Escherichia coli strain with high yield of riboflavin and construction method and application thereof

Abstract

The invention discloses an escherichia coli strain with high yield of riboflavin as well as a construction method and application of the escherichia coli strain. A series of Escherichia coli strains with high yield of riboflavin are constructed, the constructed Escherichia coli with high yield of riboflavin is safe and harmless, glucose is used as a substrate for fermentation under the aerobic condition of a shake flask, the yield of riboflavin is 1117.8 mg / L, 1330.1 mg / L, 1430.8 mg / L, 1508.7 mg / L and 1640.8 mg / L in sequence, and the yields are 112.5 mg / g of glucose, 134.1 mg / g of glucose, 146.3 mg / g of glucose, 158.8 mg / g of glucose and 168.1 mg / g of glucose in sequence; when the strain SN04M is subjected to fed-batch fermentation in a 1L fermentation tank, the yield of riboflavin reaches 8.52 g / L, and the total yield is 189.9 mg / g glucose. According to the invention, the fermentation yield of riboflavin and the substrate utilization rate of riboflavin to glucose can be obviously improved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to Escherichia coli strains that produce high levels of riboflavin, their construction methods, and applications. Background Technology

[0002] Riboflavin, also known as vitamin B2, is a yellow, naturally occurring, water-soluble vitamin. The molecular formula of riboflavin is C2. 17 H 20 N4O6, with a molecular weight of 376.4 g / mol, is used for riboflavin. The biochemical activity of riboflavin is related to the active conjugated double bond at the 1,5-position of the isochloroazide in its molecule. Riboflavin is primarily involved in the following biochemical reactions: respiratory chain energy production, amino acid and lipid oxidation, hydroxylation of aromatic compounds, and the synthesis of proteins and certain hormones. Furthermore, riboflavin plays a role in adjuvant cancer treatment by activating the immune system to kill tumor cells. Riboflavin supplementation can enhance macrophage function and reduce inflammation caused by Staphylococcus aureus infection. In addition to medical uses, riboflavin is also used in feed additives, food additives, pharmaceuticals, cosmetics, and microbial fuel cells.

[0003] Industrial riboflavin production has evolved from complex and expensive chemical synthesis to microbial fermentation. Through various mutagenesis and metabolic engineering strategies, *Ashbya gossypii* and *Bacillus subtilis* have become important hosts for riboflavin production. However, fungal fermentation involves long cycles, complex feedstock composition, the need for added unsaturated fatty acids, and high cell viscosity in the later stages of fermentation, increasing the difficulty and cost of subsequent separation and purification. *Bacillus subtilis* used for riboflavin production is mostly obtained through mutagenesis screening, which presents challenges in identifying beneficial mutations for further strain improvement. *Escherichia coli*, on the other hand, has a clear genetic background and advanced genetic tools. Liu Shuang et al. overexpressed purine pathway genes, knocked out inefficient respiratory chains, and balanced reducing power. The modified engineered strain LS72T achieved a riboflavin yield of 1339 mg / L and a glucose yield of 135.8 mg / g in shake-flask fermentation. Its fed-batch fermentation yield reached 21 g / L and a glucose yield of 110 mg / g, indicating that *E. coli* is a chassis cell with great potential for riboflavin production. A search revealed no reports of perturbing the yrfg and nrfA genes to increase riboflavin yield, nor of using metabolic engineering to simultaneously modify the *E. coli* sthA, rpe, yrfg, and nrfA genes to enhance riboflavin production. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the main objective of this invention is to provide a high-riboflavin-producing Escherichia coli strain, its construction method, and its application, which solves the problems of limited substrate conversion rate and high cost of raw materials, especially carbon sources.

