WO2022127039A1 - 一种产核黄素的枯草芽孢杆菌及其构建方法与应用 - Google Patents

一种产核黄素的枯草芽孢杆菌及其构建方法与应用 Download PDF

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WO2022127039A1
WO2022127039A1 PCT/CN2021/097024 CN2021097024W WO2022127039A1 WO 2022127039 A1 WO2022127039 A1 WO 2022127039A1 CN 2021097024 W CN2021097024 W CN 2021097024W WO 2022127039 A1 WO2022127039 A1 WO 2022127039A1
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riboflavin
leu
gly
lys
val
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吴涛
胡丹
常利斌
龚华
李岩
赵津津
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通辽梅花生物科技有限公司
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P25/00—Preparation of compounds containing alloxazine or isoalloxazine nucleus, e.g. riboflavin
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/0201—Orotate phosphoribosyltransferase (2.4.2.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01009—Riboflavin synthase (2.5.1.9)

Definitions

  • the invention belongs to the field of microbial fermentation, and in particular relates to a riboflavin-producing Bacillus subtilis and a construction method and application thereof.
  • the riboflavin-producing strains currently used in production include yeast, Bacillus, Corynebacterium and Escherichia coli, and most of them are wild strains or mutagenized strains with poor fermentation performance.
  • Traditional production strains are mainly obtained by multiple rounds of physical and chemical mutagenesis and screening of structural analogs.
  • mutation points introduced by random mutagenesis these strains often have complex genetic backgrounds.
  • the analysis of the role of relevant mutation points will consume a lot of time and energy, which also makes the subsequent metabolic engineering face greater challenges.
  • the strains obtained by mutagenesis generally have disadvantages such as difficult transformation or low transformation efficiency.
  • the mutant strains are sequenced, combined with rational analysis, the obtained beneficial mutation points are excavated, and horizontally transplanted to the Bacillus subtilis 168 strain with clear background. Bacteria.
  • Riboflavin also known as vitamin B 2 , has a molecular formula of C 17 H 20 O 6 N 4 , molecular weight: 376.36, and its chemical structure is shown in Figure 1.
  • Riboflavin is one of the 13 kinds of vitamins necessary for the human body and a member of the B vitamins. It is slightly soluble in water, soluble in sodium chloride solution, easily soluble in dilute sodium hydroxide solution, slightly soluble in ethanol, Cyclohexanol, ethyl acetate, benzyl alcohol and phenol, insoluble in ether, chloroform, acetone and benzene.
  • Riboflavin is a coenzyme component of flavin enzymes, mainly exists in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), and participates in various activities in the form of coenzyme or prosthetic group.
  • enzyme system reaction As a coenzyme of flavoprotein, it participates in the respiratory electron transport chain and redox reaction, plays an important role in respiration and biological oxidation, directly participates in the biological oxidation of carbohydrates, proteins and fats, and has various physiological functions in the living body. Essential vitamins for life activities are nutrients necessary to maintain normal metabolism and physiological functions of the body.
  • Riboflavin can promote development and cell regeneration, promote the normal growth of skin, nails, and hair, help eliminate inflammation in the mouth, lips, and tongue, improve vision, and reduce eye fatigue. Microorganisms can synthesize riboflavin by themselves, but humans and animals must obtain it from food. The concentration of serum riboflavin in normal adults is 69-98 ⁇ mol/L, and the human body needs 0.3-1.8 mg of riboflavin per day. Riboflavin has become an important feed additive, food additive, medicine and food dye, with an annual market demand of about 8,000 to 10,000 tons.
  • the semi-microbial fermentation synthesis method is to first produce D-ribose by microbial fermentation, and then use D-ribose as raw material, react with 3,4-dimethylaniline to form ribitol dimethylaniline, convert it into azo dye, and then react with barbi.
  • the acid reaction produces riboflavin.
  • the advantage of this method is that the purity of the product is high, reaching 96%.
  • the main disadvantage is that the yield is low, about 60%, and a large amount of organic solvent needs to be used, resulting in large environmental pollution.
  • the microbial fermentation method only needs one step of fermentation, and has the advantages of low production cost, small environmental pollution, short production cycle and high product purity.
  • This method is the main method for the industrial production of riboflavin at present, and the riboflavin produced accounts for more than 90% of the market share.
  • Riboflavin-producing strains are the core of microbial fermentation.
  • the early riboflavin-producing strains ranged from Clostridium acetobutylicum to Pseudomonas ashwagandha, Aschu yeast, Candida utilis, etc., which have long fermentation cycle, complex raw materials, high bacterial viscosity, and high process technology. complex issues.
  • Riboflavin engineering bacteria such as Bacillus subtilis and Corynebacterium ammoniagenes have been successfully constructed one after another, becoming the main strains for the industrial production of riboflavin.
  • Bacillus subtilis has the advantages of short fermentation cycle, high unit, easy availability of raw materials, simple process and high production efficiency.
  • Bacillus subtilis has relatively clear researches on its physiology, biochemistry and molecular genetics. It has the advantage of mature genetic engineering technology, and there is still much room for improvement in the future.
  • the present invention provides a riboflavin-producing Bacillus subtilis, characterized in that a point mutation has occurred at at least one of the following sites in the Bacillus subtilis strain: 1) purR A148D : purine operon transcription Regulatory protein PurR, whose 148-position alanine is mutated to aspartic acid; 2) ribC D144R : bifunctional flavokinase FAD synthase RibC, whose 144-position aspartic acid is mutated to arginine; 3) pyrE K104E : Orotate phosphoribosyltransferase PyrE, which mutates lysine 104 to glutamic acid.
  • the present invention provides a mutant gene purR A148D of a Bacillus subtilis purine operon transcriptional regulator protein PurR, the mutant gene is shown in SEQ NO.2, and the encoded protein is shown in SEQ NO.8;
  • the present invention also provides a mutant gene ribC D144R of Bacillus subtilis bifunctional flavin kinase FAD synthase RibC, the mutant gene is shown in SEQ NO.4, and the encoded protein is shown in SEQ NO.10;
  • the present invention Also provided is a mutant gene pyrE K104E of Bacillus subtilis orotate phosphoribosyltransferase PyrE , the mutant gene is shown in SEQ NO.6, and the encoded protein is shown in SEQ NO.12.
  • the present invention provides a riboflavin-producing engineering bacterium, characterized in that the engineering bacterium is Bacillus subtilis, and the bacterial strain has been deposited in the China Microorganism Culture Collection Management Committee on November 18, 2020 General Microbiology Center (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code: 100101), the deposit number is CGMCC No. 21202.
  • the strain has the ability to produce riboflavin, and the yield of riboflavin at the shake flask level can reach 1.65g/L.
  • the present invention provides a method for constructing a riboflavin-producing engineering bacterium, comprising the steps of: (1) integrating the genes of purR, ribC or pyrE on the chromosome of the starting strain, wherein at least one of the genes has a mutation (2) obtaining a strain with improved riboflavin yield compared to the starting strain.
  • the present invention provides a method for constructing a riboflavin-producing engineering bacterium, which comprises the following steps: (1) integrating a mutated purine operon transcriptional regulatory protein PurR on the chromosome of the starting strain, whose 148-position alanine mutated to aspartic acid; and a mutated bifunctional flavokinase FAD synthase RibC, whose aspartic acid at position 144 is mutated to arginine; and a mutated orotate phosphoribosyltransferase, PyrE, whose lysate 104 position
  • the amino acid was mutated to glutamic acid; (2) a strain with improved riboflavin yield was obtained compared with the starting strain.
  • the starting strain in the step (1) is Bacillus subtilis, preferably Bacillus subtilis 168;
  • the plasmid in the step (1) is a commonly used expression plasmid for Bacillus, preferably pKSV7 or pKSU.
  • the strain with improved riboflavin yield in the step (2) is the mutant strain CGMCC NO.21202.
  • the present invention provides a method for producing riboflavin, comprising the steps of: (1) aseptically streaking the engineering bacteria described in claim 1 stored in a glycerol tube on an LB plate, and culturing overnight at 36°C ; (2) Pick a loop of culture and inoculate it into 30 mL of seed medium, 110 rpm, and cultivate at 36 °C for 7-8 h; (3) Transfer to 30 mL of fermentation medium at 10% of the inoculum, and rotate the shaker. Incubate at 120 rpm and 36 °C for 60 h.
  • the seed medium formula (g/L) is: glucose 20, yeast powder 5, corn steep liquor 5, potassium dihydrogen phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.02, manganese sulfate 0.01, pH 7.0 ⁇ 7.2, 121 Sterilize at °C for 20 minutes;
  • the fermentation medium formula (g/L) is: glucose 60, yeast powder 3.5, potassium dihydrogen phosphate 3, ammonium sulfate 25, manganese sulfate 0.01, magnesium sulfate 5, monosodium glutamate 10, corn steep liquor 15, Calcium carbonate 25, pH 7.0-7.2, sterilized at 121°C for 20 minutes.
  • the present invention provides the application of the engineered bacteria of claim 1 in the production of riboflavin.
  • the present invention provides the application of the engineered bacteria of claim 1 in feed, medicine and food.
  • the invention provides a riboflavin-producing Bacillus subtilis and a construction method and application thereof.
  • the Bacillus subtilis of the present invention is a genetically engineered reconstituted bacterium, which carries a plurality of mutated genes that contribute to the production of riboflavin.
  • the process of obtaining the riboflavin-producing Bacillus subtilis of the present invention is as follows: first, the Bacillus subtilis 168 is subjected to multiple rounds of ultraviolet mutagenesis to obtain a mutagenic strain, and the whole genome sequencing analysis shows that the strain has multiple important mutations. It is speculated that Associated with riboflavin synthesis or breakdown.
