WO2018129980A1 - 在枯草芽孢杆菌中高效表达普鲁兰酶的方法及重组枯草芽孢杆菌 - Google Patents

在枯草芽孢杆菌中高效表达普鲁兰酶的方法及重组枯草芽孢杆菌 Download PDF

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WO2018129980A1
WO2018129980A1 PCT/CN2017/107476 CN2017107476W WO2018129980A1 WO 2018129980 A1 WO2018129980 A1 WO 2018129980A1 CN 2017107476 W CN2017107476 W CN 2017107476W WO 2018129980 A1 WO2018129980 A1 WO 2018129980A1
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signal peptide
bacillus subtilis
promoter
bacillus
amylase
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French (fr)
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李阳源
王建荣
杨玲
瞿曼
陈丽芝
黄佳乐
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广东溢多利生物科技股份有限公司
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Priority to US16/476,878 priority Critical patent/US10975366B2/en
Priority to EP17891647.4A priority patent/EP3569705A4/en
Publication of WO2018129980A1 publication Critical patent/WO2018129980A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)

Definitions

  • the invention relates to the field of genetic engineering, in particular to a method for efficiently expressing pullulanase in Bacillus subtilis and recombinant Bacillus subtilis.
  • Starch is composed of amylose and amylopectin.
  • Amylose accounts for 15%-25% of starch content, consists of linear glucose with ⁇ -1,4 glycosidic linkages; amylopectin accounts for 75%-85%, and several oligosaccharide chains with ⁇ -1,6 glycosides The bond combines to form a branched structure on the main chain. Due to the particularity of the starch structure, a variety of enzyme synergies are required to degrade the starch of the polymer into oligosaccharides, disaccharides and monosaccharides.
  • Pullulanase specifically catalyzes the ⁇ -1,6 glycosidic bond of amylopectin, which determines its effect on amylase on starch, improves starch utilization, reduces food consumption, and improves product quality. It has great value in developing new products and has important applications and good market prospects in the starch processing related industries.
  • Bacillus subtilis is a food-grade expression system, has a strong ability to secrete proteins, a good fermentation base and production technology. At present, many researchers try to express pullulanase with Bacillus subtilis, but the expression of enzyme activity is relatively low.
  • Bacillus subtilis is used as a starting strain, and the B. subtilis mutant strain Bs-vtr is obtained by gene knockout technology, which lacks the B. subtilis alkaline protease gene and the neutral protease gene.
  • Bs-vtr was used as the host, and the promoters and signal peptides of the expression factor of pullulanase were optimized.
  • a series of Bacillus subtilis strains expressing the pullulanase were obtained, which laid a foundation for the industrial application of pullulanase. .
  • the object of the present invention is to reduce the effect of subtilase on recombinant pullulanase, and obtain a mutant strain of subtilis by knocking out the subtilisin gene and the neutral protease gene, and then introducing an optimized promoter, signal peptide and Plu A recombinant vector of the orchidase gene, which expresses the pullulanase efficiently.
  • the method for efficiently expressing pullulan in Bacillus subtilis comprises the steps of:
  • the expression vector constructed above is transferred to an alkaline protease and a neutral protease-depleted Bacillus subtilis strain, and the pullulanase is cultured to obtain
  • the promoter is selected from the group consisting of: a Bacillus licheniformis high temperature alpha-amylase promoter, a Bacillus subtilis alkaline protease promoter, a Bacillus subtilis neutral protease promoter, a Bacillus subtilis medium temperature alpha-amylase promoter, One or more of a medium temperature alpha-amylase promoter of Bacillus amyloliquefaciens, and/or a promoter derived from Bacillus thuringiensis,
  • the signal peptide is selected from the group consisting of: Bacillus licheniformis high temperature alpha-amylase signal peptide, Bacillus subtilis alkaline protease signal peptide, Bacillus subtilis neutral protease signal peptide, Bacillus subtilis medium temperature alpha-amylase signal peptide, starch One or more of a Bacillus medium temperature alpha-amylase signal peptide, and/or a Bacillus licheniformis chitinase signal peptide.
  • a method for efficiently expressing pullulanase in Bacillus subtilis wherein the promoter is a Bacillus subtilis alkaline protease promoter, and the signal peptide is Bacillus subtilis medium temperature ⁇ - amylase signal peptide.