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: The method for constructing a high-riboflavin-producing Escherichia coli strain includes the following steps: S1: Prepare the sthA-tet nucleic acid fragment, integrate the sthA gene into Escherichia coli LS55 using the λ-red gene recombination method, and construct strain SN01; electroporate plasmid pLS01 into competent cells of strain SN01 to obtain the first high-riboflavin-producing Escherichia coli strain, named SN01T; the nucleotide sequence encoding the sthA gene is shown in SEQ ID NO.1, and the nucleotide sequence of the nucleic acid fragment sthA-tet is shown in SEQ ID NO.16; S2: Prepare the rpe-tet nucleic acid fragment, and replace the start codon of the rpe gene in strain SN01 described in step S1 with GTG using the λ-red gene recombination method to construct strain SN02; electroporate plasmid pLS01 into competent cells of strain SN02 to obtain a second high-riboflavin-producing Escherichia coli strain, named SN02T; encode the... rpe The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the nucleotide sequence of the nucleic acid fragment rpe-tet is shown in SEQ ID NO.23; S3: Prepare the nucleic acid fragment yrfg-tet. Replace the wild-type RBS of the gene yrfg encoding purine nucleotidase in strain SN02 with the weaker RBS1 using the λ-red gene recombination method to obtain strain SN03. Electroporate plasmid pLS01 into competent cells of strain SN03 to obtain a third high-riboflavin-producing Escherichia coli strain, named SN03T. The nucleotide sequence encoding RBS1 is shown in SEQ ID NO.4, and the nucleotide sequence of the nucleic acid fragment yrfg-tet is shown in SEQ ID NO.30. S4: The binding-nrfA of the specific target gene nrfA was ligated into plasmid p20c-MicC using a seamless cloning method to construct plasmid p20c-nrfA-MicC; the sRNA expression unit sRNA-nrfA of nrfA was ligated into plasmid pLS01 using a seamless cloning method to construct recombinant plasmid pRF-sRNA-nrfA; plasmid pRF-sRNA-nrfA was electroporated into the competent cells of strain SN03 obtained in step S3 to obtain the fourth high-riboflavin-producing Escherichia coli strain, named SN03M; the nucleotide sequence of the binding-nrfA is shown in SEQ ID NO.5, the nucleotide sequence of the sRNA-nrfA is shown in SEQ ID NO.6, and the nucleotide sequence of the recombinant plasmid pRF-sRNA-nrfA is shown in SEQ ID NO.7; S5: Prepare the RF-yajI-tet nucleic acid fragment, and integrate the artificial riboflavin operon into the strain SN03 described in step S3 by λ-red gene recombination to construct strain SN04; electroporate the plasmid pRF-sRNA-nrfA constructed in step S4 into the competent cells of strain SN04 to obtain the fifth high-riboflavin-producing Escherichia coli strain, named SN04M; the nucleotide sequence encoding the artificial riboflavin operon is shown in SEQ ID NO.8, and the nucleotide sequence of the RF-yajI-tet nucleic acid fragment is shown in SEQ ID NO.40.

[0006] The method described above constructs a high-riboflavin-producing Escherichia coli strain.

[0007] Application of the high-riboflavin-producing Escherichia coli strain in the fermentation production of riboflavin.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: The high-riboflavin-producing *E. coli* strain constructed in this invention is safe and harmless. Under shake-flask aerobic conditions using glucose as a substrate, the riboflavin yields were 1117.8 mg / L, 1330.1 mg / L, 1430.8 mg / L, 1508.7 mg / L, and 1640.8 mg / L, with yields of 112.5 mg / g glucose, 134.1 mg / g glucose, 146.3 mg / g glucose, 158.8 mg / g glucose, and 168.1 mg / g glucose, respectively. In a 1L fermenter, strain SN04M achieved a riboflavin yield of 8.52 g / L with a total yield of 189.9 mg / g glucose during fed-batch fermentation. This invention significantly improves the fermentation yield of riboflavin and the substrate utilization rate of glucose for riboflavin. Attached Figure Description

[0009] Figure 1 A map of recombinant fragments integrating the sthA gene; Figure 2 A map of the recombinant fragment in which the start codon of the rpe gene is replaced with GTG; Figure 3 A map of the recombinant fragment in which the wild-type RBS of the yrfg gene is replaced with RBS1; Figure 4 A map of the recombinant fragment integrating the artificial riboflavin operon; Figure 5 Map of the recombinant expression plasmid pRF-sRNA-nrfA; Figure 6 The fermentation curve of strain SN04M in a 500 mL shake flask is shown. Figure 7 The fermentation curve of strain SN04M in a 1L fermenter is shown. Detailed Implementation

[0010] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all the accompanying drawings and corresponding descriptions merely illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0011] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0012] The original strain of Escherichia coli MG1655 used in this invention was obtained from CGSC (Coli Genetic Stock Center, http: / / cgsc.biology.yale.edu / ).

[0013] The Escherichia coli LS55 and LS55T used in this invention were constructed according to the steps in the following article: LIU S, HU W, WANG Z, CHEN T. Rational Engineering of Escherichia coli for High-Level Production of Riboflavin [J]. Journal of Agricultural and Food Chemistry, 2021, 69(41): 12241-9. The plasmid pLS01 was derived from the following article: LIU S, KANG P, CUI ZZ, et al. Increase driboflavin production by knockout of 6-phosphofructokinase I and blocking the Entner-Doudoroff pathway in Escherichia coli [J]. Biotechnology Letters, 2016, 38(8): 1307-14. The plasmid p20c-MicC is derived from the following article: Hu W, Liu S, Wang Z, Chen T. Improving riboflavin production by knocking down ribF, purA and guaC genes using synthetic regulatory small RNA [J]. J Biotechnol, 2021, 336:25-29. The plasmids pTKRED and pTKS / CS were obtained from Addgene (https: / / www.addgene.org / ).