  • the three mutated genes are: the mutated purine operon transcriptional regulatory protein PurR, whose 148-position alanine is mutated to aspartic acid; the mutated bifunctional flavokinase FAD synthase RibC, whose 144-position aspartic acid is mutated Mutated to arginine; mutant orotate phosphoribosyltransferase PyrE, lysine 104 is mutated to glutamic acid.
  • the above three mutant genes were re-introduced into the wild starting strain subtilis 168 by reverse genetics, and tested for riboflavin production. The results showed that all three point mutations had positive effects on riboflavin production and had additive effects.
  • the present invention not only obtains a mutagenized strain that can produce high riboflavin, but also discovers for the first time three beneficial mutations of genes related to riboflavin synthesis or decomposition. It is reported in the literature that it is a unique mutation, and the most important and most innovative thing is that the present invention shows through reverse metabolic engineering that the above mutation is integrated into Bacillus subtilis, so that Bacillus subtilis can obtain the ability to produce riboflavin, and at the same time. , the mutations of these three genes all have positive effects on the production of riboflavin by Bacillus subtilis, and the mutations can be used in combination with additive effects.
  • the present invention superimposes the above-mentioned three beneficial mutations on Bacillus subtilis 168 to obtain a strain with high riboflavin production.
  • the strain is currently preserved in the General Microorganism Center of the China Microorganism Culture Collection Management Committee, and the preservation number is CGMCC NO. 21202.
  • Figure 1 Chemical structural formula of riboflavin.
  • the reagents involved in the examples of the present invention are all commercially available products, which can be purchased through commercial channels.
  • the source of the original strain B. subtilis 168 ( B. subtilis 168) used in the present invention is BGSC (Bacillus Genetic Stock Center, http://www.bgsc.org/).
  • the riboflavin standard used in the present invention was purchased from Sigma Company, and molecular biology reagents such as DNA polymerase, DNA purification kit, restriction endonuclease, dephosphorylase, DNA ligase and the like were purchased from Thermo Company.
  • Other biochemical reagents were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • the primer information used in the examples is shown in Table 1.
  • pyrE-1f, pyrE-1r and pyrE-2f, pyrE-2r using the B. subtilis 168 genome as a template, using pfu high-fidelity DNA polymerase amplification to obtain the upper and lower homology arms of pyrE K104E respectively; pyrE-1f and pyrE-2r were fused to amplify the upstream and downstream fragments to obtain the pyrE K104E fusion fragment (containing the K104E mutation, and the complete pyrE wild-type and mutant nucleotide sequences are shown in SEQ ID No. 5 and 6; the specific encoding The wild-type and mutant protein sequences are shown in SEQ ID No. 11 and 12.
  • the fusion fragment and pKSU plasmid were digested with SalI and PstI, respectively, and then ligated, and transformed into Trans1 T1 E. coli competent Finally, the recombinant plasmid pKSU- pyrE K104E was obtained.
  • a recombinant was inoculated into 5ml LB liquid medium, cultured at 42°C, 200rpm for 12h and passaged once, diluted and spread on an LB plate containing 0.8 ⁇ M 5-FU (5-fluorouracil, upp as substrate) for screening, and obtained Secondary recombinants.
  • B. subtilis MHZ-1909-5 is the starting strain, superimposed on pyrE mutation, and finally obtained in B. subtilis 168,
  • the reagents involved in the examples of the present invention are all commercially available products, which can be purchased through commercial channels.
  • the source of the original strain B. subtilis 168 ( B. subtilis 168) used in the present invention is BGSC (Bacillus Genetic Stock Center, http://www.bgsc.org/).
  • the riboflavin standard used in the present invention was purchased from Sigma Company, and molecular biology reagents such as DNA polymerase, DNA purification kit, restriction endonuclease, dephosphorylase, DNA ligase and the like were purchased from Thermo Company.
  • Other biochemical reagents were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • the primer information used in the examples is shown in Table 1.
  • Bacillus subtilis 168 Bacillus subtilis 168
  • B. subtilis 168 strain was first subjected to conventional mutagenesis treatment with UV 15W, 30 cm, 20 min, and then nitrosoguanidine was used for mutagenesis at a condition of 0.4 mg/ mL, 36°C, 20 min.
  • subtilis MHZ-1908-1 strain was obtained, which could grow on 8-azaguanine medium containing 1.0 g/L.
  • B. subtilis MHZ-1908-1 strain fermented in shake flasks for 60 hours to produce 1.2 g/L of riboflavin, and the yield to glucose was 4.00% (g/g).
  • B. subtilis MHZ-1908-1 The whole genome sequencing analysis of B. subtilis MHZ-1908-1 was performed, and the sequence information was compared with its origin strain, Bacillus subtilis 168, and its mutation rate was greater than 0.2 per thousand.
  • B. subtilis MHZ-1908-1 has three important mutations in related metabolic pathways, which are speculated to be associated with high riboflavin production.
  • the specific mutation information is: the mutated purine operon transcriptional regulator protein PurR, whose 148-position alanine is mutated to aspartic acid; the mutated bifunctional flavokinase FAD synthase RibC, whose 144-position aspartic acid is mutated to sperm amino acid; mutated orotate phosphoribosyltransferase PyrE, 104 lysine is mutated to glutamic acid.
  • the purine operon transcriptional regulator protein PurR is involved in purine synthesis, and guanosine triphosphate is an important precursor of riboflavin.
  • RibC is a bifunctional enzyme that catalyzes the synthesis of flavin adenine mononucleotide (FMN) from riboflavin and further synthesis of flavin adenine dinucleotide (FAD), both of which are downstream products of riboflavin and also important coenzyme.
  • FMN flavin adenine mononucleotide
  • FAD flavin adenine dinucleotide
  • Orotate phosphoribosyltransferase PyrE is a key gene for pyrimidine synthesis and competes with purine synthesis. Rational analysis, the mutation of these three sites may be the main factor to promote the high production of riboflavin.
  • Example 2 Construction of B. subtilis 168, ⁇ upp strain by gene-free editing method
  • the gene traceless editing method used in the present invention is based on two-step integration mediated by thermosensitive plasmids, and is screened through chloramphenicol positive screening and 5-fluorouracil (5-FU) reverse screening ( Applied Microbiology and Biotechnology, 2014, 98(21):8963-8973. Zhang W, Gao W, Feng J, et al).
  • the screening method needs to firstly delete the upp gene on the genome of the target strain, which encodes uracil phosphoribosyltransferase. When upp and 5-FU coexist, it has a lethal effect on cells.
  • the specific construction process is as follows: using primers upp-1f/1r and upp-2f/2r, using B. subtilis 168 genome as a template, using pfu DNA polymerase to amplify the upstream and downstream homology arms of 888bp and 938bp, respectively, using primer upp -1f/2r was amplified to obtain upstream and downstream fusion fragments, and the fragment and pKSV7 (tool vector) plasmid were subjected to SalI/PstI double digestion, ligation, transformation and other operations to obtain plasmid pKSV7- ⁇ upp. Transformants were transformed into B.
  • subtilis 168 by the Spizizen method, and screened on LB plates containing 2.5 ⁇ g/mL chloramphenicol at 30°C to obtain transformants.
  • the obtained transformants were inoculated into 5 ml of LB liquid medium, cultured at 42° C. and 200 rpm for 12 h and passaged once, diluted and spread on LB plates containing 5 ⁇ g/mL of chloramphenicol to obtain a recombinant.
  • a recombinant was inoculated into 5ml LB liquid medium, cultured at 42°C, 200rpm for 12h and passaged once, diluted and spread on LB plate containing 0.8 ⁇ M 5-FU (5-fluorouracil, upp as substrate) for screening, and obtained
  • the secondary recombinant, B. subtilis 168, ⁇ upp strain was named B. subtilis MHZ-1909-1.
  • Example 3 Construction of engineering strain B. subtilis 168, ⁇ upp, purR A148D
  • the fusion fragment and the pKSU plasmid were digested with SalI and PstI respectively, then ligated, and transformed into Trans1 T1 E. coli competent Finally, the recombinant plasmid pKSU-purR A148D was obtained.
  • Example 2 Subsequent transformation and screening methods were the same as those in Example 2, and finally a strain B. subtilis 168, ⁇ upp, purR A148D was obtained by introducing a purR point mutation into the B. subtilis 168, ⁇ upp strain, and the strain was named B. subtilis MHZ-1909-3.
  • Example 4 Construction of engineering strain B. subtilis 168, ⁇ upp, ribC D144R
  • ribC-1f, ribC-1r and ribC-2f, ribC-2r using B. subtilis 168 genome as a template, using pfu high-fidelity DNA polymerase to amplify, get ribC D144R upper and lower homology arms respectively; ribC-1f and ribC-2r were fused to amplify the upstream and downstream fragments to obtain the ribC D144R fusion fragment (containing the D144R mutation, and the complete ribC wild-type and mutant nucleotide sequences are shown in SEQ ID No. 3 and 4; the specific encoding The wild-type and mutant protein sequences are shown in SEQ ID No. 9 and 10.
  • the fusion fragment and pKSU plasmid were digested with SalI and PstI, respectively, and then ligated, and transformed into Trans1 T1 E. coli competent Finally, the recombinant plasmid pKSU-ribC D144R was obtained.
  • Example 2 Subsequent transformation and screening methods were the same as those in Example 2, and finally a strain B. subtilis 168, ⁇ upp, ribC D144R was obtained by introducing ribC point mutation into B. subtilis 168, ⁇ upp strain, and the strain was named B. subtilis MHZ-1909-4.
  • Example 5 Construction of engineering strains B. subtilis 168, ⁇ upp, purR A148D , ribC D144R
  • the B. subtilis MHZ-1910-3 carrying the purR point mutation was used as the starting strain, and the ribC mutation was superimposed.
