WO2018126527A1 - 一种生产核黄素的工程菌株及其应用 - Google Patents

一种生产核黄素的工程菌株及其应用 Download PDF

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WO2018126527A1
WO2018126527A1 PCT/CN2017/075585 CN2017075585W WO2018126527A1 WO 2018126527 A1 WO2018126527 A1 WO 2018126527A1 CN 2017075585 W CN2017075585 W CN 2017075585W WO 2018126527 A1 WO2018126527 A1 WO 2018126527A1
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strain
riboflavin
expression cassette
gene expression
gene
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PCT/CN2017/075585
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English (en)
French (fr)
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施明安
孙向宇
科赛科G
赛墨H
梅格戴夫斯卡V
塞瓦吉M
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上海创诺医药集团有限公司
赤峰制药股份有限公司
赤峰蒙广生物科技有限公司
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Publication of WO2018126527A1 publication Critical patent/WO2018126527A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/174—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

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  • the present invention relates to the field of biotechnology, and more particularly to an engineered strain for producing riboflavin and its use.
  • Riboflavin, vitamin B2 (VB2), is a water-soluble B vitamin that is a coenzyme component of the flavozymes and plays an important role in respiration and biooxidation. It is human and animal.
  • riboflavin engineering bacteria such as Bacillus subtilis
  • Bacillus subtilis has become a key research object because of its advantages such as short fermentation cycle, easy availability of raw materials, and mature genetic engineering technology of prokaryotic cells.
  • many strains including Bacillus subtilis have reliable safety in Bacillus, and their fermentation products have long been used in the food and feed industry.
  • the traditional method for transforming riboflavin producing bacteria is to carry out strain mutagenesis, such as physical mutagenesis and chemical mutagenesis.
  • strain mutagenesis such as physical mutagenesis and chemical mutagenesis.
  • the method of physical mutagenesis and chemical mutagenesis carries out strain modification, and the randomness is relatively large, resulting in huge screening workload, long time-consuming and slow effect.
  • the genetic stability of the strain gene is poor, and the gene may recover and mutate during the passage, resulting in a decrease in the riboflavin synthesis ability of the strain. Therefore, there is an urgent need in the art to construct stable, high-yield riboflavin-producing bacteria for riboflavin production.
  • an engineered strain for producing riboflavin the strain being Bacillus, and a genome of the strain is integrated with a foreign gene expression cassette, the exogenous source
  • the gene expression cassette contains the following elements: the P15 promoter and the riboflavin operon gene.
  • the exogenous gene expression cassette comprises a first exogenous gene expression cassette and a second exogenous gene expression cassette.
  • the first exogenous gene expression cassette further comprises an AmyE gene element.
  • the AmyE gene comprises an AmyE gene fragment.
  • the AmyE gene fragment is a nucleotide sequence of 600 bp in length at the 5' end of the AmyE gene.
  • the first exogenous gene expression cassette is integrated downstream of the AmyE gene of the engineered strain genome.
  • first exogenous gene expression cassette and the second exogenous gene expression cassette further comprise a resistance gene element.
  • the first foreign gene expression cassette contains a resistance gene element that is different from the resistance gene element contained in the second foreign gene expression cassette.
  • the resistance gene is selected from the group consisting of a tetracycline resistance gene, a kanamycin resistance gene, an ampicillin resistance gene, or a combination thereof.
  • the exogenous riboflavin operon gene is selected from the group consisting of a ribB gene, a ribG gene, a ribA gene, a ribH gene, a ribTD gene, or a combination thereof.
  • the engineered strain is a recombinant obtained by protoplast fusion.
  • the foreign gene (riboflavin operon gene) in the genome of the strain has a copy number of 10 to 50, preferably 20 to 40.
  • 10-50, preferably 20-40, of said exogenous gene expression cassettes are integrated into the genome of said strain.
  • 30-50 copies of the ribG gene are integrated into the genome of the strain.
  • 20-25 copies of the ribA gene are integrated into the genome of the strain.
  • the strain is selected from the group consisting of Bacillus subtilis, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus Halodurans and Bacillus licheniformis.
  • the strain is Bacillus subtilis, preferably Bacillus subtilis SMY.
  • Bacillus subtilis VKPM B2116 used in the examples below belongs to the genus Bacillus subtilis SMY, and is preferred.
  • a method of producing riboflavin comprising the steps of:
  • a third aspect of the invention there is provided a method of constructing the engineered strain of the first aspect of the invention, comprising the steps of:
  • the method further comprises the step (e): PCR verifying the genotype of the recombinant obtained in step (d); and/or
  • the vector is a plasmid and/or a nucleic acid fragment.
  • the vector further has a resistance gene element.
  • the vector further has a terminator element.
  • the recipient strain is Bacillus subtilis, preferably Bacillus subtilis SMY.