[0014] The methods for λ-Red gene recombination and shake-flask fermentation of the strain are derived from the following article: LIN ZQ, XU ZB, LI YF, et al. Metabolic engineering of Escherichia coli for the production of riboflavin [J]. Microbial Cell Factories, 2014, 13. The riboflavin standard used was purchased from Sigma-Aldrich (https: / / www.sigmaaldrich.cn / CN / zh). The primers used were synthesized by Genewiz (https: / / www.genewiz.com.cn / ). The seamless cloning reagent used was purchased from Abclonal (https: / / abclonal.com.cn / ). The DNA polymerase and other molecular biology reagents used were purchased from Novizan (https: / / bio.vazyme.com / ). Other biochemical reagents used were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0015] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0016] Example 1: Construction of the first high-riboflavin-producing Escherichia coli strain SN01T, comprising the following steps: In this embodiment, the gene sthA (SEQ ID NO.1) encoding NAD(P) transhydrogenase is derived from the genome of Escherichia coli MG1655.

[0017] Step 1: The sthA gene was integrated into Escherichia coli LS55 by λ-red gene recombination to construct strain SN01.

[0018] (1) Using the genome of *Escherichia coli* MG1655 as a template, PCR amplification was performed using sthA UF (SEQ ID NO.41) and sthA UR (SEQ ID NO.42), sthA GF (SEQ ID NO.43) and sthA GR (SEQ ID NO.44), sthA LF (SEQ ID NO.45) and sthA LR (SEQ ID NO.46) as upstream and downstream primers, respectively, to obtain nucleic acid fragments sthA-U (SEQ ID NO.9), sthA-G (SEQ ID NO.10) and sthA-L (SEQ ID NO.11), with sizes of 510 bp, 1645 bp and 519 bp, respectively; using plasmid pTKS / CS as a template, PCR amplification was performed using sthA TF (SEQ ID NO.47) and tet-R (SEQ ID NO.48), tet-F (SEQ ID NO.49) and sthA TR (SEQ ID NO.46), respectively. Using primers NO.50, PCR amplification was performed to obtain nucleic acid fragments sthA-T1 (SEQ ID NO.12) and sthA-T2 (SEQ ID NO.13), with sizes of 774 bp and 714 bp, respectively.

[0019] (2) Using three fragments, sthA-U (SEQ ID NO. 9), sthA-G (SEQ ID NO. 10), and sthA-T1 (SEQ ID NO. 12), as templates, and sthA UF (SEQ ID NO. 41) and tet-R (SEQ ID NO. 48) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment sthA-1 (SEQ ID NO. 14), with a size of 2889 bp; using two fragments, sthA-T2 (SEQ ID NO. 13) and sthA-L (SEQ ID NO. 11), as templates, and tet-F (SEQ ID NO. 49) and sthA LR (SEQ ID NO. 46) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment sthA-2 (SEQ ID NO. 15), with a size of 1210 bp; using two fragments, sthA-1 (SEQ ID NO. 14) and sthA-2 (SEQ ID NO. 15), as templates, and sthA UF (SEQ ID NO. 41) and tet-R (SEQ ID NO. 48), PCR fusion was performed to obtain nucleic acid fragment sthA-1 (SEQ ID NO. 14), with a size of 1210 bp; using two fragments, sthA-1 (SEQ ID NO. 14) and sthA-2 (SEQ ID NO. 15), and sthA UF (SEQ ID NO. 48) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment sthA-2 (SEQ ID NO. 15), with a size of 1210 bp; using two fragments, sthA-1 (SEQ ID NO. 14) and sthA-2 (SEQ ID NO. 15), and sthA UF (SEQ ID NO. 48) as template Using SEQ ID NO.41 and sthA LR (SEQ ID NO.46) as upstream and downstream primers, PCR fusion yielded the nucleic acid fragment sthA-tet (SEQ ID NO.16), with a size of 3955 bp.

[0020] (3) The plasmid pTKRED was electroporated into Escherichia coli strain LS55 and made into electrocompetent cells; the nucleic acid fragment sthA-tet (SEQ ID NO.16) obtained in step (2) above was electroporated into the electrocompetent cells obtained in this step; using tet-F (SEQ ID NO.49) and sthA LR (SEQ ID NO.46) as upstream and downstream primers, colony PCR was used to verify the recombination of sthA-tet (SEQ ID NO.16), and the correct band size was 1210 bp; using sthA UF (SEQ ID NO.41) and sthAL-R (SEQ ID NO.46) as upstream and downstream primers, colony PCR was used to verify the ejection of the tetracycline resistance gene, and the correct band size was 2642 bp; the strain that successfully integrated the NAD(P) transhydrogenase gene sthA was named SN01.