  • Another method for efficiently expressing pullulan in Bacillus subtilis comprises the steps of:
  • the expression vector constructed above is transferred to an alkaline protease and a neutral protease-depleted Bacillus subtilis strain, and the pullulanase is cultured to obtain
  • the promoter is one selected from the group consisting of a nucleotide sequence such as SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16 and/or SEQ ID NO. a variety,
  • the signal peptide is selected from the group consisting of: Bacillus licheniformis high temperature alpha-amylase signal peptide, Bacillus subtilis alkaline protease signal peptide, Bacillus subtilis neutral protease signal peptide, Bacillus subtilis medium temperature alpha-amylase signal peptide, starch One or more of a Bacillus medium temperature alpha-amylase signal peptide, and/or a Bacillus licheniformis chitinase signal peptide.
  • a recombinant Bacillus subtilis capable of efficiently expressing pullulan according to the present invention the recombinant Bacillus subtilis protease and neutral protease are deleted, and the bupleugenase gene, and a promoter suitable for expression of the pullulanase gene And signal peptides,
  • the promoter is selected from the group consisting of: Bacillus licheniformis high temperature alpha-amylase promoter, Bacillus subtilis alkaline protease promoter, Bacillus subtilis neutral protease promoter, Bacillus subtilis medium temperature alpha-amylase a promoter, one or more of a B. amyloliquefaciens medium-temperature alpha-amylase promoter, and/or a promoter derived from Bacillus thuringiensis,
  • the signal peptide is selected from the group consisting of: Bacillus licheniformis high temperature alpha-amylase signal peptide, Bacillus subtilis alkaline protease signal peptide, Bacillus subtilis neutral protease signal peptide, Bacillus subtilis medium temperature alpha-amylase signal peptide, starch One or more of a Bacillus medium temperature alpha-amylase signal peptide, and/or a Bacillus licheniformis chitinase signal peptide.
  • Another recombinant Bacillus subtilis capable of efficiently expressing pullulan according to the present invention the recombinant Bacillus subtilis protease and neutral protease are deleted, and the bupleugenase gene and the gene suitable for expression of the pullulanase are Promoter and signal peptide,
  • the promoter is one selected from the group consisting of a nucleotide sequence such as SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16 and/or SEQ ID NO. a variety,
  • the signal peptide is selected from the group consisting of: Bacillus licheniformis high temperature alpha-amylase signal peptide, Bacillus subtilis alkaline protease signal peptide, Bacillus subtilis neutral protease signal peptide, Bacillus subtilis medium temperature alpha-amylase signal peptide, starch One or more of a Bacillus medium temperature alpha-amylase signal peptide, and/or a Bacillus licheniformis chitinase signal peptide.
  • subtilisin gene and the neutral protease gene are first knocked out, and a subtilis mutant strain Bs-vtr is obtained.
  • the subtilis mutant strain Bs-vtr was used as the host strain, Bacillus deramificans pullulase Bdp was used as the reporter gene, and the pullulanase Bdp gene sequence was as shown in SEQ ID NO.
  • the P43+ signal peptide SacB was combined into a control, and the optimized promoter and signal peptide were separately combined.
  • promoters include: Bacillus licheniformis ⁇ - amylase promoter temperature P, the Bacillus subtilis alkaline protease promoter P base, Bacillus subtilis neutral protease promoter P in Bacillus subtilis temperature ⁇ - amylase promoter P sub starch, starch solution temperature ⁇ - amylase promoter P Bacillus solution, derived from Bacillus thuringiensis promoter P Su.
  • the sequence of the promoter is shown in SEQ ID NO. 2 to SEQ ID NO.
  • Signal peptide used include: high temperature Bacillus licheniformis ⁇ - amylase signal peptide S, the Bacillus subtilis alkaline protease signal peptide of S base, Bacillus subtilis neutral protease signal peptide S, the temperature in the ⁇ - amylase signal B. subtilis S amylin peptide, mesophilic Bacillus amyloliquefaciens ⁇ - amylase signal peptide solution S, Bacillus licheniformis chitinase signal peptide S several bacilli. The sequence of the signal peptide is shown in SEQ ID NO. 8 to SEQ ID NO.
