Method for producing riboflavin

Abstract

Provided is a method for producing riboflavin. According to the method, a wild-type expression regulatory element in the rib operon of an engineered bacterium for producing riboflavin is replaced with a strong expression regulatory element, thereby significantly improving the riboflavin yield and the carbon source conversion rate.

Description

A method for producing riboflavin

[0001] priority

[0002] This application claims the rights and priority of Chinese application No. 2024118136090, filed on December 10, 2024. The entire contents of Chinese application No. 2024118136090 are incorporated herein by reference for all purposes. Technical Field

[0003] This disclosure belongs to the field of microbial technology, and in particular relates to a method for producing riboflavin. Background Technology

[0004] Riboflavin, also known as vitamin B2, is a water-soluble B vitamin. Most microorganisms and plants can synthesize it, while humans and animals must obtain it from food. In organisms, riboflavin mainly exists in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). As a coenzyme or prosthetic group of flavoproteins, it participates in electron transport and redox reactions in the respiratory chain of body tissues, and is an essential nutrient for maintaining normal metabolism and physiological functions. Riboflavin is widely used in medicine, food, and animal feed. Due to its wide range of applications, the demand for riboflavin both domestically and internationally continues to increase.

[0005] In Bacillus subtilis, the direct precursors to riboflavin synthesis are guanine triphosphate (GTP) and ribulose 5-phosphate. GTP originates from the cellular purine synthesis pathway, while ribulose 5-phosphate is a product of glucose metabolism via the pentose phosphate pathway. All enzymes used in the synthesis process are encoded by the rib operon. Genes involved in riboflavin biosynthesis include ribG (ribD), ribB (ribE), ribA, and ribH. The ribA gene encodes two enzymatic activities: GTP cyclic hydrolase II, which catalyzes the first step in riboflavin biosynthesis, and 3,4-dihydroxy-2-butanone synthase (DHBPS), which catalyzes the conversion of 5-phosphoribosomes to 3,4-dihydroxy-2-butanone phosphate (DHBP). Deaminases and reductases are encoded by the first gene of the operon, ribG (ribD). The penultimate step of riboflavin biosynthesis is catalyzed by lumazine synthase, the gene product of ribH. The final step of the catalytic pathway, riboflavin synthase, is encoded by the second gene of the operon, ribB (ribD). The function of ribT, located at the 3' end of the rib operon, is currently unclear; however, its gene product is not essential for riboflavin synthesis.

[0006] Several methods have been developed to reduce the feedback regulation of riboflavin synthesis by mutating the ribC gene to decrease intracellular FMN concentration; or by using traditional mutagenesis to screen for high-yielding riboflavin strains. However, traditional mutagenesis breeding is labor-intensive and lacks efficient and rapid screening methods, which greatly limits the ability of Bacillus subtilis to improve riboflavin production. Furthermore, the mRNA leader region of the riboflavin operon is regulated by transcriptional attenuation mediated by FMN as a small molecule effector. It is necessary to modify the mRNA leader region of the riboflavin operon to remove the original riboswitch regulatory mechanism, enabling constitutive expression of the riboflavin operon so that cells can continuously synthesize riboflavin. This can be achieved by replacing natural / weak promoters with strong / artificial promoters, such as overexpressing the rib operon with promoters like Spo2, P43, Pveg, PgsiB, and PJ23119, or by increasing the copy number of the rib operon within the chromosome and linking it to a corresponding single promoter. However, these methods all have several drawbacks. For example, driving rib operon transcription with a single promoter may not achieve saturated mRNA levels; furthermore, the host cell chromosome multicopy expression cassette may be unstable in production, and may even prevent further expression of individual rib operon genes due to feedback inhibition, resulting in low riboflavin production and carbon source conversion. Summary of the Invention

[0007] This disclosure provides a method for producing riboflavin, comprising the following steps:

[0008] a) Cultivating engineered bacteria capable of producing riboflavin in a culture medium, wherein the nucleic acid sequence of the expression regulatory element of the rib operon in the engineered bacteria is a sequence containing SEQ ID NO: 2 or having 98% or 99% identity with SEQ ID NO: 2;

[0009] b) Riboflavin accumulates in the culture medium.