  • the specific operation method is the same as the aforementioned Example 2-4, and finally a strain B. subtilis 168, ⁇ upp, purR A148D , ribC D144R was obtained by introducing the double point mutation of purR and ribC into the B. subtilis 168, ⁇ upp strain, and the strain was named B. subtilis MHZ-1909-5.
  • the fermentation test results are shown in Table 2. It can be seen from the test results that the starting strain B. subtilis 168 does not accumulate riboflavin itself, and the mutant strain (number MHZ-1908-1) has higher riboflavin accumulation and product yield. The four engineered strains all accumulated riboflavin, indicating that the mutations of the three genes have positive effects, and the mutations can be used in combination with superimposed positive effects. This result is consistent with the previous rational analysis.
  • PurR is a purine operon transcriptional regulator with amino acid substitution at position 148, and its alanine is replaced by aspartic acid.
  • the amino acid substitution may lead to the weakening or disappearance of the effect of the transcriptional regulator on the purine operon, which is beneficial to the enhancement of purine synthesis and the accumulation of its downstream product, riboflavin.
  • RibC is a bifunctional flavin kinase FAD synthase that catalyzes the conversion of riboflavin into FMN and FAD. It has been reported in the literature that inactivation of ribC can facilitate the accumulation of riboflavin, but it will lead to the deletion of FMN and FAD, which is not conducive to the redox reaction of cells. Point mutation weakening can balance both needs. The mutations obtained in the present invention have been verified to have positive effects and are unique mutations. The combined transformation of this mutation and purR mutation has a superimposed effect, which also shows the necessity and importance of this site for riboflavin synthesis.
  • PyrE is an important gene in the pyrimidine synthesis pathway of Bacillus, encoding orotate phosphoribosyltransferase.
  • the amino acid lysine at position 104 of this enzyme is replaced by glutamic acid. From the protein conformation analysis, position 104 is the substrate binding site.
  • the amino acid substitution at this site, and the substitution of basic amino acids to acidic amino acids, will reduce the substrate binding efficiency, thereby reducing the enzymatic reaction efficiency.
  • the reduction of the flux of the pyrimidine synthesis pathway saves more substrates and energy, which is beneficial to promote the product yield of the purine pathway.
  • the inventors obtained a strain of high riboflavin-producing bacteria through traditional mutagenesis, and identified beneficial mutations of three genes. After the relevant mutations were transplanted into the type strain by reverse genetics, the type strain also acquired a riboflavin-producing phenotype. In addition, the growth and sugar consumption capacity of the related engineered strains have been improved, avoiding the defects of poor growth, poor substrate utilization, and genetic instability caused by mutagenesis.
  • the construction of the strain of the present invention does not limit the sequence of the steps. Those skilled in the art can achieve the purpose of the present invention according to the contents disclosed in the present invention, which all belong to the protection scope of the present invention.
  • the strain numbers in the present invention are for the convenience of description, but should not be construed as limiting the present invention.
  • the use of the engineering bacteria constructed by the above method comprising the purine operon transcription regulator mutant PurR A148D of Bacillus subtilis, the flavin kinase mutant RibC D144R , and the orotate phosphoribosyltransferase mutant PyrE K104E , including but not Limited to riboflavin production.
  • the invention discloses a construction method and application of a riboflavin high-yielding strain. Those skilled in the art can learn from the content of this document, or make appropriate improvements. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the method and product of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invention technology.

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Abstract

一种产核黄素的枯草芽孢杆菌及其构建方法与应用。所述的枯草芽孢杆菌其为枯草芽孢杆菌菌株中purR、ribC或pyrE中至少一个基因位点发生突变。purR、ribC或pyrE至少一个基因位点突变获得的枯草芽孢杆菌菌株在相应发酵条件下积累核黄素显著增加。所述的枯草芽孢杆菌为核黄素高产菌株，生产的核黄素显著增加，为核黄素的工业化生产奠定基础。

Description

一种产核黄素的枯草芽孢杆菌及其构建方法与应用 技术领域
本发明属于微生物发酵领域，具体涉及一种产核黄素的枯草芽孢杆菌及其构建方法与应用。
目前生产上应用的产核黄素菌种有酵母、芽孢杆菌、棒杆菌和大肠杆菌等，多为野生菌种或诱变菌种，发酵性能较差。传统生产菌种主要是由多轮物理化学诱变及结构类似物筛选获得。但由于随机诱变引入的突变点具有不确定性，使得这些菌株往往具有比较复杂的遗传背景。对于相关突变点作用的解析会耗费大量的时间和精力，也使得后续的代谢工程改造面临较大的挑战。同时，诱变获得的菌种普遍存在难转化或改造效率低等缺点。
本发明将诱变菌株进行测序，结合理性分析，将获得的有益突变点挖掘出来，水平移植到背景清晰的枯草芽孢杆菌168菌株上，同时辅以其他理性代谢工程改造，获得高产核黄素的菌种。
 背景技术
核黄素（Riboflavin），又称维生素B 2，分子式为C 17H 20O 6N 4，分子量：376.36，其化学结构式如图1所示。核黄素是人体必需的13种维生素之一，是B族维生素的成员之一，微溶于水，可溶于氯化钠溶液，易溶于稀的氢氧化钠溶液，微溶于乙醇、环己醇、乙酸乙酯、苄醇和酚，不溶于乙醚、氯仿、丙酮和苯。在中性或酸性溶液中稳定，耐热、耐氧化，但在碱溶液中不稳定，加热可被破坏，光照及紫外照射引起不可逆的分解。278~282℃时分解，饱和水溶液的pH约为6.0，水溶液呈黄色并显绿色荧光。
核黄素是黄素酶类的辅酶组成部分，在生物体内主要以黄素单核苷酸（FMN）和黄素腺嘌呤二核苷酸（FAD）的形式存在，以辅酶或辅基的形式参与各种酶系统反应。作为黄素蛋白的辅酶参与呼吸电子传递链及氧化还原反应，在呼吸和生物氧化中起着重要作用，直接参与碳水化合物、蛋白质、脂肪的生物氧化作用，在生物体内具有多种生理功能，是生命活动不可缺少的维生素，是维持机体正常代谢和生理功能所必须的营养素。核黄素具有促进发育和细胞再生，促使皮肤、指甲、毛发的正常生长，帮助消除口腔内、唇、舌炎症，增进视力，减轻眼疲劳等功效。微生物可以自身合成核黄素，但人和动物必须从食物中获取。正常成年人血清核黄素的浓度为69~98μmol/L，人体每天需要0.3~1.8mg核黄素。核黄素成为一种重要的饲料添加剂、食品添加剂、药品和食品染料，市场每年需求约8000~10000吨。
目前核黄素的生产方法主要有2种：半微生物发酵合成法和微生物发酵法。半微生物发酵合成法是先通过微生物发酵生产D-核糖，再以D-核糖为原料，与3,4-二甲代苯胺反应形成核醇二甲代苯胺，转化成偶氮染料后与巴比妥酸反应生产核黄素。该方法的优点是产品纯度较高，达到96%，主要缺点是得率较低，约60%，且需要用到大量的有机溶剂，环境污染大。微生物发酵法仅需一步发酵，具有生产成本低，环境污染小，生产周期短，产品纯度高等优点。该方法是目前核黄素工业化生产的主要方法，生产的核黄素占到市场份额的90%以上。
核黄素生产菌种是微生物发酵法的核心。早期的核黄素生产菌种从丙酮丁酸梭菌，到阿舒氏假囊酵母、棉囊阿舒酵母、解朊假丝酵母等，存在发酵周期长、原料复杂、菌体粘度大、工艺复杂等问题。枯草芽孢杆菌和产氨棒状杆菌等核黄素工程菌相继构建成功，成为核黄素工业生产的主要菌种。尤其是枯草芽孢杆菌具有发酵周期短、单位高、原料易得、工艺简单、生产效率高等优势。且枯草芽孢杆菌作为重要的模式菌，其生理、生化及分子遗传学方面的研究都比较清楚，具有基因工程改造技术成熟的优势，未来仍有较大的提升空间。
 技术问题
近年来，虽然通过代谢工程改造获得了许多高产核黄素的枯草芽孢杆菌基因工程菌，但由于我们对微生物生理及复杂代谢网络调控机制了解的局限性，运用基因工程技术对菌株继续进行改造，很难再获得核黄素产量进一步大幅度提升的菌株；而传统的诱变育种，工作量大、缺乏高效、快速的筛选方法，使得枯草芽孢杆菌在提高核黄素生产能力上受到很大程度的限制。
 技术解决方案