  • the six promoter is a hybrid promoter template constructs, to further enhance expression of pullulanase activity.
  • a series of heterozygous strong promoters were finally obtained by experiments. It can be seen from Table 2 that the relative expression activity of these heterozygous strong promoters is 400%-700% of the control combination (promoter P43 + signal peptide SacB).
  • the sequence of the heterozygous strong promoter is shown in SEQ ID NO. 14 to SEQ ID NO.
  • the present invention obtains the subtilis mutant strain Bs-vtr by gene knockout technique. Then Bs-vtr was used as the host strain, and the promoter and signal peptide expressing the pullulanase were optimized respectively. Finally, a series of promoters and signal peptides capable of expressing pullulanase were obtained, which was the pullulanase. The foundation for industrial application.
  • Figure 1 shows a schematic diagram of the structure of the knockout integration vector Puc-Ats.
  • Figure 2 shows a schematic representation of the structure of the expression vector Puc-vtr.
  • Escherichia coli strain Topl0, Bacillus subtilis 10033, Bacillus licheniformis 10037, Bacillus amyloliquefaciens 10079, Bacillus thuringiensis 20557 were purchased from the China Industrial Microbial Culture Collection Management Center, the knockout vector Puc-Ats and the expression vector Puc-vtr. Figure 1 and Figure 2.
  • High-fidelity Q5 enzyme purchased from NEB, plasmid extraction, gel purification, restriction endonuclease, and kit purchased from Shanghai Shenggong Company.
  • the E. coli medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB-Cl was added to 100 ⁇ g/mL chloramphenicol in LB medium. Enzyme-producing medium
  • Bacillus subtilis 10033 was introduced into LB medium, and cultured at 37 ° C for 24 hours, and genomic DNA was extracted.
  • the upstream and downstream homologous arms of the alkaline protease were amplified by the corresponding primers, and the amplified homologous arms were fused and amplified by overlapping PCR, and finally the amplified products were digested with restriction endonucleases BamHI and XbaI.
  • the vector Puc-Ats was ligated, and transferred to E. coli top10, and the plasmid was extracted to obtain an alkaline protease knockout vector Puc-Ats-AprE.
  • the knockout vector Puc-Ats-AprE was transferred to B. subtilis 10033 by electroporation, and the transformants were selected using tetracycline-resistant plates. The transformants grown on the resistant plates were subjected to high temperature induction at 45 ° C. The replicon of the vector Puc-Ats-AprE was inactivated, thereby causing the vector Puc-Ats-AprE and B. subtilis 10033 to undergo homologous recombination. PCR amplification of the 5-terminal homology arm and the 3-terminal homology arm was performed with the corresponding primers to determine the position where the homologous arm single exchange occurred.
  • the transformants at the position where the homologous arm single exchange occurs will be determined, and the subculture-free subculture will be carried out, and the plate experiments will be carried out 10 times in succession, which will not be long on the resistant plates, and the transformants will be obtained on the non-resistant plates. It was determined to be an alkaline protease deletion.
  • Example 2 Construction of a Bacillus subtilis neutral protease-deficient strain
  • the neutral protease gene was knocked out in the same manner as above.
  • the 5- and 3-terminal homology arms were amplified with the corresponding primers to construct a neutral protease knockout vector Puc-Ats-NprE.
  • Puc-Ats-NprE was transferred to Bs-1 by electroporation, and after induction at a high temperature of 45 ° C, a single exchange of Bacillus subtilis was obtained. After no anti-passage, a double-exchanged Bacillus subtilis was obtained.
  • the B. subtilis which was finally deficient in the neutral protease gene, was named Bs-vtr.
  • Bacillus subtilis 10033, Bacillus licheniformis 10037, Bacillus amyloliquefaciens 10079, and Bacillus thuringiensis 20557 were respectively introduced into LB medium for expansion culture.
  • the cultured cells were expanded by centrifugation, and the corresponding genomic DNA of Bacillus was extracted.
  • the corresponding promoter and signal peptide were amplified separately using the corresponding primers.