[0010] In one embodiment, the method further includes c) separating riboflavin from the riboflavin-containing liquid.

[0011] In one embodiment, the engineered bacteria is Bacillus subtilis.

[0012] In one specific embodiment, the Bacillus subtilis comprises ribC G199D .

[0013] In one specific embodiment, the Bacillus subtilis comprises ribC G199D and CCPN A44S .

[0014] In one specific embodiment, the Bacillus subtilis comprises ribC G199D And ΔcydB.

[0015] In one specific embodiment, the Bacillus subtilis comprises ribC G199D ,ccpN A44S And ΔcydB.

[0016] In one embodiment, the Bacillus subtilis comprises the amino acid sequence shown in SEQ ID NO: 4 or the nucleic acid sequence shown in SEQ ID NO: 9. In one embodiment, the Bacillus subtilis comprises the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 6 or the nucleic acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 11. In one embodiment, the Bacillus subtilis comprises the nucleic acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 13. In one embodiment, the Bacillus subtilis comprises the nucleic acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.

[0017] In one embodiment, the Bacillus subtilis is selected from Bacillus subtilis MH-1910, Bacillus subtilis MH-1914, Bacillus subtilis MH-1918 and Bacillus subtilis MH-1922.

[0018] In one embodiment, the Bacillus subtilis MH-1910 contains ribC G199D The Bacillus subtilis MH-1914 contains ribC G199D and CCPN A44S The Bacillus subtilis MH-1918 contains ribC G199D ,ccpN A44S And ΔcydB, the Bacillus subtilis MH-1922 contains ribC G199D And ΔcydB.

[0019] In one embodiment, the rib operon encodes an expression riboflavin synthesis-related enzyme, which includes ribG (ribD), ribB (ribE), ribA, and / or ribH.

[0020] On the other hand, this disclosure provides an expression regulatory element whose nucleic acid sequence contains SEQ ID NO: 2 or a sequence having 98% or 99% identity with SEQ ID NO: 2.

[0021] On the other hand, this disclosure provides an expression cassette comprising a rib operon, the rib operon including the expression control elements as described above and encoding riboflavin synthesis-related genes, the riboflavin synthesis-related genes including ribG (ribD), ribB (ribE), ribA and / or ribH.

[0022] On the other hand, this disclosure provides a carrier comprising the expression box as described above. In one specific embodiment, the carrier is an expression carrier.

[0023] On the other hand, this disclosure provides an engineered bacterium comprising the expression control element, expression cassette, or carrier as described above. In one specific embodiment, the engineered bacterium is Bacillus subtilis.

[0024] Furthermore, this disclosure provides the application of the aforementioned expression regulatory elements, expression cassettes, vectors, or engineered bacteria in the production of riboflavin. In one embodiment, the engineered bacteria is Bacillus subtilis. In a specific embodiment, the Bacillus subtilis contains ribC G199D In one specific embodiment, the Bacillus subtilis comprises ribC G199D and CCPN A44s In one specific embodiment, the Bacillus subtilis comprises ribC G199D and ΔcydB. In one specific embodiment, the Bacillus subtilis comprises ribC G199D ,ccpN A44s And ΔcydB. Attached Figure Description

[0025] This disclosure can be more fully understood with reference to the following figures.

[0026] Figure 1 shows the construction map of the pJOE8999-P1910-Riboperon plasmid. Here, Riboperon represents the operon. Detailed Implementation

[0027] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0028] The present invention will be further described below through specific embodiments. Unless otherwise specified, the terminology used herein has the same meaning as commonly understood by those skilled in the art.

[0029] As used herein, “identity” refers to sequence similarity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions in each sequence aligned for comparison purposes. When positions in the compared sequences are occupied by the same bases, then the molecules at that position are identical. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or procedures can be used to calculate identity between two sequences, including those available as part of the GCG Sequence Analysis Package (University of Wisconsin, Madison, Wis.) and which can be used, for example, by default, FASTA or BLAST. For example, those skilled in the art can expect nucleic acids that have at least 98% or 99% identity with a particular nucleic acid sequence described herein and preferably exhibit substantially the same function.