第一方面，本发明提供了一种产核黄素的枯草芽孢杆菌，其特征在于，所述枯草芽孢杆菌菌株中下述至少一个位点发生了点突变：1) purR A148D：嘌呤操纵子转录调节蛋白PurR，其148位丙氨酸突变为天冬氨酸；2) ribC D144R：双功能黄素激酶FAD合成酶RibC，其144位天冬氨酸突变为精氨酸；3) pyrE K104E：乳清酸磷酸核糖基转移酶PyrE，其104位赖氨酸突变为谷氨酸。
在此基础上，本发明提供了一种枯草芽孢杆菌嘌呤操纵子转录调节蛋白PurR的突变基因purR A148D，该突变基因如SEQ NO.2所示，其编码的蛋白如SEQ NO.8所示；本发明还提供了一种枯草芽孢杆菌双功能黄素激酶FAD合成酶RibC的突变基因ribC D144R，该突变基因如SEQ NO.4所示，其编码的蛋白如SEQ NO.10所示；本发明还提供了一种枯草芽孢杆菌乳清酸磷酸核糖基转移酶PyrE的突变基因pyrE K104E，该突变基因如SEQ NO.6所示，其编码的蛋白如SEQ NO.12所示。
第二方面，本发明提供了一株产核黄素的工程菌，其特征在于，所述工程菌为枯草芽孢杆菌，该菌株已于2020年11月18日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮政编码：100101)，保藏编号为CGMCC NO. 21202。该菌株具有核黄素生产能力，摇瓶水平核黄素产量可达1.65g/L。
第三方面，本发明提供了一种产核黄素工程菌的构建方法，其包括如下步骤：(1)在出发菌株的染色体上整合了purR、ribC或pyrE的基因，其中至少一个基因存在突变；(2)获得较出发菌株相比核黄素产量提高的菌株。
优选的，本发明提供了一种产核黄素工程菌的构建方法，其包括如下步骤： (1)在出发菌株的染色体上整合突变的嘌呤操纵子转录调节蛋白PurR，其148位丙氨酸突变为天冬氨酸；以及突变的双功能黄素激酶FAD合成酶RibC，其144位天冬氨酸突变为精氨酸；以及突变的乳清酸磷酸核糖基转移酶PyrE，其104位赖氨酸突变为谷氨酸；(2)获得较出发菌株相比核黄素产量提高的菌株。
其中，所述步骤(1)中的出发菌株为枯草芽孢杆菌，优选为Bacillus subtilis 168；所述步骤(1)中的质粒为芽孢杆菌常用表达质粒，优选为pKSV7或pKSU。
优选地，所述步骤(2)中核黄素产量提高的菌株为突变株CGMCC NO.21202。
第四方面，本发明提供了一种生产核黄素的方法，其包括如下步骤：(1)将甘油管保藏的权利要求1所述工程菌在LB平板进行无菌划线，36℃过夜培养；(2)挑一环培养物接种至30 mL种子培养基中，110 rpm，36℃培养7~8 h；(3)按10%接种量转接至30 ml 发酵培养基中，摇床转速120 rpm，36℃培养60h。
其中，种子培养基配方(g/L)为：葡萄糖20，酵母粉5，玉米浆干粉5，磷酸二氢钾3，硫酸镁0.5，硫酸亚铁0.02，硫酸锰0.01，pH 7.0～7.2，121℃灭菌20分钟；发酵培养基配方(g/L) 为：葡萄糖60，酵母粉3.5，磷酸二氢钾3，硫酸铵25，硫酸锰0.01，硫酸镁5，味精10，玉米浆干粉15，碳酸钙25，pH 7.0～7.2，121℃灭菌20分钟。
第五方面，本发明提供了权利要求1所述的工程菌在生产核黄素中的应用。
第六方面，本发明提供了权利要求1所述的工程菌在饲料、医药、食品中的应用。
 有益效果
本发明提供了一种产核黄素的枯草芽孢杆菌及其构建方法与应用。本发明所述枯草芽孢杆菌为基因工程重构菌，其携带多个有助于产核黄素的突变基因。本发明所述的产核黄素的枯草芽孢杆菌其获得过程为，首先将枯草芽孢杆菌168经多轮紫外诱变获得诱变菌，通过全基因组测序分析，该菌株有多个重要突变，推测与核黄素合成或分解相关。其中的3个突变基因为：突变的嘌呤操纵子转录调节蛋白PurR，其148位丙氨酸突变为天冬氨酸；突变的双功能黄素激酶FAD合成酶RibC，其144位天冬氨酸突变为精氨酸；突变的乳清酸磷酸核糖基转移酶PyrE，其104位赖氨酸突变为谷氨酸。其次，将上述3个突变基因分别通过反向遗传学手段重新引入到野生出发菌株枯草168菌株中，并进行核黄素生产测试。结果表明，三个点突变对核黄素生产均有正向作用，并且具有叠加的效果。
本发明不仅获得了可高产核黄素的诱变菌株，在此基础上，本发明还首次发现了三个与核黄素合成或分解相关基因的有益突变，上述有益突变由诱变获得，无文献报道，为独有突变，而且最重要且最具创新性的是，本发明通过反向代谢工程表明将上述突变整合入枯草芽孢杆菌，可使枯草芽孢杆菌获得产核黄素的能力，同时，这三个基因的突变均对枯草芽孢杆菌产核黄素有正效果，并且突变可组合使用，具有叠加效应。本发明将上述三个有益突变叠加到枯草芽孢杆菌168上获得了高产核黄素的菌种，该菌种目前保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO. 21202。
 附图说明
图1：核黄素的化学结构式。
 本发明的最佳实施方式
为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。本发明所用到的原始菌株枯草芽孢杆菌168（ B. subtilis 168）来源为BGSC (Bacillus Genetic Stock Center，http://www.bgsc.org/)。本发明所用到的核黄素标准品从Sigma公司购买，所用DNA聚合酶、DNA纯化试剂盒、限制性内切酶、去磷酸化酶、DNA连接酶等分子生物学试剂从Thermo公司购买，所用其他生化试剂从生工生物工程(上海)股份有限公司购买。
实施例中使用的引物信息见表1。
表 1 引物序列
引物 序列5'→ 3'
upp-1f acgcgtcgaccaggagcaagtgcgtatc
upp-1r atctccgagacctggaacacctttcccatactgtgttt
upp-2f aaacacagtatgggaaaggtgttccaggtctcggagat
upp-2r aaaactgcaggccacagtaatcatcagaatg
purR -1f atgaagtttcgtcgcagcggcagatGTCGACat
purR -1r cattcaaatagcttgcatctgcgtacgcaagagggatgcc
purR -2f ggcatccctcttgcgtacgcagatgcaagctatttgaatg
purR -2r tcatgattctgtctctccattctCTCGAGca
ribC -1f cttggcgatctatttccctcatcctgGTCGACat
ribC -1r ccagcttttccgtctaaatcacgcggcatggtcttcattgttc
ribC -2f gaacaatgaagaccatgccgcgtgatttagacggaaaagctgg
ribC -2r caggaagccatccgttatttcagcaCTCGAGca
pyrE -1f ctggatggagtggtctgctctgttcGTCGACat
pyrE -1r tgattgccttttccgtgcgcctccggcttgctcctcacataac
pyrE -2f gttatgtgaggagcaagccggaggcgcacggaaaaggcaatca
pyrE -2r gcaggttgccgggcttcataggtccCTCGAGca
工程菌株 B. subtilis 168,Δupp, purR A148D, ribC D144R, pyrE K104E的构建
用引物pyrE -1f、pyrE -1r和pyrE -2f、pyrE -2r，以 B. subtilis 168基因组为模板，使用pfu高保真DNA聚合酶扩增，分别得到pyrE K104E上、下游同源臂；用引物pyrE -1f、pyrE -2r融合扩增上、下游片段，得到pyrE K104E融合片段（含K104E突变，完整pyrE野生型及突变型核苷酸序列如SEQ ID No.5、6所示；具体编码的野生型及突变型蛋白序列如SEQ ID No.11、12所示。将融合片段与pKSU质粒（工具载体）分别进行SalI、PstI双酶切，然后进行连接，并转化至Trans1 T1大肠杆菌感受态细胞。最终，得到重组质粒pKSU-pyrE K104E。
通过Spizizen方法转化至 B. subtilis 168中，用含2.5μg/mL氯霉素的LB平板在30℃下筛选，获得转化子。将获得的转化子接种到5ml LB液体培养基中，42℃、200rpm培养12h并传代一次，稀释涂布于含有5μg/mL的氯霉素LB平板，获得一次重组子。将一次重组子接种到5ml LB液体培养基中，42℃、200rpm培养12h并传代一次，稀释涂布于含有0.8μM 5-FU（5-氟尿嘧啶，upp作用底物）的LB平板进行筛选，获得二次重组子。以携带purR、ribC双点突变的 B. subtilis MHZ-1909-5为出发菌株，叠加pyrE突变改造，最终得到在 B. subtilis168,Δupp菌株中引入purR、ribC、pyrE三点突变叠加的菌株 B. subtilis 168,Δupp, purR A148D, ribC D144R, pyrE K104E，该菌株命名为 B. subtilis MHZ-1910-1。
本发明的实施方式
为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。本发明所用到的原始菌株枯草芽孢杆菌168（ B. subtilis 168）来源为BGSC (Bacillus Genetic Stock Center，http://www.bgsc.org/)。本发明所用到的核黄素标准品从Sigma公司购买，所用DNA聚合酶、DNA纯化试剂盒、限制性内切酶、去磷酸化酶、DNA连接酶等分子生物学试剂从Thermo公司购买，所用其他生化试剂从生工生物工程(上海)股份有限公司购买。
实施例中使用的引物信息见表1。
表 1 引物序列
引物 序列5'→ 3'
upp-1f acgcgtcgaccaggagcaagtgcgtatc
upp-1r atctccgagacctggaacacctttcccatactgtgttt
upp-2f aaacacagtatgggaaaggtgttccaggtctcggagat
upp-2r aaaactgcaggccacagtaatcatcagaatg
purR -1f atgaagtttcgtcgcagcggcagatGTCGACat
purR -1r cattcaaatagcttgcatctgcgtacgcaagagggatgcc
purR -2f ggcatccctcttgcgtacgcagatgcaagctatttgaatg
purR -2r tcatgattctgtctctccattctCTCGAGca
ribC -1f cttggcgatctatttccctcatcctgGTCGACat
ribC -1r ccagcttttccgtctaaatcacgcggcatggtcttcattgttc
ribC -2f gaacaatgaagaccatgccgcgtgatttagacggaaaagctgg
ribC -2r caggaagccatccgttatttcagcaCTCGAGca
pyrE -1f ctggatggagtggtctgctctgttcGTCGACat
pyrE -1r tgattgccttttccgtgcgcctccggcttgctcctcacataac
pyrE -2f gttatgtgaggagcaagccggaggcgcacggaaaaggcaatca
pyrE -2r gcaggttgccgggcttcataggtccCTCGAGca
实施例1．诱变筛选获得核黄素高产菌株
以枯草芽孢杆菌168（ Bacillus subtilis 168）作为原始菌株， B. subtilis 168菌株先用紫外15W、30 cm、20 min进行常规诱变处理，再用亚硝基胍进行诱变，条件为0.4 mg/mL、36℃、20 min。然后，涂布在含有0.2 g/L 8-氮鸟嘌呤的基本培养基上（g/L：葡萄糖20、硫酸铵2、硫酸镁0.4、氯化钙0.02、硫酸亚铁0.02、磷酸氢二钠1.5、硫酸锌0.01、硫酸锰0.01、磷酸二氢钾1.5、琼脂18，pH7.0～7.2），36℃培养24h。之后，挑选长势最好的菌株进行下一轮诱变，并提高基本培养基中8-氮鸟嘌呤的浓度。经过多轮诱变筛选后，获得了 B. subtilis MHZ-1908-1菌株，该菌能在含有1.0 g/L的8-氮鸟嘌呤培养基上生长。 B. subtilis MHZ-1908-1菌株摇瓶发酵60小时产核黄素水平为1.2 g/L，对葡萄糖的得率为4.00%（g/g）。
将 B. subtilis MHZ-1908-1进行全基因组测序分析，序列信息与其出发菌株枯草芽孢杆菌168进行比对，其突变率大于千分之零点二。通过比较基因组学分析， B. subtilis MHZ-1908-1在相关代谢路径上具有三个重要突变，推测可能与核黄素高产相关。具体突变信息为：突变的嘌呤操纵子转录调节蛋白PurR，其148位丙氨酸突变为天冬氨酸；突变的双功能黄素激酶FAD合成酶RibC，其144位天冬氨酸突变为精氨酸；突变的乳清酸磷酸核糖基转移酶PyrE，其104位赖氨酸突变为谷氨酸。嘌呤操纵子转录调节蛋白PurR与嘌呤合成相关，而三磷酸鸟嘌呤核苷是核黄素的重要前体。RibC是双功能酶，可催化核黄素合成黄素腺嘌呤单核苷酸（FMN），并进一步合成黄素腺嘌呤二核苷酸（FAD），二者是核黄素的下游产物，也是重要的辅酶。乳清酸磷酸核糖基转移酶PyrE是嘧啶合成关键基因，与嘌呤合成是竞争关系。理性分析，该三个位点的突变可能是促进核黄素高产的主要因素。
实施例2：基因无痕编辑方法构建 B. subtilis168,Δupp菌株
以枯草芽孢杆菌 B. subtilis168为出发菌株，本发明所用基因无痕编辑方法是基于温敏质粒介导的两步整合，通过氯霉素正筛及5-氟尿嘧啶（5-FU）反筛(Applied Microbiology and Biotechnology,2014,98(21):8963-8973.Zhang W,Gao W,Feng J,et al)。该筛选方法需首选缺失目标菌株基因组上的upp基因，该基因编码尿嘧啶磷酸核糖转移酶，当upp与5-FU同时存在时，对细胞具有致死作用。