  • the engineered strain is integrated into the genome of the recipient strain by a method selected from the group consisting of transformation, homologous recombination.
  • an engineered strain according to the first aspect of the invention which is used as an engineered bacterium for the fermentation production of riboflavin.
  • a method of preparing a food composition comprising the steps of:
  • the riboflavin prepared by the method of the second aspect of the invention is added to the food composition to obtain a riboflavin-containing food composition.
  • the food composition comprises a feed composition.
  • Figure 1 shows the structural map of the pBR25 plasmid.
  • Figure 2 shows the structural map of the pBR31 plasmid.
  • Figure 3 shows the protoplast fusion of VBB8662 and VBB8978, and the selected fusion strains were subjected to shake flask fermentation experiments, and the riboflavin production of each fusion strain.
  • Figure 4 shows the monoclonal strain of the fusion strain with riboflavin production greater than 5g/L in the fermentation shake flask experiment, and the obtained monoclonal colonies were subjected to shake flask fermentation experiments, and the riboflavin production of each fusion strain.
  • FIG. 5 shows the fermenter experiment for the high-yield monoclonal VBB11863.
  • Figure 6 shows the fermenter experiment for the high-yield monoclonal VBB11866.
  • the present inventors have extensively and intensively studied for the first time to unexpectedly find a special structure of a foreign gene expression cassette comprising a P15 promoter and a riboflavin operon gene.
  • the exogenous gene expression cassette is used to transform Bacillus subtilis, and the protoplast fusion technique is used to perform protoplast fusion, thereby obtaining an engineering strain in which riboflavin production is significantly improved.
  • the exogenous gene of the engineering strain can be stably inherited and expressed in the genome, and has good reproducibility, and can realize large-scale industrial production.
  • the present invention provides a gene sequence of a recombinant bacterium, which preferably comprises: an exogenous riboflavin operon gene sequence carrying a P15 promoter and a tetracycline resistance gene sequence, the riboflavin
  • the operon gene sequence comprises: ribB, ribG, ribA, ribH and ribTD; comprising another exogenous riboflavin operon gene sequence carrying a P15 promoter and a kanamycin resistance gene sequence, said riboflavin
  • the operon gene sequence comprises: ribB, ribG, ribA, ribH and ribTD;
  • the gene sequence is introduced into the host cell chromosome by transformation, homologous recombination, etc.; the ribA, ribG gene exists in multiple copies in the host cell chromosome, and the copy number of the gene is ⁇ 10, and the exogenous riboflavin manipulation
  • the subgene sequences are all synthetic gene sequences.
  • Microorganisms suitable for use in the synthesis of riboflavin according to the present invention include microbial cells capable of producing riboflavin (e.g., converting a given carbon source to riboflavin) with unmodified A rib operon comprising an unmodified leader region or an equivalent thereof or homologue (subsequently mutated in a manner that results in increased riboflavin production as described herein), or into which a modified version of the rib manipulation is introduced Sub/rib leader or its equivalent.
  • Suitable microorganisms bearing such unmodified rib operon/rib leader or equivalents thereof may be selected from bacteria, such as Gram-negative and Gram-positive bacteria, which are wild-type strains, which are classically mutagenized.
  • the microorganism or host cell is selected from the group consisting of Bacillus subtilis, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus halodurans, Bacillus licheniformis. More preferred is Bacillus subtilis, especially Bacillus subtilis SMY.
  • the strain modification method of the invention integrates the engineered gene into one or more sites of the strain genome, and the gene can be stably inherited and expressed in the genome, and the transformed strain has stable riboflavin expression ability.
  • the "engineering strain”, “riboflavin producing strain”, and “recombinant bacteria” are used interchangeably and refer to the engineering strain for producing riboflavin of the present invention, that is, the first aspect of the present invention. Said strain.
  • AmyE gene is a B. subtilis amylase gene that is used to direct the site-specific integration of the exogenous gene expression cassette of the present application in the Bacillus genome.
  • the term "foreign gene expression cassette” refers to an expression cassette that expresses a foreign gene (riboflavin operon gene) with a P15 promoter.
  • the exogenous gene expression cassette includes a first exogenous gene expression cassette and a second exogenous gene expression cassette.
  • the first foreign gene expression cassette contains an AmyE gene fragment in addition to the P15 promoter and the riboflavin operon gene, and is used to direct the first foreign gene expression cassette to be integrated downstream of the AmyE gene of the Bacillus genome.
  • the second exogenous gene expression cassette is randomly integrated into the Bacillus genome.
  • the engineered strain of the present application is obtained by protoplast fusion of "recombinant bacteria integrating the first exogenous gene expression cassette" and "recombinant bacteria integrating the second exogenous gene expression cassette".
  • the engineered strain simultaneously integrates a first exogenous gene expression cassette and a second exogenous gene expression cassette.