[0030] As used herein, "expression regulatory element" refers to a nucleic acid sequence that affects the expression of an operatively linked nucleic acid. As described herein, the expression regulatory element includes a strong RBS sequence and promoter sequence in the promoter -35 region, -10 region, and gsiB mRNA leader region. In some specific embodiments of the invention, the expression regulatory element comprises SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence having 98% or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2. In this document, the "expression regulatory element" is operatively linked to the rib operon to enhance rib operon expression.

[0031] 1. Instrument Information

[0032] Spectrophotometer (Techcomp UH5300), gel imaging system (Tanon 24 TMINI / acoX-163), centrifuge (Eppendorf Centrifuge 5804R), centrifuge (Sigma 1-14), electroporator (Bio-Rad MicroPulse), gene amplification instrument (Dongsheng ETC811), shaker (Zhichu), microplate reader (BioTek SynergyLX), SGD-4 fully automated reducing sugar analyzer (SBA-40C).

[0033] 2. Culture medium formulation

[0034] LB (g / L): peptone 10, yeast extract 5, sodium chloride 10, solid culture medium with agar powder 18, pH controlled at 7.0 by NaOH.

[0035] Seed culture medium formula (g / L): glucose 20, yeast powder 5, corn steep liquor powder 5, potassium dihydrogen phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.02, manganese sulfate 0.01, pH 7.0-7.2, sterilized at 121℃ for 20 minutes.

[0036] Fermentation medium formula (g / L): glucose 50, soybean meal powder 25, potassium dihydrogen phosphate 3, urea 6, manganese sulfate 0.01, magnesium sulfate 5, monosodium glutamate 10, corn steep liquor powder 15, calcium carbonate 25, pH 7.0-7.2, sterilized at 121℃ for 20 minutes.

[0037] 3. Primers

[0038] Table 1. Primer sequences

[0039] 4. Preparation and electroconversion methods of Bacillus subtilis competent cells

[0040] Streak Bacillus subtilis culture from cryopreservation tubes onto LB agar plates and incubate at 37°C overnight (12-16 h). Transfer small, millet-grain-sized pieces of the culture to 20 ml of growth medium and inoculate. Incubate at 37°C with shaking at 200 rpm for 3 h. Based on initial OD... 600 =0.2% inoculated into 100ml of growth medium, and cultured at 37℃ with shaking at 200rpm until OD. 600 =0.8-1.0. Transfer the bacterial culture to a 50 mL centrifuge tube and incubate on ice for 30 min. Centrifuge at 4°C and 5000 rpm for 6 min, and carefully discard the supernatant. Resuspend the bacterial cells in 40 mL of pre-chilled washing buffer and centrifuge at 4°C and 5000 rpm for 6 min, and carefully discard the supernatant. Repeat the washing process twice more, discarding the supernatant each time. Resuspend the cells using the remaining washing buffer by pipetting, and aliquot 100 μL / tube into pre-chilled 1.5 mL EP tubes on ice. Use immediately or store at -80°C.

[0041] Take one tube of freshly prepared Bacillus subtilis competent cells, or take one tube of competent cells from a -80℃ freezer, thaw it on ice, add 1-2 μg of plasmid, mix gently, and incubate on ice for 2-3 min.

[0042] Competent cells were carefully transferred into a fully pre-cooled 2mm electroporation cup, fixed onto the electroporator, and electroporated at 2.5kV. The breakdown time was preferably between 4.2 and 6.0ms.

[0043] Quickly add 1 mL of resuscitation medium to the electroporation cuvette and transfer it to a 15 mL centrifuge tube. Incubate at 30°C (low temperature is required for thermosensitive plasmids) and 200 rpm for 3 hours.

[0044] Centrifuge at 6000 rpm for 2 min, resuspend the bacterial cells in the remaining 100 μL of supernatant, spread them all on solid plates with the corresponding resistance, invert the plates, and incubate at 30℃ (low temperature for temperature-sensitive plasmids) for 12-16 h.