具体构建过程如下：用引物upp-1f/1r、upp-2f/2r，以 B. subtilis 168基因组为模板，使用pfu DNA聚合酶扩增分别得到888bp和938bp的上下游同源臂，用引物upp-1f/2r扩增得到上下游融合片段，将片段与pKSV7(工具载体)质粒经SalI/PstI双酶切、连接、转化等操作，得到质粒pKSV7-Δupp。通过Spizizen方法转化至 B. subtilis 168中，用含2.5μg/mL氯霉素的LB平板在30℃下筛选，获得转化子。将获得的转化子接种到5ml LB液体培养基中，42℃、200rpm培养12h并传代一次，稀释涂布于含有5μg/mL的氯霉素LB平板，获得一次重组子。将一次重组子接种到5ml LB液体培养基中，42℃、200rpm培养12h并传代一次，稀释涂布于含有0.8μM 5-FU（5-氟尿嘧啶，upp作用底物）的LB平板进行筛选，获得二次重组子，即 B. subtilis 168,Δupp菌株，该菌株命名为 B. subtilis MHZ-1909-1。
实施例3：工程菌株 B. subtilis 168,Δupp, purR A148D的构建
用引物purR-1f、purR-1r和purR-2f、purR-2r，以 B. subtilis 168基因组为模板，使用pfu高保真DNA聚合酶扩增，分别得到purR A148D上、下游同源臂；用引物purR-1f、purR-2r融合扩增上、下游片段，得到purR A148D融合片段（含A148D突变，完整purR野生型及突变型核苷酸序列如SEQ ID No.1、2所示；具体编码的野生型及突变型蛋白序列如SEQ ID No.7、8所示。将融合片段与pKSU质粒（工具载体）分别进行SalI、PstI双酶切，然后进行连接，并转化至Trans1 T1大肠杆菌感受态细胞。最终，得到重组质粒pKSU-purR A148D。
后续转化及筛选方法同实施例2，最终得到在 B. subtilis168,Δupp菌株中引入purR点突变的菌株 B. subtilis 168,Δupp, purR A148D，该菌株命名为 B. subtilis MHZ-1909-3。
实施例4：工程菌株 B. subtilis 168,Δupp, ribC D144R的构建
用引物ribC -1f、ribC -1r和ribC -2f、ribC -2r，以 B. subtilis 168基因组为模板，使用pfu高保真DNA聚合酶扩增，分别得到ribC D144R上、下游同源臂；用引物ribC -1f、ribC -2r融合扩增上、下游片段，得到ribC D144R融合片段（含D144R突变，完整ribC野生型及突变型核苷酸序列如SEQ ID No.3、4所示；具体编码的野生型及突变型蛋白序列如SEQ ID No.9、10所示。将融合片段与pKSU质粒（工具载体）分别进行SalI、PstI双酶切，然后进行连接，并转化至Trans1 T1大肠杆菌感受态细胞。最终，得到重组质粒pKSU-ribC D144R。
后续转化及筛选方法同实施例2，最终得到在 B. subtilis168,Δupp菌株中引入ribC点突变的菌株 B. subtilis 168,Δupp, ribC D144R，该菌株命名为 B. subtilis MHZ-1909-4。
实施例5：工程菌株 B. subtilis 168,Δupp, purR A148D, ribC D144R的构建
以携带purR点突变的 B. subtilis MHZ-1910-3为出发菌株，叠加ribC突变改造。具体操作方法同前述实施例2-4，最终得到在 B. subtilis168,Δupp菌株中引入purR、ribC双点突变的菌株 B. subtilis 168,Δupp, purR A148D, ribC D144R，该菌株命名为 B. subtilis MHZ-1909-5。
工业实用性
工程菌株核黄素生产性能测试
1. 培养基：
(1) 种子培养基配方(g/L)：葡萄糖20，酵母粉5，玉米浆干粉5，磷酸二氢钾3，硫酸镁0.5，硫酸亚铁0.02，硫酸锰0.01，pH 7.0～7.2，121℃灭菌20分钟。
(2) 发酵培养基配方(g/L)：葡萄糖50，酵母粉3.5，磷酸二氢钾3，硫酸铵25，硫酸锰0.01，硫酸镁5，味精10，玉米浆干粉15，碳酸钙25，pH 7.0～7.2，121℃灭菌20分钟。
2. 培养方法：
（1）将甘油管保藏的菌株LB平板进行无菌划线，36℃过夜培养；
（2）挑一环培养物接种至30 mL种子培养基中，110 rpm，36℃培养7~8 h；
（3）按10%接种量转接至30 ml 发酵培养基中，摇床转速120 rpm，36℃培养40h；
发酵测试结果见表2。从测试结果可以看出，出发菌株 B. subtilis168本身不积累核黄素，诱变菌（编号MHZ-1908-1）具有较高核黄素积累量和产物得率。4种工程菌株均积累核黄素，说明三个基因的突变均有正效果，并且突变可组合使用，具有叠加的正效果。该结果与前期理性分析相符。
PurR为嘌呤操纵子转录调节因子，其148位发生氨基酸取代，其丙氨酸被天冬氨酸取代。该氨基酸替换可能导致该转录调节因子对嘌呤操纵子的作用减弱或消失，有利于强化嘌呤合成，进而利于其下游产物核黄素的积累。
RibC为双功能黄素激酶FAD合成酶，可催化核黄素转变为FMN及FAD。有文献报道失活ribC可利于核黄素的积累，但会导致缺失FMN和FAD，对细胞的氧化还原反应不利。点突变弱化可以平衡两种需求。本发明获得的突变经验证具有正的效果，且为独有突变。该突变与purR突变的组合改造具有叠加的效果，也说明该位点对于核黄素合成的必要性和重要性。
PyrE是芽孢杆菌嘧啶合成途径重要基因，编码乳清酸磷酸核糖基转移酶。该酶的第104位氨基酸赖氨酸被谷氨酸取代。从蛋白构象上解析，104位为其底物结合位点，该位点发生氨基酸取代，且由碱性氨基酸取代为酸性氨基酸，会造成底物结合效率降低，进而降低了酶促反应效率。而嘧啶合成途径通量的减少，节省了更多底物和能量，有利于促进嘌呤途径的产物得率。
综上所述，发明人通过传统诱变方式获得了一株核黄素高产菌，并鉴定得到三个基因的有益突变。将相关突变通过反向遗传学手段移植到模式菌株后，模式菌株也获得了产核黄素表型。并且，相关工程菌株的生长和耗糖能力有所改善，避免了诱变带来的生长差、底物利用差、遗传不稳定等缺陷。
表 2  核黄素发酵测试
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本发明的菌株构建，其步骤的前后顺序不限定，本领域的技术人员按本发明公开的内容达到本发明的目的均属于本发明的保护范围。本发明中的菌株编号是为了方便描述，但不应理解为对本发明的限定。上述方法构建的包含有枯草芽孢杆菌的嘌呤操纵子转录调节因子突变体PurR A148D、黄素激酶突变体RibC D144R、乳清酸磷酸核糖基转移酶突变体PyrE K104E的工程菌的用途，包括但不局限于核黄素生产。
本发明公开了一种核黄素高产菌株构建方法与应用。本领域技术人员可以借鉴本文内容，或进行适当改进。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及产品已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或适当变更与组合，来实现和应用本发明技术。
 序列表自由内容
<110>  申请人
<120>  一种产核黄素的枯草芽孢杆菌及其构建方法与应用
<130>   一种产核黄素的枯草芽孢杆菌及其构建方法与应用
<160>  12   
<170>  PatentIn version 3.3
<210>  1
<211>  858
<212>  DNA
<213>  B. subtilis 168
<400>  1
atgaagtttc gtcgcagcgg cagattggtg gacttaacaa attatttgtt aacccatccg     60
cacgagttaa taccgctaac ctttttctct gagcggtatg aatctgcaaa atcatcgatc    120
agtgaagatt taacaattat taaacaaacc tttgaacagc aggggattgg tactttgctt    180
actgttcccg gagatgccgg aggcgttaaa tatattccga aaatgaagca ggctgaagct    240
gaagagtttg tgcagacact tggacagtcg ctggcaaatc ctgagcgtat ccttccgggc    300
ggttatgtat atttaacgga tatcttagga aagccatctg tactctccaa ggtagggaag    360
ctgtttgctt ccgtgtttgc agagcgcgaa attgatgttg tcatgaccgt tgccacgaaa    420
ggcatccctc ttgcgtacgc agctgcaagc tatttgaatg tgcctgttgt gatcgttcgt    480
aaagacaata aggtaacaga gggctccaca gtcagcatta attacgtttc aggctcctca    540
aaccgcattc aaacaatgtc acttgcgaaa agaagcatga aaacgggttc aaacgtactc    600
attattgatg actttatgaa agcaggcggc accattaatg gtatgattaa cctgttggat    660
gagtttaacg caaatgtggc gggaatcggc gtcttagttg aagccgaagg agtagatgaa    720
cgtcttgttg acgaatatat gtcacttctt actctttcaa ccatcaacat gaaagagaag    780
tccattgaaa ttcagaatgg caattttctg cgttttttta aagacaatct tttaaagaat    840
ggagagacag aatcatga                                                  858
<210>  2
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<213>  B. subtilis 168
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atgaagtttc gtcgcagcgg cagattggtg gacttaacaa attatttgtt aacccatccg     60
cacgagttaa taccgctaac ctttttctct gagcggtatg aatctgcaaa atcatcgatc    120
agtgaagatt taacaattat taaacaaacc tttgaacagc aggggattgg tactttgctt    180
actgttcccg gagatgccgg aggcgttaaa tatattccga aaatgaagca ggctgaagct    240
gaagagtttg tgcagacact tggacagtcg ctggcaaatc ctgagcgtat ccttccgggc    300
ggttatgtat atttaacgga tatcttagga aagccatctg tactctccaa ggtagggaag    360
ctgtttgctt ccgtgtttgc agagcgcgaa attgatgttg tcatgaccgt tgccacgaaa    420
ggcatccctc ttgcgtacgc agatgcaagc tatttgaatg tgcctgttgt gatcgttcgt    480
aaagacaata aggtaacaga gggctccaca gtcagcatta attacgtttc aggctcctca    540
aaccgcattc aaacaatgtc acttgcgaaa agaagcatga aaacgggttc aaacgtactc    600
attattgatg actttatgaa agcaggcggc accattaatg gtatgattaa cctgttggat    660
gagtttaacg caaatgtggc gggaatcggc gtcttagttg aagccgaagg agtagatgaa    720
cgtcttgttg acgaatatat gtcacttctt actctttcaa ccatcaacat gaaagagaag    780
tccattgaaa ttcagaatgg caattttctg cgttttttta aagacaatct tttaaagaat    840
ggagagacag aatcatga                                                  858
<210>  3
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gtgaagacga tacatattac acatcctcat catttaataa aagaggagca ggcaaaatca     60
gtgatggcgt taggttattt tgacggcgtt catctcgggc atcaaaaggt aatcggcaca    120
gcgaagcaaa tagccgaaga aaaaggtctg acattagctg tgatgacctt tcatccccat    180
ccttctcacg tgttgggcag agataaggaa ccaaaggatc tgattacgcc tcttgaagac    240
aaaataaacc aaattgaaca attaggcaca gaagttctgt atgtcgttaa atttaatgaa    300
gtgtttgctt ctctttctcc taagcagttt atagaccagt atattatcgg ccttaatgtg    360
cagcacgcag tggcaggctt tgactttacg tacggcaaat acggcaaggg aacaatgaag    420