  • the first exogenous gene expression cassette is integrated downstream of the AmyE gene and the second exogenous gene expression cassette is integrated at any position.
  • the foreign gene (riboflavin operon gene) in the genome of the engineered strain has a copy number of 10-50, preferably 20-40, and the copy number of the ribA and ribG genes are ⁇ 10.
  • the VKPM B2116 strain is transferred into the pBR25 plasmid, which contains a riboflavin synthesis gene and a tetracycline resistance gene with a P15 promoter, and the transformed strain is subjected to monoclonal screening, and the high-yield monoclonal is subjected to EMS. Mutagenesis, using different concentrations of tetracycline, screened the riboflavin high-yield strain VBB8662.
  • the pBR31 plasmid was transformed into the strain containing the AmyE 5'-end 600 bp gene fragment, the riboflavin synthesis gene with the P15 promoter, and kanamycin.
  • the resistance gene, the transformed strain were screened by monoclonal screening and the concentration of kanamycin was screened, and the high-yield strain VBB8978 was screened.
  • the protoplasts of VBB8662 and VBB8978 were fused, and the fused strains were screened by monoclonal screening to screen out the high-yield strains VBB11863 and VBB11866.
  • a promoter is a component of a gene that controls the start and expression of gene expression (transcription) and activates RNA polymerase to bind precisely to the template DNA and has the specificity of transcription initiation.
  • the P15 promoter is a strong promoter with high affinity for RNA polymerase. It can guide the synthesis of large amounts of mRNA, promote the expression of riboflavin gene, and improve the riboflavin production ability of cells.
  • the foreign gene contained in the engineered strain of the present invention is integrated into the genome, and has high genetic stability, and the strain can stably produce riboflavin;
  • the engineered strain of the present invention adopts the P15 promoter, which is a strong promoter, can efficiently initiate transcription of genes, and improve gene expression efficiency.
  • the riboflavin synthesis gene (ribB, ribG, ribA, ribH and ribTD) carrying the P15 promoter was synthesized, and the gene was introduced into the pUC19 plasmid using a restriction enzyme site, and the tetracycline resistance gene was named.
  • the plasmid was named pBR25. Plasmid (see Figure 1).
  • the pBR25 plasmid was transformed into a host cell (commercially available Bacillus subtilis VKPM B2116 strain, Bacillus subtilis VKPM B2116), and the gene was introduced into the host cell genome by homologous recombination, and the concentration of the antibiotic was ranged by using different concentrations of tetracycline. 10-100 mg/L, strain VBB8662 with high copy number gene was obtained.
  • the riboflavin synthesis gene (ribB, ribG, ribA, ribH and ribTD) carrying the P15 promoter was synthesized, and the gene was introduced into the pUC19 plasmid using a cleavage site with a kanamycin resistance gene and a part of AmyE. Gene, this plasmid was named pBR31 plasmid (see Figure 2).
  • the pBR31 plasmid was transformed into a host cell (B2116 strain) in which the riboflavin synthesis gene was knocked out, and the gene was introduced into the host cell genome by homologous recombination, and the concentration of the antibiotic was screened by using different concentrations of kanamycin.
  • the strain VBB8978 which has a high copy number gene, is obtained in the range of 10-100 mg/L.
  • Regeneration medium 81 g of sodium succinate, 5 g of complex protein amino acid, 10 g of yeast powder, 4 g of K2HPO, 1.5 g of KH2PO4, 0.1 g of L-tryptophan, 0.1 g of MgCl2.6H2O, 0.1 g of BSA, 10 g of glucose, and 10 g of glucose were added.
  • water after dissolution, add water to dissolve to 1L (solid medium is added 15-15g agar powder per liter), autoclave at 121 °C for 20min.
  • Lysozyme 20mg/mL was prepared by using sterile double distilled water, filtered and sterilized, and stored at -20 °C after dispensing.
  • Example 2 The two strains VBB8662 and VBB8978 obtained in Example 1 were inoculated into 4 ml of GM liquid medium, respectively, and cultured overnight at 37 ° C with shaking.
  • the protoplast fusion technique is used to increase the riboflavin gene copy number of the cells, and the riboflavin production ability of the cells is improved, and the riboflavin expression of the cells is obviously improved.
  • 1 L solid medium Tryptone: 5-10 g; Yeast Extract: 1-5 g; NaCl: 1-5 g; maltose: 10-20 g; agar: 20 g; tetracycline: 10-100 mg; kanamycin: 10-100 mg/L .