[0045] 5.OD 600 Detection methods

[0046] Mix the fermentation broth thoroughly, dilute it with 0.8% NaCl solution to an appropriate concentration, and mix well. Using 0.8% NaCl solution as a blank, measure the absorbance at 600 nm using a visible spectrophotometer (with the displayed value controlled between 0.2 and 0.8). Calculate the OD using the following formula. 600 Value: OD 600 = Dilution factor * Absorbance.

[0047] 6. Methods for detecting riboflavin

[0048] Mix the fermentation broth thoroughly, dilute it to an appropriate factor with 0.01 mol / L NaOH, mix well, and dissolve in the light for 20 min. Centrifuge at 12000 rpm for 2 min, take the supernatant, use 0.01 mol / L NaOH as a blank, and measure the absorbance at 444 nm using a visible spectrophotometer (the displayed value should be controlled between 0.2 and 0.8). Calculate the riboflavin content using the following formula: FB (mg / L) = (dilution factor * absorbance) / 0.0321.

[0049] Example

[0050] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0051] Example 1: Construction of a plasmid for a strong expression regulatory element overexpressing the Riboperon operon

[0052] 1.1 Construction of the pJOE8999-PgsiB-Riboperon vector

[0053] Primers OPERON-N20-1F and OPERON-N20-2F (containing N20) and gRNA-R were designed. Using plasmid pJOE8999 (HonorGene, product code: HG-VCH1431) as a template, a 244bp long gRNA sequence with N20 was amplified to obtain the gRNA sequence.

[0054] Primers 2434-F and 2434-R were designed to amplify the 862bp length of Bacillus subtilis 168 genome using it as a template, and the upstream homologous arm was obtained.

[0055] Primers 2435-2F, 2435-1F and 2436-R were designed to amplify a length of 1063 bp using the Bacillus subtilis 168 genome as a template, and downstream homologous arms were obtained.

[0056] The pJOE8999 plasmid was double-digested with Bsa I and Xba I, dephosphorylated with FastAP, and then recovered by gel extraction.

[0057] The fragments obtained in the above steps and the linearized vector were mixed in the same system. Following the instructions of the Novizan ClonExpress II One Step Cloning Kit C112, the target vector plasmid pJOE8999-PgsiB-Riboperon was obtained by one-step cloning. The plasmid was transformed into E. coli Top10, and positive clones were grown. After enzyme digestion to verify that the clones were correct, they were sent for sequencing. The plasmids with correct sequencing results were stored at -20℃.

[0058] 1.2 Construction of the pJOE8999-P1910-Riboperon vector

[0059] Primers OPERON-N20-1F and OPERON-N20-2F (containing N20) and gRNA-R were designed. Using plasmid pJOE8999 as a template, a 244bp long gRNA sequence with N20 was amplified to obtain the gRNA sequence.

[0060] Primers 2434-F and p1910-LR were designed to amplify a length of 1163 bp using the Bacillus subtilis 168 genome as a template, and upstream homologous arms were obtained.

[0061] Primers p1910-RF and 2436-R were designed to amplify a length of 1015 bp using the Bacillus subtilis 168 genome as a template, and downstream homologous arms were obtained.

[0062] The pJOE8999 plasmid was double-digested with Bsa I and Xba I, dephosphorylated with FastAP, and then recovered by gel extraction.

[0063] The fragments obtained in the above steps and the linearized vector were mixed in the same system. Following the instructions of the Novizan ClonExpress II One Step Cloning Kit C112, the target vector plasmid pJOE8999-P1910-Riboperon was obtained by one-step cloning. The plasmid was transformed into E. coli Top10, and positive clones were grown. After enzyme digestion to verify that the clones were correct, they were sent for sequencing. The plasmids with correct sequencing results were stored at -20℃.

[0064] Table 2. Sequence Information

[0065] Example 2: Construction of Bacillus subtilis MH-1910

[0066] 2.1 Construction of the pJOE8999-ribC-G199D vector

[0067] Primers ribC-G199D-N20 and gRNA-R were designed, and plasmid pJOE8999 was used as a template to amplify a 244bp long gRNA sequence with N20.