  • the resulting promoters include: Bacillus licheniformis ⁇ - amylase promoter temperature P, the Bacillus subtilis alkaline protease promoter P base, Bacillus subtilis neutral protease promoter P in Bacillus subtilis temperature ⁇ - amylase promoter P sub starch, starch solution temperature ⁇ - amylase promoter P Bacillus solution, derived from Bacillus thuringiensis promoter P Su.
  • the sequence of the promoter is shown in SEQ ID NO. 2 to SEQ ID NO.
  • the resulting signal peptide comprising: a high temperature Bacillus licheniformis ⁇ - amylase signal peptide S, the Bacillus subtilis alkaline protease signal peptide of S base, Bacillus subtilis neutral protease signal peptide S, the temperature in the ⁇ - amylase signal B. subtilis S amylin peptide, mesophilic Bacillus amyloliquefaciens ⁇ - amylase signal peptide solution S, Bacillus licheniformis chitinase signal peptide S several bacilli.
  • the sequence of the signal peptide is shown in SEQ ID NO. 8 to SEQ ID NO.
  • P alkali, P, P is precipitated, P and P Su solutions were combined as template.
  • the combination of good and hybrid promoters are different from the S signal peptide, a base S, S in, S lake, S and S, several solutions are combined, filtered to obtain a high table da Rouland four hybrid enzyme strong promoter promoter, were named P 1, P 2, P 3 , P 4, wherein P. 1 is constituted by P to, P base and P solution; wherein P 2 by a P-to, P solution and P Su configuration; P. 3 by a P to , P and P Su configuration; P. 4, P and P solution composed of P base.
  • the Bacillus subtilis medium temperature ⁇ -amylase was used as the target integration site, and the promoter and signal peptide combination obtained in Examples 3 and 4, the pullulanase gene sequence and the partial medium temperature ⁇ -amylase as the homology arm were respectively respectively.