  • 1 L shake flask seed medium glucose: 5-10 g; corn syrup: 10-20 g; waste molasses: 10-20 g; yeast extract: 5-10 g; MgSO4: 1-5 g; (NH4)2SO4: 5-10 g;
  • 1L shake flask fermentation medium glucose: 50-100g; corn syrup: 1-10g; soybean meal: 10-50g; MgSO4.7H2O: 1-5g; urea: 5-10g;
  • Example 2 The monoclonal colony glycerol solution of the recombinant bacteria obtained in Example 2 was applied to a plate medium for culture, and the culture conditions were: 37 ° C, 14-16 hr;
  • the cultured overnight culture solution was inoculated into the fermentation medium at a 10% inoculation amount, and cultured, and the culture conditions were: 37 ° C, 220 rpm, 48 hr;
  • Example 7 Using the method of Example 2, VBB8662 and VBB8978 were fused to obtain a plurality of riboflavin-producing fusion strains, and the fused strain was subjected to shake flask fermentation experiment. The experimental results are shown in Fig. 3, and the riboflavin yield was up to 6 g/L.
  • 1 L solid medium Tryptone: 5-10 g; Yeast Extract: 1-5 g; NaCl: 1-5 g; maltose: 10-20 g; agar: 20 g; tetracycline: 10-100 mg; kanamycin: 10-100 mg/L .
  • 1 L shake flask seed medium glucose: 5-10 g; corn syrup: 10-20 g; waste molasses: 10-20 g; yeast extract: 5-10 g; MgSO4: 1-5 g; (NH4)2SO4: 5-10 g;
  • 1L shake flask fermentation medium glucose: 50-100g; corn syrup: 1-10g; soybean meal: 10-50g; MgSO4.7H2O: 1-5g; urea: 5-10g;
  • Example 2 The recombinant bacteria VBB11863 and VBB11866 obtained in Example 2 were cultured in a blister culture medium, and culture conditions were 37 ° C, 14-16 hr;
  • the cultured overnight culture solution is inoculated into a 5 L fermentor at a 10% inoculum for culture, the culture condition is 37 ° C, the dissolved oxygen is controlled by 15-20%, and the fermentation time is 48 hr;

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Abstract

提供一种生产核黄素的工程菌株及其应用，该菌株是一种通过基因修饰获得的工程细菌，其中基因修饰包括对核黄素合成基因及其启动子的改造。

Description

一种生产核黄素的工程菌株及其应用 技术领域
本发明涉及生物技术领域，更具体地涉及一种生产核黄素的工程菌株及其应用。
背景技术
核黄素(Riboflavin)，即维生素B2(VB2)，是一种水溶性的B族维生素，为黄素酶类的辅酶组成部分，在呼吸和生物氧化中起着重要的作用，是人类和动物必需的一种维生素，主要应用于医药、食品及饲料工业作为食品、饲料添加剂和食用天然色素等。