[0068] Primers ribC-FF and ribC-G199D-FR were designed to amplify 898 bp length using Bacillus subtilis 168 genome as a template, and upstream homologous arms were obtained.

[0069] Primers ribC-G199D-RF and ribC-RR were designed and amplified to a length of 533 bp using the Bacillus subtilis 168 genome as a template to obtain downstream homologous arms.

[0070] The pJOE8999 plasmid was double-digested with Bsa I and Xba I, dephosphorylated with FastAP, and then recovered by gel extraction.

[0071] The fragments obtained in the above steps and the linearized vector were mixed in the same system. Following the instructions of the Novizan ClonExpress II One Step Cloning Kit C112, the target vector plasmid pJOE8999-ribC-D144R was obtained by one-step cloning. The plasmid was transformed into E. coli Top10, and positive clones were grown. After enzyme digestion to verify that the clones were correct, they were sent for sequencing. The plasmids with correct sequencing results were stored at -20℃.

[0072] 2.2 Electroporation and Obtaining Recombinant Strains

[0073] The pJOE8999-ribC-G199D plasmid was electroporated into Bacillus subtilis 168 to induce editing and recombination. Verification was performed using primers ribC-FF and ribC-RR. After PCR amplification and correct sequencing, the strain was named MH-1910 (genotype B. subtilis 168: ribC). G199D );

[0074] Example 3: Replacement of strongly expressed regulatory elements using Bacillus subtilis MH-1910 as the starting strain.

[0075] 3.1 Construction of Bacillus subtilis MH-1911

[0076] The pJOE8999-PgsiB-Riboperon plasmid was electroporated into Bacillus subtilis MH-1910 (genotype B. subtilis168:ribC). G199D ), induced editing and recombination, designed primers 2436-YZ-R and pgsiB-YZ-F for verification, and after PCR verification and correct sequencing, named strain MH-1911 (genotype B. subtilis168: ribC G199D (PgsiB-Riboperon).

[0077] 3.2 Construction of Bacillus subtilis MH-1913

[0078] The pJOE8999-P1910-Riboperon plasmid was electroporated into Bacillus subtilis MH-1910 (genotype B. subtilis168:ribC). G199D ), induced editing and recombination, verified by 2436-YZ-R and p1910-YZ-F, and after PCR verification and sequencing confirmation, the strain was named MH-1913 (genotype B. subtilis168: rbCG). 199D (P1910-Riboperon).

[0079] Example 4: Construction of Bacillus subtilis MH-1914

[0080] 4.1 Construction of the pJOE8999-ccpN-A44S vector

[0081] Primers ccpN-N20 and gRNA-R were designed, and plasmid pJOE8999 was used as a template to amplify a 244bp long gRNA sequence with N20.

[0082] Primers ccpN-FF and ccpN-FR were designed to amplify a length of 1010 bp using the Bacillus subtilis 168 genome as a template, and upstream homologous arms were obtained.

[0083] Primers ccpN-RF and ccpN-RR were designed and used as a template of Bacillus subtilis 168 genome to amplify 818 bp in length, obtaining downstream homologous arms.

[0084] The pJOE8999 plasmid was double-digested with Bsa I and Xba I, dephosphorylated with FastAP, and then recovered by gel extraction.

[0085] The fragments obtained in the above steps and the linearized vector were mixed in the same system. Following the instructions of the Novizan ClonExpress II One Step Cloning Kit C112, the target vector plasmid pJOE8999-ccpN-A44S was obtained by one-step cloning. The plasmid was transformed into E. coli Top10, and positive clones were grown. After enzyme digestion to verify that the clones were correct, they were sent for sequencing. The plasmids with correct sequencing results were stored at -20℃.

[0086] 4.2 Electroporation and Obtaining Recombinant Strains

[0087] The pJOE8999-ccpN-A44S plasmid was electroporated into Bacillus subtilis-derived strain MH-1910 to induce editing and recombination. Verification was performed using primers ccpN-FF and ccpN-RR. After PCR amplification and correct sequencing, the strain was named MH-1914 (genotype B. subtilis168: ribC). G199D CCPN A44S ).