  • the gene sequence clone was ligated to the knockout integration vector Puc-Ats to obtain the corresponding knockout integration vector.
  • the construction of the pullulanase-integrating type Bacillus subtilis was carried out in accordance with the method described in Example 1, to obtain the corresponding pullulanase-integrating type Bacillus subtilis.

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Abstract

提供了在枯草芽孢杆菌中高效表达普鲁兰酶的方法及重组枯草芽孢杆菌。所述方法包括构建碱性蛋白酶和中性蛋白酶缺失的枯草芽孢杆菌菌种；构建包含启动子、信号肽和普鲁兰酶基因的表达载体；将上述载体导入上述枯草芽孢杆菌中。

Description

在枯草芽孢杆菌中高效表达普鲁兰酶的方法及重组枯草芽孢杆菌 技术领域
本发明涉及基因工程领域，具体涉及在枯草芽孢杆菌中高效表达普鲁兰酶的方法及重组枯草芽孢杆菌。
背景技术
淀粉由直链淀粉和支链淀粉组成。直链淀粉占淀粉含量的15％-25％，由线性的葡萄糖以α-1,4糖苷键构成；支链淀粉占75％-85％，由若干个寡糖链以α-1,6糖苷键结合在主链上形成分支结构。由于淀粉结构的特殊性，需要多种酶协同作用才能把高分子的淀粉降解为寡糖、二糖和单糖。普鲁兰酶因其能专一性地水解支链淀粉的α-1,6糖苷键的性质决定了其在增强淀粉酶对淀粉的作用效果、提高淀粉利用率、降低粮耗、提高产品质量和开发新产品方面有着巨大的价值，在淀粉加工相关行业有着重要的用途和良好的市场前景。
目前限制普鲁兰酶产业化应用的主要问题是普鲁兰酶生产菌的发酵活力低，生产成本高。将普鲁兰酶进行异源表达是提高普鲁兰酶发酵酶活的有效方法之一。相对于大肠和毕赤酵母表达系统，枯草芽孢杆菌具有以下优点：枯草芽孢杆菌是食品级的表达系统、具有较强的分泌蛋白质的能力、良好的发酵基础和生产技术等。目前许多研究人员尝试用枯草芽孢杆菌表达普鲁兰酶，但表达酶活都比较低。通过分析发现以下几个因素影响了普鲁兰酶的表达：(1)枯草芽孢杆菌自身会分泌大量的蛋白酶，这些蛋白酶会分解表达的普鲁兰酶；(2)没有找到合适的启动子和信号肽；(3)表达质粒随着传代次数的增加，容易丢失。为了提高普鲁兰酶在枯草芽孢杆菌的表达，必须解决好以上所提到的问题。
本专利以枯草芽孢杆菌为出发菌，通过基因敲除技术缺失了枯草芽孢碱性蛋白酶基因和中性蛋白酶基因得到枯草芽孢突变菌株Bs-vtr。其次以Bs-vtr为宿主，组合优化了普鲁兰酶的表达元件启动子和信号肽，最终得到一系列高效表达普鲁兰酶的枯草芽孢菌种，为普鲁兰酶的工业化应用奠定基础。
发明内容
本发明的目的是减少枯草蛋白酶对重组普鲁兰酶的影响，通过敲除了枯草碱性蛋白酶基因和中性蛋白酶基因，得到了枯草突变菌株，然后导入包含优化的启动子、信号肽和普鲁兰酶基因的重组载体，高效表达普鲁兰酶。
根据本发明的枯草芽孢杆菌中高效表达普鲁兰酶的方法包括步骤：
构建碱性蛋白酶和中性蛋白酶缺失的枯草芽孢杆菌菌种；
构建包含启动子、信号肽和普鲁兰酶基因的表达载体；
将上述构建的表达载体转入碱性蛋白酶和中性蛋白酶缺失的枯草芽孢杆菌菌种，培养获得普鲁兰酶，
