L NaCl and 10. mu.g/m L chloramphenicol.

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

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

A supplemented medium: 600g/L glucose, 10g/L yeast powder, 6g/L (NH) ₄ ) ₂ HPO ₄ ，5g/L K ₂ HPO ₄ ，0.5g/L MgSO ₄ ·7H ₂ O。

Minimum medium contains glucose: 20.0g/L, (NH4) ₂ SO ₄ 2.0g/L、KH ₂ PO ₄ 13.1g/L、K ₂ HPO ₄ 6.0g/L、NaC ₆ H ₅ O ₇ ·2H ₂ O 1.2g/L、MgSO ₄ ·7H ₂ O0.05 g/L, and supplemented with tryptophan, phenylalanine and tyrosine (25 mg/L each).

The detection methods referred to in the following examples are as follows:

fluorescence values of EGFP and mCherry were determined to assess the expression range of pMTG libraries. Escherichia coli and Bacillus subtilis containing the pMTG library were inoculated in a sterile 96-well plate (Corning 3603) containing 200. mu.l of LB medium and cultured at 37 ℃ for 10 hours. In addition, EGFP fluorescence (excitation, 490 nm; emission, 530nm), mCherry fluorescence (excitation, 588 nm; emission, 633nm), optical density (absorbance at 600 nm) were measured at the end of the incubation with a microplate multimode reader (BIOTEK, rotation 3). The relative fluorescence density was calculated using equation (1). FPbg represents the fluorescence value of the strain containing no fluorescent protein, and ODbg represents the absorbance of the medium.

The riboflavin emits yellow-green fluorescence under the irradiation of light with the wavelength of 440-500 nm, and the fluorescence intensity is in direct proportion to the concentration of the riboflavin in the dilute solution. For the standard curve of riboflavin, 10mM riboflavin were usedThe mother liquors were diluted to different concentrations, including 10mM, 5mM, 2.5mM, 1.25mM, 0.625mM, 0.313mM and 0.1562 mM. Fluorescence values were measured at an excitation wavelength of 444nm and an absorption wavelength of 500nm, and a standard curve was plotted. Clones from the engineered strain pool were inoculated into 96-well plates containing 200. mu.l minimum medium and cultured with shaking at 220rpm at 37 ℃ for 24 hours. The cell culture was diluted to the appropriate concentration using 0.01m naoh and the fluorescence intensity was measured by a microplate multi-mode reader (BIOTEK, rotation 3). OD monitoring with spectrophotometer ₆₀₀ Growth of cells at nm.

The prepared fermentation broth was diluted with 0.01M NaOH, and then centrifuged at 12000rpm for 2min, and the supernatant was taken to determine the riboflavin concentration. The supernatant was transferred to a new EP tube and diluted to the appropriate concentration range (0.3-0.8) at OD using a spectrophotometer ₄₄₄ The absorbance values were measured at nm. The riboflavin concentration was calculated according to the standard curve of riboflavin concentration. The formula is calculated according to the riboflavin standard curve as follows: 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 No. CN 104531745A.

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

TABLE 1 primer sequences

Example 1: heterologous expression of Gene rne in Bacillus subtilis

The E.coli-derived RNase E Gene rne was integrated into the genome of B.subtilis 168 at Gene amyE (Gene ID:938356) by homologous recombination.

The method comprises the following specific steps:

(1) amplifying an upstream homology arm (1000bp) and a downstream homology arm (1000bp) of the gene amyE according to the primer sequences in the table 1 to respectively obtain upstream and downstream homology arm fragments of the amyE; and separating the PCR product by agarose gel electrophoresis, and cutting the gel to recover the target PCR product. And then fusing the upstream and downstream homologous arms, the Pveg-rne fragment (the fragment nucleotide sequence is shown as SEQ ID NO. 1) and a resistant Marker (bleomycin resistant gene, the nucleotide sequence is shown as SEQ ID NO. 2) by a fusion PCR strategy.

Mixing an upstream and downstream homologous arm, a Pveg-rne fragment and a resistant Marker fragment according to a volume ratio of 1:1, adding PCR enzyme with the same volume for fusion PCR reaction (under the conditions of 98 ℃ for 3min, 98 ℃ for 8s, 61 ℃ for 5s, 72 ℃ for 2min, and amplification for 13 cycles), and taking the product obtained by the reaction as a template, and amplifying the fusion fragment by using primers amyE-veg-rne-F1 and amyE-veg-rne-R3 (under the reaction conditions of 98 ℃ for 3min, 98 ℃ for 10s, 58 ℃ for 15s, 72 ℃ for 1min, and amplification for 34 cycles). And purifying and recovering a PCR product for knockout reaction, wherein the fused fragment contains a bleomycin resistance gene and lox66-lox71 recombination sites, so that the elimination of the later-stage resistance Marker is facilitated.