二十世纪七十年代前，微生物发酵法生产核黄素主要以棉囊阿舒氏酵母(Ashbya gossypii)、解朊假丝酵母(Candida famata)、阿舒氏假囊酵母(Eremothecium ashbyii)等真菌为主。相继构建成功的Bacillus subtilis等产核黄素工程菌，因其具有发酵周期短、原料价廉易得、原核细胞基因工程技术成熟等优点，成为重点研究对象。此外，在芽孢杆菌属中，包括Bacillus subtilis在内的许多菌株具有可靠的安全性，它们的发酵产物在食品与饲料工业中已有长期的应用。
目前传统的核黄素生产菌改造方法是进行菌种诱变，比如物理诱变，化学诱变等。物理诱变、化学诱变的方法进行菌种改造，随机性比较大，造成筛选工作量巨大，耗时长，见效慢。而且菌株基因的遗传稳定性差，传代过程中基因可能恢复突变，造成菌种核黄素合成能力下降。因此，本领域迫切需要构建稳定、高产的核黄素生产菌，从而进行核黄素生产。
发明内容
本发明的目的在于提供一种稳定、高产的核黄素生产菌及其应用。
在本发明的第一方面，提供了一种生产核黄素的工程菌株，所述菌株为芽孢杆菌(Bacillus)，并且所述菌株的基因组中整合有外源基因表达盒，所述的外源基因表达盒含有以下元件：P15启动子和核黄素操纵子基因。
在另一优选例中，所述的外源基因表达盒包含第一外源基因表达盒和第二外源基因表达盒。
在另一优选例中，所述的第一外源基因表达盒还含有AmyE基因元件。
在另一优选例中，所述的AmyE基因包括AmyE基因片段。
在另一优选例中，所述的AmyE基因片段为AmyE基因5’端的长度为600bp的核苷酸序列。
在另一优选例中，所述的第一外源基因表达盒整合于所述工程菌株基因组的AmyE基因下游。
在另一优选例中，所述的第一外源基因表达盒和第二外源基因表达盒还含有抗性基因元件。
在另一优选例中，所述的第一外源基因表达盒含有的抗性基因元件与第二外源基因表达盒含有的抗性基因元件不同。
在另一优选例中，所述抗性基因选自下组：四环素抗性基因、卡那霉素抗性基因、氨苄霉素抗性基因、或其组合。
在另一优选例中，所述的外源核黄素操纵子基因选自下组：ribB基因、ribG基因、ribA基因、ribH基因、ribTD基因、或其组合。
在另一优选例中，所述的工程菌株是经原生质体融合获得的重组子。
在另一优选例中，所述菌株的基因组中外源基因(核黄素操纵子基因)的拷贝数为10-50个，较佳地为20-40个。
在另一优选例中，所述菌株的基因组中整合有10-50个，较佳地20-40个所述的外源基因表达盒。
在另一优选例中，所述菌株的基因组中整合有30-50个拷贝的ribG基因。
在另一优选例中，所述菌株的基因组中整合有20-25个拷贝的ribA基因。
在另一优选例中，所述的菌株选自下组：枯草芽胞杆菌Bacillus subtilis、蜡样芽孢杆菌Bacillus cereus、解淀粉芽孢杆菌Bacillus amyloliquefaciens、嗜热脂肪芽胞杆菌Bacillus stearothermophilus、嗜碱芽孢杆菌属Bacillus halodurans和地衣形芽孢杆菌Bacillus licheniformis。
在另一优选例中，所述的菌株为枯草芽胞杆菌Bacillus subtilis，较佳地为Bacillus subtilis SMY。下文实施例中使用的Bacillus subtilis VKPM B2116属于Bacillus subtilis SMY菌属，为优选。
在本发明的第二方面，提供了一种生产核黄素的方法，包括步骤：
(i)培养本发明第一方面所述的工程菌株，从而获得含核黄素的发酵产物；和
(ii)从所述发酵产物中分离出核黄素。
在本发明的第三方面，提供了一种构建本发明第一方面所述工程菌株的方法，包括步骤：
(a)构建含有第一外源基因表达盒的载体，所述第一外源基因表达盒具有以下元件：AmyE基因、P15启动子和核黄素操纵子基因；
(b)构建含有第二外源基因表达盒的载体，所述第二外源基因表达盒具有以下元件：P15启动子和核黄素操纵子基因；
(c)将步骤(a)和步骤(b)获得的载体分别转入受体菌株，获得基因组整合有第一外源基因表达盒的第一重组菌株和基因组整合有第二外源基因表达盒的第二重组菌株；
(d)对第一重组菌株和第二重组菌株进行原生质体融合，获得基因组中整合有第一外源基因表达盒和第二外源基因表达盒的重组子，所述的重组子即为本发明第一方面所述的工程菌株。
在另一优选例中，所述方法还包括步骤(e)：PCR验证步骤(d)得到的重组子的基因型；和/或
步骤(f)：发酵检测步骤(d)得到的重组子的核黄素产量。
在另一优选例中，所述的载体为质粒和/或核酸片段。
在另一优选例中，所述的载体还具有抗性基因元件。
在另一优选例中，所述的载体还具有终止子元件。
在另一优选例中，所述的受体菌株为枯草芽胞杆菌Bacillus subtilis，较佳地为Bacillus subtilis SMY。
在另一优选例中，所述的工程菌株是用选自下组的方法将所述载体整合到受体菌株的基因组中：转化、同源重组。