其中，所述启动子为选自：地衣芽孢杆菌高温α-淀粉酶启动子，枯草芽孢杆菌碱性蛋白酶启动子，枯草芽孢杆菌中性蛋白酶启动子，枯草芽孢杆菌中温α-淀粉酶启动子，解淀粉芽孢杆菌中温α-淀粉酶启动子，和/或来源于苏云金芽孢杆菌的启动子中的一种或多种，
所述信号肽为选自：地衣芽孢杆菌高温α-淀粉酶信号肽，枯草芽孢杆菌碱性蛋白酶信号肽，枯草芽孢杆菌中性蛋白酶信号肽，枯草芽孢杆菌中温α-淀粉酶信号肽，解淀粉芽孢杆菌中温α-淀粉酶信号肽，和/或地衣芽孢杆菌几丁质酶信号肽中的一种或多种。
根据本发明的具体实施方式的在枯草芽孢杆菌中高效表达普鲁兰酶的方法，其特中，所述启动子为枯草芽孢杆菌碱性蛋白酶启动子，所述信号肽为枯草芽孢杆菌中温α-淀粉酶信号肽。
根据本发明的另一在枯草芽孢杆菌中高效表达普鲁兰酶的方法包括步骤：
构建碱性蛋白酶和中性蛋白酶缺失的枯草芽孢杆菌菌种；
构建包含启动子、信号肽和普鲁兰酶基因的表达载体；
将上述构建的表达载体转入碱性蛋白酶和中性蛋白酶缺失的枯草芽孢杆菌菌种，培养获得普鲁兰酶，
其中，所述启动子为选自核苷酸序列如SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16和/或SEQ ID NO.17所示杂合启动子中的一种或多种，
所述信号肽为选自：地衣芽孢杆菌高温α-淀粉酶信号肽，枯草芽孢杆菌碱性蛋白酶信号肽，枯草芽孢杆菌中性蛋白酶信号肽，枯草芽孢杆菌中温α-淀粉酶信号肽，解淀粉芽孢杆菌中温α-淀粉酶信号肽，和/或地衣芽孢杆菌几丁质酶信号肽中的一种或多种。
根据本发明的能够高效表达普鲁兰酶的重组枯草芽孢杆菌，该重组枯草芽孢杆菌的蛋白酶和中性蛋白酶缺失，并且包普鲁兰酶基因、以及适于普鲁兰酶基因表达的启动子和信号肽，
其中，所述启动子为选自：地衣芽孢杆菌高温α-淀粉酶启动子，枯草芽孢杆菌碱性蛋白酶启动子，枯草芽孢杆菌中性蛋白酶启动子，枯草芽孢杆菌中温α-淀粉酶 启动子，解淀粉芽孢杆菌中温α-淀粉酶启动子，和/或来源于苏云金芽孢杆菌的启动子中的一种或多种，
所述信号肽为选自：地衣芽孢杆菌高温α-淀粉酶信号肽，枯草芽孢杆菌碱性蛋白酶信号肽，枯草芽孢杆菌中性蛋白酶信号肽，枯草芽孢杆菌中温α-淀粉酶信号肽，解淀粉芽孢杆菌中温α-淀粉酶信号肽，和/或地衣芽孢杆菌几丁质酶信号肽中的一种或多种。
根据本发明的另一能够高效表达普鲁兰酶的重组枯草芽孢杆菌，该重组枯草芽孢杆菌的蛋白酶和中性蛋白酶缺失，并且包普鲁兰酶基因、以及适于普鲁兰酶基因表达的启动子和信号肽，
其中，所述启动子为选自核苷酸序列如SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16和/或SEQ ID NO.17所示杂合启动子中的一种或多种，
所述信号肽为选自：地衣芽孢杆菌高温α-淀粉酶信号肽，枯草芽孢杆菌碱性蛋白酶信号肽，枯草芽孢杆菌中性蛋白酶信号肽，枯草芽孢杆菌中温α-淀粉酶信号肽，解淀粉芽孢杆菌中温α-淀粉酶信号肽，和/或地衣芽孢杆菌几丁质酶信号肽中的一种或多种。
根据本发明的具体实施方式，首先敲除了枯草碱性蛋白酶基因和中性蛋白酶基因，得到了枯草突变菌株Bs-vtr。以枯草突变菌株Bs-vtr为宿主菌，以脱支芽孢杆菌(Bacillus deramificans)普鲁兰酶Bdp为报告基因，普鲁兰酶Bdp基因序列如SEQ ID NO.1所示，以表达元件启动子P43+信号肽SacB组合为对照，分别组合优化启动子和信号肽。所用的启动子包括：地衣芽孢杆菌高温α-淀粉酶启动子P地，枯草芽孢杆菌碱性蛋白酶启动子P碱，枯草芽孢杆菌中性蛋白酶启动子P中，枯草芽孢杆菌中温α-淀粉酶启动子P淀，解淀粉芽孢杆菌中温α-淀粉酶启动子P解，来源于苏云金芽孢杆菌的启动子P苏。启动子的序列如SEQ ID NO.2至SEQ ID NO.7所示。所用的信号肽包括：地衣芽孢杆菌高温α-淀粉酶信号肽S地，枯草芽孢杆菌碱性蛋白酶信号肽S碱，枯草芽孢杆菌中性蛋白酶信号肽S中，枯草芽孢杆菌中温α-淀粉酶信号肽S淀，解淀粉芽孢杆菌中温α-淀粉酶信号肽S解，地衣芽孢杆菌几丁质酶信号肽S几。信号肽的序列如SEQ ID NO.8至SEQ ID NO.13所示。得到一系列高效表达普鲁兰酶的启动子和信号肽的组合。由表1可知这些组合的相对表达酶活是对照组合(启动子P43+信号肽SacB)的150％-300％。