(2) And (2) transforming the fusion PCR product obtained in the step (1) into Bacillus subtilis168 competent cells by using a transformation method, coating the cells on an LB plate containing bleomycin resistance, culturing the cells at 37 ℃ for 12h, verifying whether the integration of the strains growing on the resistance plate is successful by using a colony PCR method, verifying primers such as amyE-veg-rne-F1 and amyE-veg-rne-R3, and obtaining a positive transformant which is successfully integrated after PCR verification.

(3) And (3) granulating the pDG148 to transfer to the correct positive transformant prepared in the step (2), performing shake culture at 180rpm and 37 ℃ for 24h, then taking part of culture solution to coat on an LB plate, performing culture at 37 ℃ for 12h until a single colony grows out, using a sterile toothpick to correspondingly spot the single colony on the LB plate to another resistance plate containing the bleomycin one by one, performing culture at 37 ℃ for 12h, and obtaining a colony which can grow on the LB plate and cannot grow on the bleomycin resistance plate as a knock-out strain for eliminating the bleomycin resistance.

The resulting knockout strain was shake-cultured at 180rpm at 42 ℃ for 24 hours to eliminate pDG148 plasmid. And coating part of the culture solution on an LB solid culture medium, culturing at 37 ℃ for 12h, then using a sterile toothpick to correspondingly spot colonies on an LB plate to another ampicillin-containing resistant plate one by one, culturing at 37 ℃ for 12h, and allowing the colonies to grow on the LB plate, wherein the colonies which cannot grow on the bleomycin resistant plate are knockout strains for eliminating pDG148 particles. The obtained strain is a traceless knockout target strain BSF 01.

Example 2: construction of TIGR library containing dual-fluorescence reporter gene

TIGRs libraries include mRNA secondary structure, RNase E cleavage sites, and RBS sequence control elements. The TIGRs library utilizes the secondary structure of mRNA, RNase E cleavage site, and RBS sequence to control mRNA processing and stabilization, thereby affecting gene expression on the operon (fig. 1).

To further amplify the synthetic bio-tool applicable to Bacillus subtilis, a M-TIGR-G library (FIG. 2) was constructed based on TIGR and introduced into strain BSF01, which overexpresses gene rne derived from E.coli at the amyE site. Fluorescence values of the two reporter genes were determined to assess the regulatory ability of the TIGR library in bacillus subtilis.

The method comprises the following specific steps:

1. preparation of TIGR library

(1) TIGR is synthesized by using PCR, and oligonucleotide sequences TIGR-A, TIGR-B, TIGR-C and TIGR-D (nucleotide sequences are respectively shown as SEQ ID NO. 3-6) are assembled into a chimeric DNA sequence (the nucleotide sequence is shown as SEQ ID NO. 7). Five nucleotides in each oligonucleotide sequence were replaced with random nucleotides "N" to synthesize a diversity sequence. The mCherry gene, the TIGR sequence (chimeric DNA sequence) and the GFP gene are integrated through fusion PCR to construct a fusion fragment M-TITRs-G. Then, the fusion PCR fragment M-TIGRs-G was inserted into the plasmid pP43NMK by Gibson assembly, and the reporter plasmid pMTG for screening TIGR was constructed such that transcription of the reporter operon was controlled by the strong promoter P43. The plasmid pMTG was transformed into competent E.coli JM109 cells, and transformants were selected with ampicillin. Coli library colonies were scraped and plasmids were extracted, collected from three separate plates, and transformed into BSF 01.

(2) And (2) inoculating the polyclonal library obtained in the step (1) into a 96-well plate, and measuring GFP and mCherry fluorescence values to construct a TIGR library.

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

Example 3: construction of TIGR library for screening high-yield riboflavin engineering strain

In the manner as described in example 1, the strain RF1-R was constructed by integrating the E.coli-derived RNase E gene rne into amyE gene of the riboflavin-producing strain RF 1.

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

The method comprises the following specific steps:

(1) gene fragments containing TIGR libraries were constructed by fusion PCR. First, primers ZRY-F2/ZRY-R2, ZRY-F4/ZRY-R4 were designed, and TIGR libraries between genes zwf, ribBA and ywlf were amplified using the TIGR library (the chimeric DNA sequence of which nucleotide sequence is shown in SEQ ID NO. 7) in example 2 as a template. And performing fusion PCR on the PCR fragment zwf and TIGR library to obtain zwf-TIGR (the nucleotide sequence is shown as SEQ ID NO. 8). A PCR fragment of ribBA-TIGR (nucleotide sequence shown in SEQ ID NO. 9) was obtained according to the above-mentioned method. Plasmid pMA5-sat was treated with restriction enzymes EcoRI and KpnI at 37 ℃ for 30min and purified for recovery, then the purified PCR fragments zwf-TIGR, ribBA-TIGR and ywlf were ligated into one PCR fragment by the fusion PCR method, and the plasmid pMA5-sat was inserted, a zwf-TIGR-ribBA-TIGR-ywlf library was obtained by Gibson assembly and transformed into E.coli DH5 α competence, cultured at 37 ℃ for 16h, and the correct transformants were selected by colony PCR. The obtained transformants were mixed and plasmid-extracted to construct a library of engineered strains, which were transformed into RF1-R, and the clones from the library of engineered strains were inoculated into a 96-well plate containing 200. mu.l of minimal medium and cultured at 37 ℃ for 24 hours with shaking at 220 rpm. The cell culture was diluted to an appropriate concentration using 0.01M NaOH, and the mutant strain with the highest fluorescence intensity was screened by measuring the fluorescence intensity with a microplate multimode reader (BIOTEK, rotation 3).

Example 4: production of riboflavin by fermentation of genetically engineered strains in the shake flask stage

The mutant strains with the highest fluorescence intensity screened in example 3 were rescreened using shake flask fermentation.

Individual colonies were inoculated in 10ml-LB medium and cultured with shaking at 180rpm at 37 ℃. After 16 hours, the culture was transferred to a 250ml baffle flask containing 50ml of LBG medium (40g/L glucose, 5g/L yeast extract, 10g/L peptone and 10g/L NaCl) in an inoculum size of 1% (v/v) and shaken at 220rpm for 24 hours at 41 ℃ to obtain a seed culture. Seed culture (OD) ₆₀₀ 25.21) were transferred to a 500ml baffled shake flask containing 50ml fermentation medium (3% (v/v) inoculum size) and then incubated at 41 ℃ for 48 hours with shaking at 220 rpm. During the shake flask fermentation, 1 ml of cell suspension was sampled every 12 hours to measure OD ₆₀₀ And riboflavin concentration.

Results show that the riboflavin titer of the five engineering bacteria is remarkably improved, wherein the titer advantage of the engineering bacteria RF1-L3 is the most obvious. The riboflavin titer of the engineering bacteria RF1-L3 reaches 2.7g/L and is improved by 64.35 percent compared with that of the parent strain of 1.67g/L (the riboflavin yield of each strain after 48h of fermentation is shown in figure 4), and the riboflavin titers of the engineering bacteria RF1-L1, RF1-L2, RF1-L4 and RF1-L5 are respectively 1.94g/L, 2.01g/L, 2.16g/L and 2.24 g/L.

TABLE 2 TIGR sequences from strains RF 1-L1-RF 1-L5

Example 5: production of riboflavin by fermentation of genetically engineered strains in horizontal fermentation tank

The method comprises the following specific steps:

(1) respectively inoculating bacillus subtilis strains RF1-aPaGaTgV and RF1 which are cultured in 10mL LB culture medium for 24h into 100mL of seed culture medium (the seed culture medium comprises 20g/L glucose, 20g/L yeast powder, 4g/L ammonium citrate and 1g/L K) according to the inoculation amount of 3% (v/v) in volume ratio ₂ HPO ₄ ，1g/L KH ₂ PO ₄ ,，2g/L MgSO ₄ ·7H ₂ O，0.04g/L MnCl ₂ ，0.06g/L CaCl ₂ ，2g/L CuSO ₄ ) Culturing at 41 deg.C and 180rpm for 16h to obtain seed solution (OD) ₆₀₀ ＝23.5)；

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

The residual glucose concentration in the fermentation liquor is kept to be not less than 5g/L by controlling the flow of the feed culture medium. In the fermentation process, 1M H is used ₂ SO ₄ And 50% ammonia water to maintain the pH of the fermentation broth at 6.8. Before starting the batch feed, the rotation speed was maintained at 400rpm, and then gradually increased to 900rpm until the end of the fermentation, the temperature being maintained at 41 ℃.

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

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> BAA220441A

<130> a method for efficiently synthesizing riboflavin based on balanced gene expression

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 3263

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gcaccgaagc gttaacggcc atcgacatca actccgcacg cgcgacccgc ggcggcgata 1020

tcgaagaaac cgcgtttaac actaacctcg aagctgccga tgagattgct cgtcagctgc 1080

gcctgcgtga cctcggcggc ctgattgtta tcgacttcat cgacatgacg ccagtacgcc 1140

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 sequence

<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 sequence

<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 sequence

<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 sequence

<220>

<221> misc_feature

<222> (31)..(35)

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

<400> 5

gccttgaatt tacagtattt tagtggcctt nnnnnatact attcggtcac cttatccgct 60

caaga 65

<210> 6

<211> 71

<212> DNA

<213> Artificial sequence

<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 sequence

<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 sequence

<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 aagaccgcgc 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 sequence

<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 nnnaaggtca 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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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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