在本发明的第四方面，提供了一种本发明第一方面所述工程菌株的用途，所述菌株被用作发酵生产核黄素的工程菌。
在本发明的第五方面，提供了一种制备食品组合物的方法，包括步骤：
将本发明第二方面所述方法制备的核黄素添加到食品组合物中，从而获得含有核黄素的食品组合物。
在另一优选例中，所述的食品组合物包括饲料组合物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了pBR25质粒的结构图谱。
图2显示了pBR31质粒的结构图谱。
图3显示了VBB8662和VBB8978进行原生质体融合，筛选的融合菌株进行摇瓶发酵实验，各融合菌株的核黄素产量。
图4显示了发酵摇瓶实验中，核黄素产量大于5g/L的融合菌株进行单克隆筛选，获得的单克隆菌落进行摇瓶发酵实验，各融合菌株的核黄素产量。
图5显示了高产单克隆VBB11863的发酵罐实验情况。
图6显示了高产单克隆VBB11866的发酵罐实验情况。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现一种特殊结构的外源基因表达盒，所述的外源基因表达盒包含P15启动子和核黄素操纵子基因。利用所述外源基因表达盒转化枯草芽胞杆菌，再利用原生质体融合技术进行原生质体的融合，能够得到核黄素产量显著提高的工程菌株。该工程菌株的外源基因可以稳定的在基因组中进行遗传、表达，且重复性好，可实现规模化工业生产。
具体地，本发明提供了一种重组细菌的基因序列，所述基因序列优选：包含带有一个P15启动子的外源核黄素操纵子基因序列及四环素抗性基因序列，所述核黄素操纵子基因序列包含：ribB、ribG、ribA、ribH及ribTD；包含另一个带有一个P15启动子的外源核黄素操纵子基因序列及卡那霉素抗性基因序列，所述核黄素操纵子基因序列包含：ribB、ribG、ribA、ribH及ribTD；上 述基因序列是通过转化、同源重组等方式引入到宿主细胞染色体中；ribA、ribG基因在宿主细胞染色体中以多拷贝形式存在，基因的拷贝数均≥10，所述外源核黄素操纵子基因序列均为合成基因序列。
本发明涉及的适合用于合成核黄素的微生物包括下述微生物细胞，所述微生物细胞能够生产核黄素(例如将给定的碳源转化成核黄素)，并且带有未经修饰的rib操纵子，包括未经修饰的前导区或其等同物或同源物(随后以如本文所述导致核黄素生产提高的方式被突变)，或向其中引入经修饰版本的所述rib操纵子/rib前导区或其等同物。带有此类未经修饰的rib操纵子/rib前导区或其等同物的合适的微生物可选自细菌，例如革兰氏阴性和革兰氏阳性细菌，其为野生型菌株、通过经典诱变和选择方法获得的突变体菌株，或重组菌株。优选地，微生物或宿主细胞选自由以下组成的组：枯草芽胞杆菌Bacillus subtilis、蜡样芽孢杆菌Bacillus cereus、解淀粉芽孢杆菌Bacillus amyloliquefaciens、嗜热脂肪芽胞杆菌Bacillus stearothermophilus、嗜碱芽孢杆菌属Bacillus halodurans、地衣形芽孢杆菌Bacillus licheniformis。更优选的是Bacillus subtilis，特别是Bacillus subtilis SMY。
本发明的菌种改造方法将改造的基因整合到菌种基因组一个或多个位点中，基因可以稳定的在基因组中进行遗传、表达，改造后的菌种具有稳定的核黄素表达能力。
如本文所用，所述的“工程菌株”、“核黄素生产菌株”、“重组细菌”可互换使用，均指本发明的用于生产核黄素的工程菌株，即本发明第一方面所述的菌株。
如本文所用，“AmyE基因”是枯草芽孢杆菌淀粉酶基因，该基因用于指导本申请的外源基因表达盒在芽孢杆菌基因组定点整合。
外源基因表达盒
如本文所用，术语“外源基因表达盒”是指带有P15启动子的表达外源基因(核黄素操纵子基因)的表达盒。该外源基因表达盒包括第一外源基因表达盒和第二外源基因表达盒。其中，第一外源基因表达盒除含有P15启动子和核黄素操纵子基因外，还含有AmyE基因片段，用于指导第一外源基因表达盒定点整合于芽孢杆菌基因组AmyE基因的下游。第二外源基因表达盒随机整合于芽孢杆菌基因组。
本申请的工程菌株是通过将“整合第一外源基因表达盒的重组菌”和“整合第二外源基因表达盒的重组菌”进行原生质体融合得到的。该工程菌株同时整合有第一外源基因表达盒和第二外源基因表达盒。其中，在AmyE基因的下游整合第一外源基因表达盒和任意位置整合第二外源基因表达盒。该工程菌株的基因组中外源基因(核黄素操纵子基因)的拷贝数为10-50个，较佳地为20-40个，ribA、ribG基因的拷贝数均≥10。
本申请的优选实施方式中，VKPM B2116菌种转入pBR25质粒，该质粒含有带P15启动子的核黄素合成基因及四环素抗性基因，转化后的菌株进行单克隆筛选，高产单克隆进行EMS诱变，利用不同浓度的四环素，筛选出核黄素高产菌株VBB8662。VKPM B2116菌种菌株的核黄素合成基因敲除后，将pBR31质粒转化入该菌株中，该质粒含有AmyE 5’端600bp基因片段、带P15启动子的核黄素合成基因及卡那霉素抗性基因，转化的菌株进行单克隆筛选及卡那霉素浓度筛选，筛选出高产菌株VBB8978。将VBB8662与VBB8978进行原生质体融合，融合后的菌株进行单克隆筛选，筛选出高产菌株VBB11863，VBB11866。