以P地，P碱，P中，P淀，P解和P苏这6种启动子为模板构建杂合启动子，进一步提高普鲁兰酶的表达活性。通过实验最终得到一系列杂合强启动子，由表2可知这些杂合强启动子的相对表达酶活是对照组合(启动子P43+信号肽SacB)的 400％-700％。杂合强启动子的序列如SEQ ID NO.14至SEQ ID NO.17所示。
本发明通过基因敲除技术得到枯草突变菌株Bs-vtr。再以Bs-vtr为宿主菌，分别组合优化了表达普鲁兰酶的启动子和信号肽，最终得到了一系列能高效表达普鲁兰酶的启动子和信号肽，为普鲁兰酶的工业化应用奠定基础。
附图说明
图1显示敲除整合载体Puc-Ats的结构示意图。
图2显示表达载体Puc-vtr的结构示意图。
具体实施方式
以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
实验材料和试剂：
1、菌株与载体
大肠杆菌菌株Topl0、枯草芽胞杆菌10033、地衣芽胞杆菌10037、解淀粉芽胞杆菌10079、苏云金芽胞杆菌20557购自中国工业微生物菌种保藏管理中心、敲除载体Puc-Ats和表达载体Puc-vtr示意图如图1和图2所示。
2、酶与试剂盒
高保真Q5酶，购自NEB公司，质粒提取，胶纯化，限制性内切酶、试剂盒购自上海生工公司。
3、培养基
大肠杆菌培养基为LB(1％蛋白胨，0.5％酵母提取物，1％NaCl，pH7.0)。LB-Cl为LB培养基加100μg/mL氯霉素。产酶培养基
实施例1、构建枯草芽孢杆菌碱性蛋白酶缺失菌种
将枯草芽胞杆菌10033接入LB培养基，37℃培养24h后，提取基因组DNA。用相应引物，分别扩增碱性蛋白酶的上下游同源臂，将扩增好的同源臂用重叠PCR进行融合扩增，最后将扩增产物用限制性内切酶BamHI和XbaI酶切，纯化回收后连入载体Puc-Ats，转至大肠杆菌top10，提取质粒得到碱性蛋白酶敲除载体Puc-Ats-AprE。
采用电转化的方法将敲除载体Puc-Ats-AprE转入枯草芽胞杆菌10033，转化子用四环素抗性平板筛选。将在抗性平板上长出的转化子，在45℃下进行高温诱导， 使载体Puc-Ats-AprE的复制子失活，从而促使载体Puc-Ats-AprE和枯草芽孢杆菌10033进行同源重组。用相应引物分别进行5端同源臂和3端同源臂PCR扩增，确定同源臂单交换所发生的位置。
将确定好同源臂单交换所发生位置的转化子，进行无抗传代培养，连续传代10次进行点板实验，将在抗性板上不长，在无抗性板上长得转化子初步确定为碱性蛋白酶缺失。提取初步确定碱性蛋白酶缺失的枯草芽孢杆菌的基因组，用相应引物进行PCR扩增，对扩增产物进行测序，最终确定碱性蛋白酶缺失的枯草芽孢杆菌，命名为Bs-1。
实施例2、构建枯草芽孢杆菌中性蛋白酶缺失菌种
以实施例1构建的Bs-1为出发菌，按照上述相同的方法，将中性蛋白酶基因进行敲除。用相应引物进行5端和3端同源臂扩增，构建中性蛋白酶敲除载体Puc-Ats-NprE。通过电转化方法将Puc-Ats-NprE转入Bs-1，经过45℃高温诱导后，得到单交换的枯草芽孢杆菌。再经过无抗传代，得到双交换的枯草芽孢杆菌。经过PCR测序，最终得到中性蛋白酶基因缺失的枯草芽孢杆菌命名为Bs-vtr。
实施例3、启动子和信号肽组合优化表达普鲁兰酶Bdp
1、启动子及信号肽克隆
分别将枯草芽胞杆菌10033、地衣芽胞杆菌10037、解淀粉芽胞杆菌10079、苏云金芽胞杆菌20557接入LB培养基进行扩大培养。离心收集扩大培养的菌体，提取相应芽孢杆菌的基因组DNA。用相应引物分别扩增相应的启动子和信号肽。得到的启动子包括：地衣芽孢杆菌高温α-淀粉酶启动子P地，枯草芽孢杆菌碱性蛋白酶启动子P碱，枯草芽孢杆菌中性蛋白酶启动子P中，枯草芽孢杆菌中温α-淀粉酶启动子P淀，解淀粉芽孢杆菌中温α-淀粉酶启动子P解，来源于苏云金芽孢杆菌的启动子P苏。启动子的序列如SEQ ID NO.2至SEQ ID NO.7所示。得到的信号肽包括：地衣芽孢杆菌高温α-淀粉酶信号肽S地，枯草芽孢杆菌碱性蛋白酶信号肽S碱，枯草芽孢杆菌中性蛋白酶信号肽S中，枯草芽孢杆菌中温α-淀粉酶信号肽S淀，解淀粉芽孢杆菌中温α-淀粉酶信号肽S解，地衣芽孢杆菌几丁质酶信号肽S几。信号肽的序列如SEQ ID NO.8至SEQ ID NO.13所示。