accatgccgg atgatttaga cggaaaagct gggtgcacaa tggtagaaaa attaacggag    480
caggataaaa aaatcagttc ttcgtatatc cgtaccgcgc ttcaaaacgg agatgttgaa    540
ttggcgaatg tcttgcttgg acaaccttat tttattaaag gaattgtcat tcatggtgat    600
aaaagagggc ggaccatcgg gtttccgaca gcgaatgtcg gtttaaataa cagctatatc    660
gttccgccca caggtgtata tgccgtaaaa gcggaagtga acggcgaagt ttacaatggc    720
gtttgcaata ttggctataa gccaacgttt tatgaaaagc gccctgaaca gccttccatc    780
gaggtcaatc tgtttgattt caatcaagag gtatatggag ccgctataaa aatcgaatgg    840
tacaaacgga ttcggagcga gcggaaattc aatggcatca aagaattaac tgagcaaatt    900
gagaaagata agcaggaagc catccgttat ttcagcaatt tgcggaaata a             951
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gtgaagacga tacatattac acatcctcat catttaataa aagaggagca ggcaaaatca     60
gtgatggcgt taggttattt tgacggcgtt catctcgggc atcaaaaggt aatcggcaca    120
gcgaagcaaa tagccgaaga aaaaggtctg acattagctg tgatgacctt tcatccccat    180
ccttctcacg tgttgggcag agataaggaa ccaaaggatc tgattacgcc tcttgaagac    240
aaaataaacc aaattgaaca attaggcaca gaagttctgt atgtcgttaa atttaatgaa    300
gtgtttgctt ctctttctcc taagcagttt atagaccagt atattatcgg ccttaatgtg    360
cagcacgcag tggcaggctt tgactttacg tacggcaaat acggcaaggg aacaatgaag    420
accatgccgc gtgatttaga cggaaaagct gggtgcacaa tggtagaaaa attaacggag    480
caggataaaa aaatcagttc ttcgtatatc cgtaccgcgc ttcaaaacgg agatgttgaa    540
ttggcgaatg tcttgcttgg acaaccttat tttattaaag gaattgtcat tcatggtgat    600
aaaagagggc ggaccatcgg gtttccgaca gcgaatgtcg gtttaaataa cagctatatc    660
gttccgccca caggtgtata tgccgtaaaa gcggaagtga acggcgaagt ttacaatggc    720
gtttgcaata ttggctataa gccaacgttt tatgaaaagc gccctgaaca gccttccatc    780
gaggtcaatc tgtttgattt caatcaagag gtatatggag ccgctataaa aatcgaatgg    840
tacaaacgga ttcggagcga gcggaaattc aatggcatca aagaattaac tgagcaaatt    900
gagaaagata agcaggaagc catccgttat ttcagcaatt tgcggaaata a             951
<210>  5
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<212>  DNA
<213>  B. subtilis 168
<400>  5
atgggaggga atcaaatctt gaaacaaatc atcgcaaaac atctattaga catccaagct     60
gtatttttac gcccgaacga gccgttcaca tgggcaagcg gcattttatc accgatctac    120
tgtgacaacc gccttacgct atctttccca gaggtcagaa acgatgttgc ttcaggtatc    180
agcaagcttg ttaaagagca ttttcctgaa gctgaaatga ttgcgggaac agcaactgcc    240
ggtattcctc atgctgctct tgcggcggac catttgaatc ttccgatgtg ttatgtgagg    300
agcaagccga aggcgcacgg aaaaggcaat cagattgagg gagctgtgca agaagggcaa    360
aaaacagtcg tcattgaaga cttaatttcc acaggaggca gcgtgcttga agcttgtgca    420
gctttacaag cggccggctg tgaagtgctt ggtgtcgtct caatctttac gtacggactt    480
cctaaagcgg aggaagcctt cgcaaaggca gaactgccat actactcatt aaccgattat    540
gatacgctca cagaggtcgc gcttgaaaac ggaaatattc attcagatga tctaaaaaag    600
ctgcaaacat ggaaacgaaa tcccgagtca aaagattggt ttaaaaaata a             651
<210>  6
<211>  651
<212>  DNA
<213>  B. subtilis 168
<400>  6
atgggaggga atcaaatctt gaaacaaatc atcgcaaaac atctattaga catccaagct     60
gtatttttac gcccgaacga gccgttcaca tgggcaagcg gcattttatc accgatctac    120
tgtgacaacc gccttacgct atctttccca gaggtcagaa acgatgttgc ttcaggtatc    180
agcaagcttg ttaaagagca ttttcctgaa gctgaaatga ttgcgggaac agcaactgcc    240
ggtattcctc atgctgctct tgcggcggac catttgaatc ttccgatgtg ttatgtgagg    300
agcaagccgg aggcgcacgg aaaaggcaat cagattgagg gagctgtgca agaagggcaa    360
aaaacagtcg tcattgaaga cttaatttcc acaggaggca gcgtgcttga agcttgtgca    420
gctttacaag cggccggctg tgaagtgctt ggtgtcgtct caatctttac gtacggactt    480
cctaaagcgg aggaagcctt cgcaaaggca gaactgccat actactcatt aaccgattat    540
gatacgctca cagaggtcgc gcttgaaaac ggaaatattc attcagatga tctaaaaaag    600
ctgcaaacat ggaaacgaaa tcccgagtca aaagattggt ttaaaaaata a             651
<210>  7
<211>  285
<212>  PRT
<213>  B. subtilis 168
<400>  7
Met Lys Phe Arg Arg Ser Gly Arg Leu Val Asp Leu Thr Asn Tyr Leu
1               5                   10                  15     
Leu Thr His Pro His Glu Leu Ile Pro Leu Thr Phe Phe Ser Glu Arg
            20                  25                  30          
Tyr Glu Ser Ala Lys Ser Ser Ile Ser Glu Asp Leu Thr Ile Ile Lys
        35                  40                  45             
Gln Thr Phe Glu Gln Gln Gly Ile Gly Thr Leu Leu Thr Val Pro Gly
    50                  55                  60                 
Asp Ala Gly Gly Val Lys Tyr Ile Pro Lys Met Lys Gln Ala Glu Ala
65                  70                  75                  80 
Glu Glu Phe Val Gln Thr Leu Gly Gln Ser Leu Ala Asn Pro Glu Arg
                85                  90                  95     
Ile Leu Pro Gly Gly Tyr Val Tyr Leu Thr Asp Ile Leu Gly Lys Pro
            100                 105                 110        
Ser Val Leu Ser Lys Val Gly Lys Leu Phe Ala Ser Val Phe Ala Glu
        115                 120                 125            
Arg Glu Ile Asp Val Val Met Thr Val Ala Thr Lys Gly Ile Pro Leu
    130                 135                 140                
Ala Tyr Ala Ala Ala Ser Tyr Leu Asn Val Pro Val Val Ile Val Arg
145                 150                 155                 160
Lys Asp Asn Lys Val Thr Glu Gly Ser Thr Val Ser Ile Asn Tyr Val
                165                 170                 175    
Ser Gly Ser Ser Asn Arg Ile Gln Thr Met Ser Leu Ala Lys Arg Ser
            180                 185                 190        
Met Lys Thr Gly Ser Asn Val Leu Ile Ile Asp Asp Phe Met Lys Ala
        195                 200                 205             
Gly Gly Thr Ile Asn Gly Met Ile Asn Leu Leu Asp Glu Phe Asn Ala
    210                 215                 220                
Asn Val Ala Gly Ile Gly Val Leu Val Glu Ala Glu Gly Val Asp Glu
225                 230                 235                 240
Arg Leu Val Asp Glu Tyr Met Ser Leu Leu Thr Leu Ser Thr Ile Asn
                245                 250                 255    
Met Lys Glu Lys Ser Ile Glu Ile Gln Asn Gly Asn Phe Leu Arg Phe
            260                 265                 270        
Phe Lys Asp Asn Leu Leu Lys Asn Gly Glu Thr Glu Ser
        275                 280                 285
<210>  8
<211>  285
<212>  PRT
<213>  B. subtilis 168
<400>  8
Met Lys Phe Arg Arg Ser Gly Arg Leu Val Asp Leu Thr Asn Tyr Leu
1               5                   10                  15     
Leu Thr His Pro His Glu Leu Ile Pro Leu Thr Phe Phe Ser Glu Arg