Figure PCTCN2017075585-appb-000001
P15启动子
启动子是基因(gene)的一个组成部分，控制基因表达(转录)的起始时间和表达的程度，能活化RNA聚合酶，使之与模板DNA准确的结合并具有转录起始的特异性。P15启动子是一种强启动子，对RNA聚合酶有很高亲和力，它能指导合成大量的mRNA，促进核黄素基因的表达，提高菌体的核黄素生成能力。
本发明的主要优点包括：
(a)本发明工程菌株中包含的外源基因整合在基因组中，遗传稳定性高，该菌株可稳定的进行核黄素的生产；
(b)经过改造的核黄素工程菌株其核黄素产量得到明显的提高；
(c)本发明工程菌株采用P15启动子，该启动子为强启动子，可高效启动基因的转录，提高基因的表达效率。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
核黄素重组菌的构建
合成带有P15启动子的核黄素合成基因(ribB、ribG、ribA、ribH和ribTD)，利用酶切位点将该基因引入pUC19质粒中，并带有四环素抗性基因，该质粒命名为pBR25质粒(见图1)。将pBR25质粒转化入宿主细胞(市售Bacillus subtilis VKPM B2116菌种，Bacillus subtilis VKPM B2116)中，利用同源重组的方法将基因引入宿主细胞基因组中，利用不同浓度的四环素进行筛选，抗生素浓度范围为10-100mg/L，获得高拷贝数基因的菌株VBB8662。
合成带有P15启动子的核黄素合成基因(ribB、ribG、ribA、ribH和ribTD)，利用酶切位点将该基因引入pUC19质粒中，并带有卡那霉素抗性基因及部分AmyE基因，该质粒命名为pBR31质粒(见图2)。将pBR31质粒转化入敲除核黄素合成基因的宿主细胞(B2116菌种)中，利用同源重组的方法将基因定向引入宿主细胞基因组中，利用不同浓度的卡那霉素进行筛选，抗生素浓度范围为10-100mg/L，获得高拷贝数基因的菌株VBB8978。
表1.pBR25质粒和pBR31质粒的结构
Figure PCTCN2017075585-appb-000002
实施例2
原生质体融合
1)培养基的制备：
再生培养基：称取琥珀酸钠81g，络蛋白氨基酸5g，酵母粉10g，K2HPO44.6g，KH2PO4 1.5g，L-色氨酸0.1g，Mgcl2.6H2O 4.06g，BSA 0.1g，葡萄糖10g，加入水中，溶解后加水定溶至1L(固体培养基每升加15～16g琼脂粉)，121℃高温高压灭菌20min。
SMM溶液：山梨醇0.75M，pH6.5马来酸溶液0.02M，Mgcl2.6H2O 4.06g/L。
溶菌酶：用无菌双蒸水配制成20mg/mL，过滤除菌，分装后-20℃保存。
2)将实施例1获得的两株菌株VBB8662和VBB8978，分别接种于4ml GM液体培养基中，37℃振荡培养过夜。
3)取4ml培养菌液转接到50ml GM液体培养基中，37℃振荡培养3-4h至OD600为0.6-0.8。
4)5000rpm离心10min，弃上清后菌体悬浮于4-5ml SMM溶液中，加入终浓度为0.25mg/mL的溶菌酶，在37℃水浴中保温20-30min(10min左右观察一次)。立即以4000rpm离心10min，弃上清以去除溶菌酶，再用3-5ml SMM洗涤菌体。
5)4000rpm离心10min，弃上清，将原生质体重新悬浮于1-2ml SMM中，制得VBB8662和VBB8978的原生质体高渗悬浮液。
6)取以上两种原生质体高深悬浮液各1ml混合静止5-10min，4000rpm离心10min，弃上清，加入10％PEG溶液，36℃水浴，促融10min，4000rpm离心10min，弃上清，加入2ml再生培养基，涂布于含四环素和卡那霉素的固体再生培养基，36℃，倒置培养3-4天。
7)将固体培养基上生长的单克隆菌落分别保存于10％甘油，-80℃保藏。
本实施例利用原生质体融合技术增加菌体的核黄素基因拷贝数，提高菌体的核黄素生成能力，菌体的核黄素表达量得到明显的提高。
实施例3
高产核黄素重组细菌的筛选
1)培养基的配制：
1L固体培养基：Tryptone：5-10g；Yeast Extract：1-5g；NaCl：1-5g；麦芽糖：10-20g；琼脂：20g；四环素：10-100mg；卡那霉素：10-100mg/L。
1L摇瓶种子培养基：葡萄糖：5-10g；玉米浆：10-20g；废糖蜜：10-20g；酵母膏：5-10g；MgSO4：1-5g；(NH4)2SO4:5-10g；
1L摇瓶发酵培养基：葡萄糖：50-100g；玉米浆：1-10g；豆粕粉：10-50g；MgSO4.7H2O：1-5g；尿素:5-10g；