[0088] Example 5: Replacement of strongly expressed regulatory elements using Bacillus subtilis MH-1914 as the starting strain.

[0089] 5.1 Construction of Bacillus subtilis MH-1915

[0090] The pJOE8999-PgsiB-Riboperon plasmid was electroporated into Bacillus subtilis MH-1914 (genotype B. subtilis168:ribC). G199D CCPN A44S Induced editing and recombination were performed, and primers 2436-YZ-R and pgsiB-YZ-F were designed for verification. After PCR verification and correct sequencing, the strain was named MH-1915 (genotype B. subtilis168: ribC). G199D CCPN A44S (PgsiB-Riboperon).

[0091] 5.2 Construction of Bacillus subtilis MH-1917, a strain derived from Bacillus subtilis 168

[0092] The pJOE8999-P1910-Riboperon plasmid was electroporated into Bacillus subtilis MH-1914 (genotype B. subtilis168:ribC). G199D CCPN A44S ), induced editing and recombination, verified by 2436-YZ-R and p1910-YZ-F, and after PCR verification and sequencing confirmation, the strain was named MH-1917 (genotype B. subtilis168: ribC). G199D CCPN A44s (P1910-Riboperon).

[0093] Example 6: Construction of Bacillus subtilis MH-1918

[0094] 6.1 Construction of the pJOE8999-ΔcydB vector

[0095] Primers cydB-N20 and gRNA-R were designed, and plasmid pJOE8999 was used as a template to amplify a 244bp long gRNA sequence containing N20.

[0096] Primers cydB-FF and cydB-FR were designed and amplified to a length of 916 bp using the Bacillus subtilis 168 genome as a template to obtain the upstream homologous arm.

[0097] Primers cydB-RF and cydB-RR were designed and amplified to a length of 1044 bp using the Bacillus subtilis 168 genome as a template to obtain downstream homologous arms.

[0098] The pJOE8999 plasmid was double-digested with Bsa I and Xba I, dephosphorylated with FastAP, and then recovered by gel extraction.

[0099] The fragments obtained in the above steps and the linearized vector were mixed in the same system. Following the instructions of the Novizan ClonExpress II One Step Cloning Kit C112, the target vector plasmid pJOE8999-ΔcydB was obtained by one-step cloning. The plasmid was transformed into E. coli Top10, and positive clones were grown. After enzyme digestion to verify that the clones were correct, they were sent for sequencing. The plasmids with correct sequencing were stored at -20℃.

[0100] 6.2 Electroporation and Obtaining Recombinant Strains

[0101] The pJOE8999-ΔcydB plasmid was electroporated into Bacillus subtilis-derived strain MH-1914 (genotype B. subtilis168:ribC). G199D CCPN A44S The strain was induced to undergo editing and recombination, verified using primers cydB-FF and cydB-RR, and named strain MH-1918 (genotype B. subtilis168: ribC) after PCR amplification and correct sequencing. G199D CCPN A44s ,ΔcydB).

[0102] Example 7: Replacement of strongly expressed regulatory elements using Bacillus subtilis MH-1918 as the starting strain.

[0103] 7.1 Construction of Bacillus subtilis MH-1919

[0104] The pJOE8999-PgsiB-Riboperon plasmid was electroporated into Bacillus subtilis MH-1918 (genotype B. subtilis168:ribC). G199D CCPN A44s ΔcydB) induced editing and recombination, primers 2436-YZ-R and pgsiB-YZ-F were designed for verification, and after PCR verification and correct sequencing, the strain was named MH-1919 (genotype B. subtilis168: ribC). G199D CCPN A44S , ΔcydB, PgsiB-Riboperon).

[0105] 7.2 Construction of Bacillus subtilis MH-1921

[0106] The pJOE8999-P1910-Riboperon plasmid was electroporated into Bacillus subtilis MH-1918 (genotype B.subtilis168:ribC). G199D CCPN A44s ΔcydB) induced editing and recombination, 2436-YZ-R and p1910-YZ-F were used for verification, and after PCR verification and sequencing confirmation, the strain was named MH-1921 (genotype B. subtilis168: ribC). G199D CCPN A44S , ΔcydB, P1910-Riboperon).