[0021] Step 2: The plasmid pLS01 was electroporated into the competent cells of strain SN01 to obtain the first high-riboflavin-producing Escherichia coli strain, which was named strain SN01T.

[0022] Example 2: Construction of a second high-riboflavin-producing Escherichia coli strain SN02T, comprising the following steps: In this embodiment, the gene rpe (SEQ ID NO.2) encoding ribulose phosphate-3-epimerase was derived from the genome of Escherichia coli MG1655.

[0023] Step 1: Prepare the rpe-tet nucleic acid fragment, and replace the start codon of the rpe gene in strain SN01 with GTG by λ-red gene recombination to construct strain SN02.

[0024] (1) Using the genome of Escherichia coli MG1655 as a template, PCR amplification was performed using rpe UF (SEQ ID NO.51) and rpe UR (SEQ ID NO.52), rpe LF (SEQ ID NO.53) and rpe LR (SEQ ID NO.54) as upstream and downstream primers, respectively, to obtain nucleic acid fragments rpe-U (SEQ ID NO.17) and rpe-L (SEQ ID NO.18), with sizes of 547 bp and 658 bp, respectively; using plasmid pTKS / CS as a template, PCR amplification was performed using rpe TF (SEQ ID NO.55) and tet-R (SEQ ID NO.48), tet-F (SEQ ID NO.49) and rpe TR (SEQ ID NO.56) as upstream and downstream primers, respectively, to obtain nucleic acid fragments rpe-T1 (SEQ ID NO.19) and rpe-T2 (SEQ ID NO.20), with sizes of 786 bp and 719 bp, respectively.

[0025] (2) Using rpe-U (SEQ ID NO.17) and rpe-T1 (SEQ ID NO.19) as templates, and rpe UF (SEQ ID NO.51) and tet-R (SEQ ID NO.48) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment rpe-1 (SEQ ID NO.21), with a size of 1303 bp; using rpe-T2 (SEQ ID NO.20) and rpe-L (SEQ ID NO.18) as templates, and tet-F (SEQ ID NO.49) and rpe LR (SEQ ID NO.54) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment rpe-2 (SEQ ID NO.22), with a size of 1357 bp; using rpe-1 (SEQ ID NO.21) and rpe-2 (SEQ ID NO.22) as templates, and rpe UF (SEQ ID NO.51) and rpe LR (SEQ ID NO.19 ...1 (SEQ ID NO.21), with a size of 1303 bp; using rpe-T2 (SEQ ID NO.21) and rpe-L (SEQ ID NO.18) as templates, and rpe UF (SEQ ID NO.51) and rpe LR (SEQ ID NO.19) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment rpe-2 (SEQ ID NO.22), with a size of 1357 bp; using rpe-T2 (SEQ ID NO.21 Using NO.54 as upstream and downstream primers, PCR fusion yielded the nucleic acid fragment rpe-tet (SEQ ID NO.23), which is 2516 bp in size.

[0026] (3) The plasmid pTKRED was electroporated into strain SN01 and made into electrocompetent cells; the nucleic acid fragment rpe-tet (SEQ ID NO.23) obtained in step (2) above was electroporated into the electrocompetent cells obtained in this step; using tet-F (SEQ ID NO.49) and rpe LR (SEQ ID NO.54) as upstream and downstream primers, colony PCR was used to verify the recombination of rpe-tet (SEQ ID NO.23), and the correct band size was 1357 bp; using rpe UF (SEQ ID NO.51) and rpe LR (SEQ ID NO.54) as upstream and downstream primers, colony PCR was used to verify the ejection of the tetracycline resistance gene, and the correct band size was 1203 bp; the strain that successfully replaced the start codon of ribulose phosphate-3-epimerase rpe with GTG was named SN02.

[0027] Step 2: The plasmid pLS01 was electroporated into the competent cells of strain SN02 to obtain a second high-riboflavin-producing Escherichia coli strain, which was named strain SN02T.

[0028] Example 3: Construction of a third high-riboflavin-producing Escherichia coli strain SN03T, comprising the following steps: In this embodiment, the gene encoding purine nucleotidase yrfg (SEQ ID NO.3) is derived from the genome of Escherichia coli MG1655.

[0029] Step 1: Prepare the nucleic acid fragment yrfg-tet, and replace the wild-type RBS of the gene yrfg encoding purine nucleotidase in strain SN02 with the weaker RBS1 (SEQ ID NO.4) by λ-red gene recombination to obtain strain SN03.