2)将实施例2获得的重组细菌的单克隆菌落甘油菌液涂布平板培养基进行培养，培养条件：37℃，14-16hr；
3)利用5ml无菌水收集平板上的菌体，混合均匀后备用；
4)以1％接种量接种到种子摇瓶中进行培养，培养条件：37℃，220rpm，14-16hr；
5)将培养过夜的菌液以10％接种量接种到发酵培养基中，进行培养，培养条件：37℃，220rpm，48hr；
6)利用HPLC检测摇瓶中核黄素含量；
7)利用实施例2的方法，VBB8662和VBB8978融合后获得多个核黄素高产融合菌株，融合后的菌株进行摇瓶发酵实验，实验结果见图3，核黄素产量最高可达6g/L
8)将7)获得的核黄素含量高于5g/L的菌株涂布于固体培养基上，进行单克隆筛选，重复步骤2)-6)；
9)利用HPLC检测摇瓶中核黄素含量；
10)经过单克隆筛选，获得多个核黄素高产单克隆菌株，单克隆菌落进行摇瓶发酵实验，实验结果见图4，核黄素产量最高可达8-9g/L。
实施例4
核黄素重组细菌的发酵罐实验
1)培养基的配制：
1L固体培养基：Tryptone：5-10g；Yeast Extract：1-5g；NaCl：1-5g；麦芽糖：10-20g；琼脂：20g；四环素：10-100mg；卡那霉素：10-100mg/L。
1L摇瓶种子培养基：葡萄糖：5-10g；玉米浆：10-20g；废糖蜜：10-20g；酵母膏：5-10g；MgSO4：1-5g；(NH4)2SO4:5-10g；
1L摇瓶发酵培养基：葡萄糖：50-100g；玉米浆：1-10g；豆粕粉：10-50g；MgSO4.7H2O：1-5g；尿素:5-10g；
2)将实施例2获得的重组细菌VBB11863、VBB11866的甘油菌液涂布平板培养基进行培养，培养条件：37℃，14-16hr；
3)利用5ml 20％甘油收集平板上的菌体，混合均匀后备用；
4)以1％接种量接种到种子摇瓶中进行培养，培养条件：37℃，220rpm， 14-16hr；
5)将培养过夜的菌液以10％接种量接种到5L发酵罐中进行培养，培养条件：37℃，溶氧控制15-20％，发酵时间为48hr；
6)利用HPLC检测摇瓶中核黄素含量。
结果如图5-6所示，经前述所改造的核黄素菌株，在5L发酵罐中，发酵70小时，核黄素产量可达到20g/L以上，其中重组菌株VBB11863在P15启动子的作用下，70hr时，核黄素产量可达22.5g/L(见图5)，重组菌株VBB11866在P15启动子的作用下，50hr时，核黄素产量可达20.8g/L(见图6),菌体的核黄素生成得到明显的提高。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种生产核黄素的工程菌株，其特征在于，所述菌株为芽孢杆菌(Bacillus)，并且所述菌株的基因组中整合有外源基因表达盒，所述的外源基因表达盒含有以下元件：P15启动子和核黄素操纵子基因。
  2. 如权利要求1所述的工程菌株，其特征在于，所述的外源基因表达盒包含第一外源基因表达盒和第二外源基因表达盒。
  3. 如权利要求1所述的工程菌株，其特征在于，所述的第一外源基因表达盒还含有AmyE基因元件。
  4. 如权利要求1所述的工程菌株，其特征在于，所述的工程菌株是经原生质体融合获得的重组子。
  5. 如权利要求1所述的工程菌株，其特征在于，所述菌株的基因组中外源基因(核黄素操纵子基因)的拷贝数为10-50个，较佳地为20-40个。
  6. 一种生产核黄素的方法，其特征在于，包括步骤：
    (i)培养权利要求1所述的工程菌株，从而获得含核黄素的发酵产物；和
    (ii)从所述发酵产物中分离出核黄素。
  7. 一种构建权利要求1所述工程菌株的方法，其特征在于，包括步骤：
    (a)构建含有第一外源基因表达盒的载体，所述第一外源基因表达盒具有以下元件：AmyE基因、P15启动子和核黄素操纵子基因；
    (b)构建含有第二外源基因表达盒的载体，所述第二外源基因表达盒具有以下元件：P15启动子和核黄素操纵子基因；
    (c)将步骤(a)和步骤(b)获得的载体分别转入受体菌株，获得基因组整合有第一外源基因表达盒的第一重组菌株和基因组整合有第二外源基因表达盒的第二重组菌株；
    (d)对第一重组菌株和第二重组菌株进行原生质体融合，获得基因组中整合有第一外源基因表达盒和第二外源基因表达盒的重组子，所述的重组子即为权利要求1所述的工程菌株。
  8. 如权利要求7所述的方法，其特征在于，所述的受体菌株为枯草芽胞杆菌Bacillus subtilis，较佳地为Bacillus subtilis SMY。
  9. 一种权利要求1所述工程菌株的用途，其特征在于，所述菌株被用作发 酵生产核黄素的工程菌。
  10. 一种制备食品组合物的方法，其特征在于，包括步骤：
    将权利要求2所述方法制备的核黄素添加到食品组合物中，从而获得含有核黄素的食品组合物。
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