[0107] Example 8: Construction of Bacillus subtilis MH-1922

[0108] 8.1 Electroporation and Obtaining Recombinant Strains

[0109] The pJOE8999-ΔcydB plasmid was electroporated into Bacillus subtilis-derived strain MH-1910 (genotype B. subtilis168:ribC). G199D The strain was induced to undergo editing and recombination, verified using primers cydB-FF and cydB-RR, and named MH-1922 (genotype B. subtilisl68: ribC) after PCR amplification and correct sequencing. G199D ,ΔcydB).

[0110] Example 9: Replacement of strongly expressed regulatory elements using Bacillus subtilis MH-1922 as the starting strain.

[0111] 9.1 Construction of Bacillus subtilis MH-1923

[0112] The pJOE8999-PgsiB-Riboperon plasmid was electroporated into Bacillus subtilis MH-1922 (genotype B. subtilis168:ribC). G199D ΔcydB) induced editing and recombination, primers 2436-YZ-R and pgsiB-YZ-F were designed for verification, and after PCR verification and correct sequencing, the strain was named MH-1923 (genotype B. subtilis168: ribC). G199D , ΔcydB, PgsiB-Riboperon).

[0113] 9.2 Construction of Bacillus subtilis MH-1925

[0114] The pJOE8999-P1910-Riboperon plasmid was electroporated into Bacillus subtilis MH-1922 (genotype B. subtilis168:ribCG). 199D ΔcydB) induced editing and recombination, 2436-YZ-R and p1910-YZ-F were used for verification, and after PCR verification and sequencing confirmation, the strain was named MH-1925 (genotype B. subtilis168: ribC). G199D ,ΔcydB,P1910-Riboperon).

[0115] Example 10: Verification of riboflavin fermentation by mutant strain

[0116] The glycerol-preserved strains MH-1911, MH-1913, and the starting strain MH-1910 were aseptically streaked on LB agar plates and cultured overnight at 37°C. A loopful of culture was inoculated into 30 mL of seed culture medium and cultured at 110 rpm at 37°C for 7-8 h. Then, a 10% inoculum was transferred to 30 mL of fermentation medium and cultured at 120 rpm at 37°C for 46 h. The fermentation results are shown in Table 2. The results show that the starting strain MH-1910 had a higher riboflavin accumulation and product yield. Both engineered strains accumulated more riboflavin than the starting strain, indicating that the genetic engineering modification of the riboflavin promoter had a positive effect. Compared to the expression regulatory element PgsiB (SEQ ID NO: 1) reported in the existing patent, the strong expression regulatory element P1910 protected in this patent significantly increased riboflavin production. The fermentation results are shown in Table 3.

[0117] Table 3. Results of riboflavin fermentation test

[0118] The glycerol-preserved strains MH-1915, MH-1917, and the starting strain MH-1914 were aseptically streaked on LB agar plates and incubated overnight at 37°C. A loopful of the culture was inoculated into 30 mL of seed culture medium and incubated at 110 rpm at 37°C for 7-8 h. Then, a 10% inoculum was transferred to 30 mL of fermentation medium and incubated at 120 rpm at 37°C for 46 h. The fermentation results are shown in Table 3. The results show that the starting strain MH-1914 had a higher riboflavin accumulation and product yield. Both engineered strains accumulated more riboflavin than the starting strain, indicating that the genetic engineering modification of the riboflavin promoter had a positive effect. Compared to the expression regulatory element PgsiB (SEQ ID NO: 1) reported in the existing patent, the strong expression regulatory element P1910 protected in this patent significantly increased riboflavin production. The fermentation results are shown in Table 4.