[0030] (1) Using the genome of *Escherichia coli* MG1655 as a template, PCR amplification was performed using yrfg UF (SEQ ID NO. 57) and yrfg UR (SEQ ID NO. 58), yrfg LF (SEQ ID NO. 59) and yrfg LR (SEQ ID NO. 60) as upstream and downstream primers, respectively, to obtain nucleic acid fragments yrfg-U (SEQ ID NO. 24) and yrfg-L (SEQ ID NO. 25), with sizes of 680 bp and 702 bp, respectively; using plasmid pTKS / CS as a template, PCR amplification was performed using yrfg TF (SEQ ID NO. 61) and tet-R (SEQ ID NO. 48), tet-F (SEQ ID NO. 49) and yrfg TR (SEQ ID NO. 62) as upstream and downstream primers, respectively, to obtain nucleic acid fragments yrfg-T1 (SEQ ID NO. 26) and yrfg-T2 (SEQ ID NO. 68). NO.27), with sizes of 768 bp and 714 bp.

[0031] (2) Using yrfg-U (SEQ ID NO.24) and yrfg-T1 (SEQ ID NO.26) fragments as templates, and yrfgU-F (SEQ ID NO.57) and tet-R (SEQ ID NO.48) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment yrfg-1 (SEQ ID NO.28), with a size of 1428 bp; using yrfg-T2 (SEQ ID NO.27) and yrfg-L (SEQ ID NO.25) fragments as templates, and tet-F (SEQ ID NO.49) and yrfg LR (SEQ ID NO.60) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment yrfg-2 (SEQ ID NO.29), with a size of 1395 bp; using yrfg-1 (SEQ ID NO.28) and yrfg-2 (SEQ ID NO.29) fragments as templates, and yrfg UF (SEQ ID NO.26) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment yrfg-2 (SEQ ID NO.29), with a size of 1395 bp; using yrfg-U (SEQ ID NO.28) and yrfg-T1 (SEQ ID NO.26) fragments as templates, and yrfg UF (SEQ ID NO.40) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment yrfg-2 (SEQ ID NO.29), with a size of 1395 bp; using yrfg-T2 (SEQ ID NO.28) and yrfg-T1 (SEQ ID NO.26) fragments as templates, and yrfg UF (SEQ ID NO.40) as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment yrfg- Using NO.57 and yrfg LR (SEQ ID NO.60) as upstream and downstream primers, PCR fusion yielded the nucleic acid fragment yrfg-tet (SEQ ID NO.30), with a size of 2679 bp.

[0032] (3) The plasmid pTKRED was electroporated into strain SN02 and made into electrocompetent cells; the nucleic acid fragment yrfg-tet (SEQ ID NO.30) obtained in step (2) above was electroporated into the electrocompetent cells obtained in this step; using tet-F (SEQ ID NO.49) and yrfg LR (SEQ ID NO.60) as upstream and downstream primers, colony PCR was used to verify the recombination of yrfg-tet (SEQ ID NO.30), and the correct band size was 1395 bp; using yrfg UF (SEQ ID NO.57) and yrfg LR (SEQ ID NO.60) as upstream and downstream primers, colony PCR was used to verify the ejection of the tetracycline resistance gene, and the correct band size was 1366 bp; the strain that successfully replaced the wild-type RBS of purine nuclease yrfg with the weaker RBS1 (SEQ ID NO.4) was named SN03.

[0033] Step 2: The plasmid pLS01 was electroporated into the competent cells of strain SN03 to obtain the third high-riboflavin-producing Escherichia coli strain, which was named strain SN03T.

[0034] Example 4: Construction of a fourth high-riboflavin-producing Escherichia coli strain SN03M, comprising the following steps Step 1: Binding-nrfA, which specifically targets the gene nrfA, is ligated into plasmid p20c-MicC using a seamless cloning method to construct plasmid p20c-nrfA-MicC; Using plasmid p20c-MicC as a template, and p20c-F (SEQ ID NO. 63) and p20c-R (SEQ ID NO. 64) as upstream and downstream primers, PCR amplification was performed to obtain the nucleic acid fragment nrfA-MicC (including the binding-nrfA sequence (SEQ ID NO. 5), SEQ ID NO. 31), with a size of 2878 bp; the nrfA-MicC fragment was circularized using a seamless cloning method to construct plasmid p20c-nrfA-MicC; Step 2: The sRNA expression unit sRNA-nrfA of nrfA is ligated into plasmid pLS01 using a seamless cloning method to construct the recombinant plasmid pRF-sRNA-nrfA; Using plasmid p20c-nrfA-MicC as a template, and nrfA-F (SEQ ID NO. 65) and nrfA-R (SEQ ID NO. 66) as upstream and downstream primers, PCR amplification was performed to obtain the linear sRNA-nrfA fragment (SEQ ID NO. 6), with a size of 320 bp. Using plasmid pLS01 as a template, and pRF-F (SEQ ID NO. 67) and pRF-R (SEQ ID NO. 68) as upstream and downstream primers, PCR amplification was performed to obtain the pRF vector fragment (SEQ ID NO. 32), with a size of 7245 bp. The linear sRNA-nrfA fragment (SEQ ID NO. 6) and the pRF vector fragment (SEQ ID NO. 32) were combined using a seamless cloning method to construct the plasmid pRF-sRNA-nrfA (SEQ ID NO. 7). Step 3: Electroporate the plasmid pRF-sRNA-nrfA (SEQ ID NO.7) obtained in Step 2 into the competent cells of strain SN03 obtained in Example 3 to obtain the fourth high-riboflavin-producing Escherichia coli strain, named strain SN03M.