            20                  25                  30         
Tyr Glu Ser Ala Lys Ser Ser Ile Ser Glu Asp Leu Thr Ile Ile Lys
        35                  40                  45             
Gln Thr Phe Glu Gln Gln Gly Ile Gly Thr Leu Leu Thr Val Pro Gly
    50                  55                  60                 
Asp Ala Gly Gly Val Lys Tyr Ile Pro Lys Met Lys Gln Ala Glu Ala
65                  70                  75                  80 
Glu Glu Phe Val Gln Thr Leu Gly Gln Ser Leu Ala Asn Pro Glu Arg
                85                  90                  95     
Ile Leu Pro Gly Gly Tyr Val Tyr Leu Thr Asp Ile Leu Gly Lys Pro
            100                 105                 110        
Ser Val Leu Ser Lys Val Gly Lys Leu Phe Ala Ser Val Phe Ala Glu
        115                 120                 125            
Arg Glu Ile Asp Val Val Met Thr Val Ala Thr Lys Gly Ile Pro Leu
    130                 135                 140                
Ala Tyr Ala Asp Ala Ser Tyr Leu Asn Val Pro Val Val Ile Val Arg
145                 150                 155                 160
Lys Asp Asn Lys Val Thr Glu Gly Ser Thr Val Ser Ile Asn Tyr Val
                165                 170                 175    
Ser Gly Ser Ser Asn Arg Ile Gln Thr Met Ser Leu Ala Lys Arg Ser
            180                 185                 190         
Met Lys Thr Gly Ser Asn Val Leu Ile Ile Asp Asp Phe Met Lys Ala
        195                 200                 205            
Gly Gly Thr Ile Asn Gly Met Ile Asn Leu Leu Asp Glu Phe Asn Ala
    210                 215                 220                 
Asn Val Ala Gly Ile Gly Val Leu Val Glu Ala Glu Gly Val Asp Glu
225                 230                 235                 240
Arg Leu Val Asp Glu Tyr Met Ser Leu Leu Thr Leu Ser Thr Ile Asn
                245                 250                 255    
Met Lys Glu Lys Ser Ile Glu Ile Gln Asn Gly Asn Phe Leu Arg Phe
            260                 265                 270        
Phe Lys Asp Asn Leu Leu Lys Asn Gly Glu Thr Glu Ser
        275                 280                 285
<210>  9
<211>  316
<212>  PRT
<213>  B. subtilis 168
<400>  9
Val Lys Thr Ile His Ile Thr His Pro His His Leu Ile Lys Glu Glu
1               5                   10                  15     
Gln Ala Lys Ser Val Met Ala Leu Gly Tyr Phe Asp Gly Val His Leu
            20                  25                  30         
Gly His Gln Lys Val Ile Gly Thr Ala Lys Gln Ile Ala Glu Glu Lys
        35                  40                  45             
Gly Leu Thr Leu Ala Val Met Thr Phe His Pro His Pro Ser His Val
    50                  55                  60                 
Leu Gly Arg Asp Lys Glu Pro Lys Asp Leu Ile Thr Pro Leu Glu Asp
65                  70                  75                  80 
Lys Ile Asn Gln Ile Glu Gln Leu Gly Thr Glu Val Leu Tyr Val Val
                85                  90                  95     
Lys Phe Asn Glu Val Phe Ala Ser Leu Ser Pro Lys Gln Phe Ile Asp
            100                 105                 110        
Gln Tyr Ile Ile Gly Leu Asn Val Gln His Ala Val Ala Gly Phe Asp
        115                 120                 125            
Phe Thr Tyr Gly Lys Tyr Gly Lys Gly Thr Met Lys Thr Met Pro Asp
    130                 135                 140                
Asp Leu Asp Gly Lys Ala Gly Cys Thr Met Val Glu Lys Leu Thr Glu
145                 150                 155                 160
Gln Asp Lys Lys Ile Ser Ser Ser Tyr Ile Arg Thr Ala Leu Gln Asn
                165                 170                 175    
Gly Asp Val Glu Leu Ala Asn Val Leu Leu Gly Gln Pro Tyr Phe Ile
            180                 185                 190        
Lys Gly Ile Val Ile His Gly Asp Lys Arg Gly Arg Thr Ile Gly Phe
        195                 200                 205             
Pro Thr Ala Asn Val Gly Leu Asn Asn Ser Tyr Ile Val Pro Pro Thr
    210                 215                 220                
Gly Val Tyr Ala Val Lys Ala Glu Val Asn Gly Glu Val Tyr Asn Gly
225                 230                 235                 240
Val Cys Asn Ile Gly Tyr Lys Pro Thr Phe Tyr Glu Lys Arg Pro Glu
                245                 250                 255    
Gln Pro Ser Ile Glu Val Asn Leu Phe Asp Phe Asn Gln Glu Val Tyr
            260                 265                 270        
Gly Ala Ala Ile Lys Ile Glu Trp Tyr Lys Arg Ile Arg Ser Glu Arg
        275                 280                 285            
Lys Phe Asn Gly Ile Lys Glu Leu Thr Glu Gln Ile Glu Lys Asp Lys
    290                 295                 300                 
Gln Glu Ala Ile Arg Tyr Phe Ser Asn Leu Arg Lys
305                 310                 315    
<210>  10
<211>  316
<212>  PRT
<213>  B. subtilis 168
<400>  10
Val Lys Thr Ile His Ile Thr His Pro His His Leu Ile Lys Glu Glu
1               5                   10                  15     
Gln Ala Lys Ser Val Met Ala Leu Gly Tyr Phe Asp Gly Val His Leu
            20                  25                  30         
Gly His Gln Lys Val Ile Gly Thr Ala Lys Gln Ile Ala Glu Glu Lys
        35                  40                  45             
Gly Leu Thr Leu Ala Val Met Thr Phe His Pro His Pro Ser His Val
    50                  55                  60                 
Leu Gly Arg Asp Lys Glu Pro Lys Asp Leu Ile Thr Pro Leu Glu Asp
65                  70                  75                  80 
Lys Ile Asn Gln Ile Glu Gln Leu Gly Thr Glu Val Leu Tyr Val Val
                85                  90                  95     
Lys Phe Asn Glu Val Phe Ala Ser Leu Ser Pro Lys Gln Phe Ile Asp
            100                 105                 110        
Gln Tyr Ile Ile Gly Leu Asn Val Gln His Ala Val Ala Gly Phe Asp
        115                 120                 125            
Phe Thr Tyr Gly Lys Tyr Gly Lys Gly Thr Met Lys Thr Met Pro Arg
    130                 135                 140                
Asp Leu Asp Gly Lys Ala Gly Cys Thr Met Val Glu Lys Leu Thr Glu
145                 150                 155                 160
Gln Asp Lys Lys Ile Ser Ser Ser Tyr Ile Arg Thr Ala Leu Gln Asn
                165                 170                 175    