[0119] Table 4. Results of Riboflavin Fermentation Test

[0120] The glycerol-preserved strains MH-1919, MH-1921, and the starting strain MH-1918 were aseptically streaked on LB agar plates and incubated overnight at 37°C. A loopful of the culture was inoculated into 30 mL of seed culture medium and incubated at 110 rpm at 37°C for 7-8 h. Then, a 10% inoculum was transferred to 30 mL of fermentation medium and incubated at 120 rpm at 37°C for 46 h. The fermentation results are shown in Table 3. The results show that the starting strain MH-1918 had a higher riboflavin accumulation and product yield. Both engineered strains accumulated more riboflavin than the starting strain, indicating that the genetic engineering modification of the riboflavin promoter had a positive effect. Compared to the expression regulatory element PgsiB (SEQ ID NO: 1) reported in the existing patent, the strong expression regulatory element P1910 protected in this patent significantly increased riboflavin production. The fermentation results are shown in Table 5.

[0121] Table 5. Results of riboflavin fermentation test

[0122] The glycerol-preserved strains MH-1923, MH-1925, and the starting strain MH-1922 were aseptically streaked on LB agar plates and cultured overnight at 37°C. A loopful of culture was inoculated into 30 mL of seed culture medium and cultured at 110 rpm at 37°C for 7-8 h. Then, a 10% inoculum was transferred to 30 mL of fermentation medium and cultured at 120 rpm at 37°C for 46 h. The fermentation results are shown in Table 3. The results show that the starting strain MH-1922 had a higher riboflavin accumulation and product yield. Both engineered strains accumulated more riboflavin than the starting strain, indicating that the genetic engineering modification of the riboflavin promoter had a positive effect. Compared to the expression regulatory element PgsiB (SEQ ID NO: 1) reported in the existing patent, the strong expression regulatory element P1910 protected in this patent significantly increased riboflavin production. The fermentation results are shown in Table 6.

[0123] Table 6. Results of Riboflavin Fermentation Test

[0124] By incorporating via reference

[0125] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0126] Equivalence

[0127] This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. A method for producing riboflavin, comprising the following steps: a) Cultivating engineered bacteria that produce riboflavin in a culture medium, wherein the nucleic acid sequence of the expression regulatory element of the rib operon in the engineered bacteria is a sequence containing SEQ ID NO: 2 or having 98% or 99% identity with SEQ ID NO: 2; b) Riboflavin accumulates in the culture medium.

2. The method of claim 1, further comprising c) separating riboflavin from the riboflavin-containing liquid.

3. The method according to claim 1 or 2, wherein the engineered bacteria is Bacillus subtilis.

4. The method of claim 3, wherein the 199th amino acid sequence of the Bacillus subtilis containing the flavin kinase / FAD synthase bifunctional enzyme RibC is replaced with aspartic acid (ribC). G199D ).

5. The method of claim 4, wherein the Bacillus subtilis further comprises a transcriptional regulatory protein CcpN in which the amino acid sequence at position 44 is replaced by alanine with serine (ccpN). A44S ).

6. The method of claim 4 or 5, wherein the Bacillus subtilis further comprises the deletion of the cytochrome bd oxidase CydB subunit (ΔcydB).

7. The method of claim 1, wherein the rib operon encodes a riboflavin synthesis-related gene, the riboflavin synthesis-related gene comprising ribG (ribD), ribB (ribE), ribA, and / or ribH.

8. An expression regulatory element having a nucleic acid sequence comprising SEQ ID NO: 2 or a sequence having 98% or 99% identity with SEQ ID NO:

2.

9. An expression cassette comprising a rib operon, the rib operon comprising the expression control element as described in claim 8 and encoding an expression riboflavin synthesis-related gene, the riboflavin synthesis-related gene comprising ribG (ribD), ribB (ribE), ribA and / or ribH.

10. A carrier comprising the expression cassette as described in claim 9.

11. The carrier of claim 10, wherein the carrier is an expression carrier.

12. An engineered bacterium comprising the expression control element as claimed in claim 8, the expression cassette as claimed in claim 9, or the vector as claimed in claim 10 or 11.

13. The engineered bacteria as described in claim 12, wherein the engineered bacteria is Bacillus subtilis.

14. The application of the expression regulation element as described in claim 8, the expression cassette as described in claim 9, the carrier as described in claim 10 or 11, or the engineered bacteria as described in claim 12 or 13 in riboflavin production.

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