[0035] Example 5: Construction of the fifth high-riboflavin-producing Escherichia coli strain SN04M, including the following steps. In this embodiment, the nucleotide fragment (SEQ ID NO.8) encoding the artificial riboflavin operon is derived from plasmid pLS01.

[0036] Step 1: Prepare the RF-yajI-tet nucleic acid fragment, and integrate the artificial riboflavin operon into strain SN03 through λ-red gene recombination to construct strain SN04.

[0037] (1) Using the genome of *Escherichia coli* MG1655 as a template, PCR amplification was performed using yajI UF (SEQ ID NO. 69) and yajI UR (SEQ ID NO. 70), yajI LF (SEQ ID NO. 71) and yajI LR (SEQ ID NO. 72) as upstream and downstream primers, respectively, to obtain nucleic acid fragments yajI-U (SEQ ID NO. 33) and yajI-L (SEQ ID NO. 34), with sizes of 595 bp and 545 bp, respectively; using plasmid pTKS / CS as a template, PCR amplification was performed using TF (SEQ ID NO. 73) and tet-R (SEQ ID NO. 48), tet-F (SEQ ID NO. 49) and TR (SEQ ID NO. 74) as upstream and downstream primers, respectively, to obtain nucleic acid fragments T1 (SEQ ID NO. 35) and T2 (SEQ ID NO. 36), with sizes of 774 bp and 728 bp, respectively. Using plasmid pLS01 as a template, and with RF-F (SEQ ID NO.75) and RF-R (SEQ ID NO.76) as upstream and downstream primers respectively, PCR amplification was performed to obtain the nucleic acid fragment GENE-RF (SEQ ID NO.37), which is 4091 bp in size. (2) Using three fragments, yajI-U (SEQ ID NO.33), GENE-RF (SEQ ID NO.37), and T1 (SEQ ID NO.35), as templates, and yajI UF (SEQ ID NO.69) and tet-R (SEQ ID NO.48), as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment UGT1 (SEQ ID NO.38), with a size of 5459 bp; using two fragments, T2 (SEQ ID NO.36) and yajI-L (SEQ ID NO.34), as templates, and tet-F (SEQ ID NO.49) and yajI LR (SEQ ID NO.72), as upstream and downstream primers, PCR fusion was performed to obtain nucleic acid fragment T2L (SEQ ID NO.39), with a size of 1238 bp; using two fragments, UGT1 (SEQ ID NO.38) and T2L (SEQ ID NO.39), as templates, and yajI UF (SEQ ID NO.69) and yajI LR (SEQ ID NO.35 ... Using NO.72 as upstream and downstream primers, PCR fusion yielded the nucleic acid fragment RF-yajI-tet (SEQ ID NO.40) integrating the artificial riboflavin operon, with a size of 6553 bp.

[0038] (3) The plasmid pTKRED was electroporated into strain SN03 and made into electrocompetent cells; the nucleic acid fragment RF-yajI-tet (SEQ ID NO.40) obtained in step (2) above was electroporated into the electrocompetent cells obtained in this step; using tet-F (SEQ ID NO.49) and yajI LR (SEQ ID NO.72) as upstream and downstream primers, colony PCR was used to verify the recombination of RF-yajI-tet (SEQ ID NO.40), and the correct band size was 1238 bp; using yajI UF (SEQ ID NO.69) and yajI LR (SEQ ID NO.72) as upstream and downstream primers, colony PCR was used to verify the ejection of the tetracycline resistance gene, and the correct band size was 5240 bp; the strain that successfully integrated the artificial riboflavin operon was named SN04.

[0039] Step 2: The plasmid pRF-sRNA-nrfA (SEQ ID NO.7) constructed in Example 4 was electroporated into the competent cells of strain SN04 to obtain the fifth high-riboflavin-producing Escherichia coli strain, which was named strain SN04M.