Gly Asp Val Glu Leu Ala Asn Val Leu Leu Gly Gln Pro Tyr Phe Ile
            180                 185                 190        
Lys Gly Ile Val Ile His Gly Asp Lys Arg Gly Arg Thr Ile Gly Phe
        195                 200                 205            
Pro Thr Ala Asn Val Gly Leu Asn Asn Ser Tyr Ile Val Pro Pro Thr
    210                 215                 220                 
Gly Val Tyr Ala Val Lys Ala Glu Val Asn Gly Glu Val Tyr Asn Gly
225                 230                 235                 240
Val Cys Asn Ile Gly Tyr Lys Pro Thr Phe Tyr Glu Lys Arg Pro Glu
                245                 250                 255    
Gln Pro Ser Ile Glu Val Asn Leu Phe Asp Phe Asn Gln Glu Val Tyr
            260                 265                 270        
Gly Ala Ala Ile Lys Ile Glu Trp Tyr Lys Arg Ile Arg Ser Glu Arg
        275                 280                 285            
Lys Phe Asn Gly Ile Lys Glu Leu Thr Glu Gln Ile Glu Lys Asp Lys
    290                 295                 300                
Gln Glu Ala Ile Arg Tyr Phe Ser Asn Leu Arg Lys
305                 310                 315    
<210>  11
<211>  216
<212>  PRT
<213>  B. subtilis 168
<400>  11
Met Gly Gly Asn Gln Ile Leu Lys Gln Ile Ile Ala Lys His Leu Leu
1               5                   10                  15     
Asp Ile Gln Ala Val Phe Leu Arg Pro Asn Glu Pro Phe Thr Trp Ala
            20                  25                  30         
Ser Gly Ile Leu Ser Pro Ile Tyr Cys Asp Asn Arg Leu Thr Leu Ser
        35                  40                  45             
Phe Pro Glu Val Arg Asn Asp Val Ala Ser Gly Ile Ser Lys Leu Val
    50                  55                  60                 
Lys Glu His Phe Pro Glu Ala Glu Met Ile Ala Gly Thr Ala Thr Ala
65                  70                  75                  80 
Gly Ile Pro His Ala Ala Leu Ala Ala Asp His Leu Asn Leu Pro Met
                85                  90                  95     
Cys Tyr Val Arg Ser Lys Pro Lys Ala His Gly Lys Gly Asn Gln Ile
            100                 105                 110        
Glu Gly Ala Val Gln Glu Gly Gln Lys Thr Val Val Ile Glu Asp Leu
        115                 120                 125            
Ile Ser Thr Gly Gly Ser Val Leu Glu Ala Cys Ala Ala Leu Gln Ala
    130                 135                 140                
Ala Gly Cys Glu Val Leu Gly Val Val Ser Ile Phe Thr Tyr Gly Leu
145                 150                 155                 160
Pro Lys Ala Glu Glu Ala Phe Ala Lys Ala Glu Leu Pro Tyr Tyr Ser
                165                 170                 175     
Leu Thr Asp Tyr Asp Thr Leu Thr Glu Val Ala Leu Glu Asn Gly Asn
            180                 185                 190        
Ile His Ser Asp Asp Leu Lys Lys Leu Gln Thr Trp Lys Arg Asn Pro
        195                 200                 205            
Glu Ser Lys Asp Trp Phe Lys Lys
    210                 215    
<210>  12
<211>  216
<212>  PRT
<213>  B. subtilis 168
<400>  12
Met Gly Gly Asn Gln Ile Leu Lys Gln Ile Ile Ala Lys His Leu Leu
1               5                   10                  15     
Asp Ile Gln Ala Val Phe Leu Arg Pro Asn Glu Pro Phe Thr Trp Ala
            20                  25                  30         
Ser Gly Ile Leu Ser Pro Ile Tyr Cys Asp Asn Arg Leu Thr Leu Ser
        35                  40                  45             
Phe Pro Glu Val Arg Asn Asp Val Ala Ser Gly Ile Ser Lys Leu Val
    50                  55                  60                 
Lys Glu His Phe Pro Glu Ala Glu Met Ile Ala Gly Thr Ala Thr Ala
65                  70                  75                  80 
Gly Ile Pro His Ala Ala Leu Ala Ala Asp His Leu Asn Leu Pro Met
                85                  90                  95     
Cys Tyr Val Arg Ser Lys Pro Glu Ala His Gly Lys Gly Asn Gln Ile
            100                 105                 110        
Glu Gly Ala Val Gln Glu Gly Gln Lys Thr Val Val Ile Glu Asp Leu
        115                 120                 125             
Ile Ser Thr Gly Gly Ser Val Leu Glu Ala Cys Ala Ala Leu Gln Ala
    130                 135                 140                
Ala Gly Cys Glu Val Leu Gly Val Val Ser Ile Phe Thr Tyr Gly Leu
145                 150                 155                 160
Pro Lys Ala Glu Glu Ala Phe Ala Lys Ala Glu Leu Pro Tyr Tyr Ser
                165                 170                 175    
Leu Thr Asp Tyr Asp Thr Leu Thr Glu Val Ala Leu Glu Asn Gly Asn
            180                 185                 190        
Ile His Ser Asp Asp Leu Lys Lys Leu Gln Thr Trp Lys Arg Asn Pro
        195                 200                 205            
Glu Ser Lys Asp Trp Phe Lys Lys
    210                 215

Claims (12)

  1. 一种产核黄素的枯草芽孢杆菌，其特征在于，所述枯草芽孢杆菌菌株中存在下述至少一个位点发生了点突变：
    1)purR A148D：嘌呤操纵子转录调节蛋白PurR，其148位丙氨酸突变为天冬氨酸；
    2)ribC D144R：双功能黄素激酶FAD合成酶RibC，其144位天冬氨酸突变为精氨酸；
    3)pyrE K104E：乳清酸磷酸核糖基转移酶PyrE，其104位赖氨酸突变为谷氨酸。
  2. 如权利要求1所述的产核黄素的枯草芽孢杆菌，其特征在于，其保藏编号为CGMCC NO.21202。
  3. 一种枯草芽孢杆菌嘌呤操纵子转录调节蛋白PurR的突变基因，其特征在于，该突变基因如SEQ NO.2所示；编码的蛋白如SEQ NO.8所示。
  4. 一种枯草芽孢杆菌双功能黄素激酶FAD合成酶RibC的突变基因，其特征在于，该突变基因如SEQ NO.4所示；编码的蛋白如SEQ NO.10所示。
  5. 一种枯草芽孢杆菌乳清酸磷酸核糖基转移酶PyrE的突变基因，其特征在于，该突变基因如SEQ NO.6所示；编码的蛋白如SEQ NO.12所示。
  6. 一种产核黄素工程菌的构建方法，包括如下步骤：
    (1)在出发菌株的染色体上整合了purR、ribC或pyrE的基因，其中至少一个基因存在突变；
    (2)获得较出发菌株相比核黄素产量提高的菌株。
  7. 如权利要求6所述方法，其特征在于，所述基因突变优选为：1）PurR的148位丙氨酸突变为天冬氨酸；2）RibC的144位天冬氨酸突变为精氨酸；3）PyrE的104位赖氨酸突变为谷氨酸；所述步骤(1)中的出发菌株为枯草芽孢杆菌，优选为Bacillus subtilis 168；所述步骤(1)中的质粒为芽孢杆菌常用表达质粒，优选为pKSV7或pKSU。
  8. 一种制备核黄素的方法，包括如下步骤：
    (1)将甘油管保藏的权利要求1所述工程菌在LB平板进行无菌划线，36℃过夜培养；
    (2)挑一环培养物接种至30 mL种子培养基中，110 rpm，36℃培养7~8 h；
    (3)按10%接种量转接至30 ml 发酵培养基中，摇床转速120 rpm，36℃培养40h。
  9. 如权利要求8所述方法，其特征在于，种子培养基配方(g/L)为：葡萄糖20，酵母粉5，玉米浆干粉5，磷酸二氢钾3，硫酸镁0.5，硫酸亚铁0.02，硫酸锰0.01，pH 7.0～7.2，121℃灭菌20分钟；发酵培养基配方(g/L)为：葡萄糖50，酵母粉3.5，磷酸二氢钾3，硫酸铵25，硫酸锰0.01，硫酸镁5，味精10，玉米浆干粉15，碳酸钙25，pH 7.0～7.2，121℃灭菌20分钟。
  10. 权利要求1-2所述的工程菌在生产核黄素中的应用。
  11. 权利要求1-2所述的工程菌在饲料、医药、食品中的应用。
  12. 权利要求3-5所述的突变基因或蛋白在生产核黄素中的应用。
PCT/CN2021/097024 2020-12-15 2021-05-29 一种产核黄素的枯草芽孢杆菌及其构建方法与应用 WO2022127039A1 (zh)

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CN117660577A (zh) * 2023-12-08 2024-03-08 苏州华赛生物工程技术有限公司 LtaSA蛋白或其编码基因在核黄素生产中的应用

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