[0040] Example 6: Preparation of shake-flask fermentation medium, including the following steps: Take 1 mL of the mixture of component I, 1 mL of component II, 1 mL of component III and IV, add glucose to make a final concentration of 10 g / L, add yeast extract to make a final concentration of 5 g / L, and add distilled water to 50 mL; thus obtaining the fermentation medium. Component I is: take 10g (NH4)2SO4 and 2g MgSO4, make up to 200mL with distilled water, and sterilize at 121℃ for 20min. Component II is: Take 38.3g Na2HPO4 and 15g KH2PO4, dilute to 200mL with distilled water, and sterilize at 121℃ for 20min; Component III is: Take 5g of ferric ammonium citrate, 2g of CaCl2·2H2O, and 41.7mL of HCl aqueous solution with a concentration of 12mol / L, and dilute to 1000mL with distilled water. The component IV is as follows: Take 1g ZnSO4·7H2O, 0.3g MnCl2·4H2O, 3g H3BO3, 2g CoCl2·6H2O, 0.1g CuSO4·5H2O, 0.2g NiCl2·6H2O, and 0.3g NaMoO4·2H2O, and dilute to 1000mL with distilled water; The mixture of components III and IV is prepared by taking 100 ml of component III and 1 ml of component IV, making up to 200 mL with distilled water, adjusting the pH to 4.5-5.5 with 5 M NaOH aqueous solution, and sterilizing at 121℃ for 20 min.

[0041] Example 7: Shake-flask fermentation method for a high-riboflavin-producing Escherichia coli strain, comprising the following steps (1) Activation of strains: The strains SN01T, SN02T, SN03T, SN03M and SN04M obtained in Examples 1, 2, 3, 4 and 5 were streaked on LB solid medium and cultured at 37℃ for 12-16 h to revitalize the strains; (2) Seed culture: The single colony obtained in step (1) was inoculated into LB liquid medium and cultured at 37℃ and 220rpm for 12-16 h. (3) Shake flask fermentation: The seed liquid obtained in step (2) is fermented according to the initial OD. 600 The inoculum was 0.025, and the inoculum was added to the shake flask fermentation medium. The volume of the shake flask was 50 mL / 500 mL. Chloramphenicol was added to bring the final concentration to 10 mg / L. The cultures were incubated at 37℃ and 220 rpm for 48 hours. Recombinant Escherichia coli strains SN01T, SN02T, SN03T, SN03M, and SN04M fermented under aerobic conditions in shake flasks using glucose as a substrate. The riboflavin yields were 1117.8 mg / L, 1330.1 mg / L, 1430.8 mg / L, 1508.7 mg / L, and 1640.8 mg / L, respectively. Compared to the starting strain LS55T (1020.2 mg / L), the riboflavin yields increased by 9.6%, 30.4%, 40.2%, 47.9%, and 60.8%, respectively. The yields were 112.5 mg / g glucose, 134.1 mg / g glucose, 146.3 mg / g glucose, 158.8 mg / g glucose, and 168.1 mg / g glucose, respectively.

[0042] Example 8: Preparation of fermentation medium for fermentation tank, including the following steps: Take 10 mL of a mixture of components I, II, III, and IV, add glucose to a final concentration of 18 g / L, add yeast extract to a final concentration of 20 g / L, and add distilled water to a final volume of 500 mL to obtain the initial fermentation medium; take 20 mL of a mixture of components I, II, III, and IV, add glucose to a final concentration of 500 g / L, add yeast extract to a final concentration of 20 g / L, and add distilled water to a final volume of 500 mL to obtain the fed-batch fermentation medium; Component I is: take 10g (NH4)2SO4 and 2g MgSO4, make up to 200mL with distilled water, and sterilize at 121℃ for 20min. Component II is: Take 38.3g Na2HPO4 and 15g KH2PO4, dilute to 200mL with distilled water, and sterilize at 121℃ for 20min; Component III is: Take 5g of ferric ammonium citrate, 2g of CaCl2·2H2O, and 41.7mL of HCl aqueous solution with a concentration of 12mol / L, and dilute to 1000mL with distilled water. The component IV is as follows: Take 1g ZnSO4·7H2O, 0.3g MnCl2·4H2O, 3g H3BO3, 2g CoCl2·6H2O, 0.1g CuSO4·5H2O, 0.2g NiCl2·6H2O, and 0.3g NaMoO4·2H2O, and dilute to 1000mL with distilled water; The mixture of components III and IV is prepared by taking 100 ml of component III and 1 ml of component IV, making up to 200 mL with distilled water, adjusting the pH to 4.5-5.5 with 5 M NaOH aqueous solution, and sterilizing at 121℃ for 20 min.

[0043] Example 9: A fermentation method for a high-riboflavin-producing Escherichia coli strain SN04M in a fermenter, comprising the following steps: (1) Activation of strain: The strain SN04M obtained in Example 5 was streaked on LB solid medium and cultured at 37°C for 12-16 h to rejuvenate the strain; (2) Seed culture: The single colony obtained in step (1) was inoculated into LB liquid medium and cultured at 37℃ and 220rpm for 12-16 h. (3) Fermentation in a fermenter: The seed culture obtained in step (2) was inoculated into a 1L fermenter containing 500mL of initial fermentation medium at a volume ratio of 10%. Chloramphenicol was added to bring the final concentration to 10 mg / L. Fermentation was carried out at 37℃, pH=6.8±0.2, with stirring speed and dissolved oxygen as the parameters. The dissolved oxygen was set at 15%~80%, and the stirring speed was set at 300~1100 rpm. When the glucose concentration in the medium dropped below 1 g / L, fed-batch medium was used to control the glucose concentration to maintain 2~5 g / L. Fermentation was stopped when the riboflavin concentration in the fermentation broth no longer increased. After 40 h of fermentation, the riboflavin yield reached 8.52 g / L, and the maximum cell density OD was 8.52 g / L. 600 The concentration was 95.4%, and the total yield was 189.9 mg / g glucose. Fermentation results are shown in [see figure]. Figure 7 .

[0044] The above embodiments only describe a portion of the specific implementation methods of the present invention in detail, and are not limited to the embodiments disclosed herein. Furthermore, the substantive content protected by the present invention is not limited thereto. Any other modifications, equivalent substitutions, improvements, etc., made based on the principles and techniques of the present invention without departing from its design scope are all within the protection scope of the present invention.

Claims

1. A method for constructing a high-riboflavin-producing *Escherichia coli* strain, characterized in that, Includes the following steps: S1: Prepare the sthA-tet nucleic acid fragment, integrate the sthA gene into Escherichia coli LS55 using the λ-red gene recombination method, and construct strain SN01; electroporate plasmid pLS01 into competent cells of strain SN01 to obtain the first high-riboflavin-producing Escherichia coli strain, named SN01T; the nucleotide sequence encoding the sthA gene is shown in SEQ ID NO.1, and the nucleotide sequence of the nucleic acid fragment sthA-tet is shown in SEQ ID NO.16; S2: Prepare the rpe-tet nucleic acid fragment, and replace the start codon of the rpe gene in strain SN01 described in step S1 with GTG using the λ-red gene recombination method to construct strain SN02; electroporate plasmid pLS01 into competent cells of strain SN02 to obtain a second high-riboflavin-producing Escherichia coli strain, named SN02T; encode the... rpe The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the nucleotide sequence of the nucleic acid fragment rpe-tet is shown in SEQ ID NO.23; S3: Prepare the nucleic acid fragment yrfg-tet. Replace the wild-type RBS of the gene yrfg encoding purine nucleotidase in strain SN02 with the weaker RBS1 using the λ-red gene recombination method to obtain strain SN03. Electroporate plasmid pLS01 into competent cells of strain SN03 to obtain a third high-riboflavin-producing Escherichia coli strain, named SN03T. The nucleotide sequence encoding RBS1 is shown in SEQ ID NO.4, and the nucleotide sequence of the nucleic acid fragment yrfg-tet is shown in SEQ ID NO.

30. S4: The binding-nrfA of the specific target gene nrfA was ligated into plasmid p20c-MicC using a seamless cloning method to construct plasmid p20c-nrfA-MicC; the sRNA expression unit sRNA-nrfA of nrfA was ligated into plasmid pLS01 using a seamless cloning method to construct recombinant plasmid pRF-sRNA-nrfA; plasmid pRF-sRNA-nrfA was electroporated into the competent cells of strain SN03 obtained in step S3 to obtain the fourth high-riboflavin-producing Escherichia coli strain, named SN03M; the nucleotide sequence of the binding-nrfA is shown in SEQ ID NO.5, the nucleotide sequence of the sRNA-nrfA is shown in SEQ ID NO.6, and the nucleotide sequence of the recombinant plasmid pRF-sRNA-nrfA is shown in SEQ ID NO.7; S5: Prepare the RF-yajI-tet nucleic acid fragment, and integrate the artificial riboflavin operon into the strain SN03 described in step S3 by λ-red gene recombination to construct strain SN04; electroporate the plasmid pRF-sRNA-nrfA constructed in step S4 into the competent cells of strain SN04 to obtain the fifth high-riboflavin-producing Escherichia coli strain, named SN04M; the nucleotide sequence encoding the artificial riboflavin operon is shown in SEQ ID NO.8, and the nucleotide sequence of the RF-yajI-tet nucleic acid fragment is shown in SEQ ID NO.

40.

2. The Escherichia coli strain with high riboflavin production constructed by the construction method of claim 1.

3. The application of the high-riboflavin-producing Escherichia coli strain of claim 2 in the fermentation production of riboflavin.

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