2、启动子和信号肽组合
采用重叠PCR的方法将不同的启动子、不同的信号肽和普鲁兰酶Bdp融合至一起后连接到表达载体Puc-vtr得到相应的普鲁兰酶表达载体。将构建好的表达载体，通过电转化的方法转入宿主菌Bs-vtr。将转化子接入产酶培养基，培养24h后进行普鲁兰酶活性测定。普鲁兰酶活性测定参照国标GB 1886.174-2016进行。
根据酶活测定结果，得到有效的启动子和信号肽组合。不同启动子和信号肽组合表达普鲁兰酶的相对酶活如表1所示。由表1可知，相对于P43+SacB的组合，所有优化组合的普鲁兰酶相对酶活均有一定的提高。其中以P碱+S淀的效果最好，相对酶活性为300％，其次为P淀+S中，相对酶活性为210％。
表1
启动子+信号肽 普鲁兰酶相对表达活性(％)
P43+SacB 100
P碱+S淀 300
P碱+S地 225
P碱+S中 178
P淀+S中 210
P淀+S几 198
P淀+S碱 168
P地+S解 190
P地+S几 180
P中+S地 160
P中+S淀 189
P淀+S碱 175
P淀+S地 180
P苏+S地 169
P苏+S碱 186
实施例4、杂合启动子构建
为了进一步筛选表达普鲁兰酶的强启动子，以P地，P碱，P中，P淀，P解和P苏为模板分别进行组合。将组合好的杂合启动子分别和不同的信号肽S地，S碱，S中，S淀，S解和S几进行组合，经过筛选得到四个高表达普鲁兰酶的杂合强启动子，分别命名为P1，P2，P3，P4，其中P1由P地，P碱和P解构成；其中P2由P地，P解和P苏构成；P3由P地，P中和P苏构成；P4由P碱，P解和P中构成。这四个杂合启动子的序列如SEQ ID NO.14至SEQ ID NO.17所示。这四个杂合启动子和不同信号肽S地，S碱，S中，S淀，S解和S几组合的表达普鲁兰酶活性如表2所示。
表2
启动子+信号肽 普鲁兰酶相对表达活性(％)
P43+SacB 100
P1+S淀 400
P1+S地 620
P1+S中 580
P1+S解 450
P1+S几 620
P2+S碱 480
P2+S解 450
P2+S几 470
P2+S地 490
P2+S淀 430
P3+S碱 560
P3+S地 550
P3+S几 630
P3+S解 650
P3+S淀 590
P4+S几 600
P4+S碱 580
P4+S解 610
P4+S淀 570
P4+S地 620
实施例5、普鲁兰酶整合型枯草芽孢杆菌的构建
以枯草芽孢杆菌中温α-淀粉酶为目标整合位点，分别将实施例3和4所得到的启动子和信号肽组合、普鲁兰酶基因序列以及作为同源臂的部分中温α-淀粉酶基因序列克隆连接到敲除整合载体Puc-Ats，得到相应的敲除整合载体。在根据实施例1所描述的方法进行普鲁兰酶整合型枯草芽孢杆菌的构建，得到相应的普鲁兰酶整合型枯草芽孢杆菌。

Claims (6)

  1. 一种在枯草芽孢杆菌中高效表达普鲁兰酶的方法，其特征在于，所述方法包括步骤：
    构建碱性蛋白酶和中性蛋白酶缺失的枯草芽孢杆菌菌种；
    构建包含启动子、信号肽和普鲁兰酶基因的表达载体；
    将上述构建的表达载体转入碱性蛋白酶和中性蛋白酶缺失的枯草芽孢杆菌菌种，培养获得普鲁兰酶，
    其中，所述启动子为选自：地衣芽孢杆菌高温α-淀粉酶启动子，枯草芽孢杆菌碱性蛋白酶启动子，枯草芽孢杆菌中性蛋白酶启动子，枯草芽孢杆菌中温α-淀粉酶启动子，解淀粉芽孢杆菌中温α-淀粉酶启动子，和/或来源于苏云金芽孢杆菌的启动子中的一种或多种，
    所述信号肽为选自：地衣芽孢杆菌高温α-淀粉酶信号肽，枯草芽孢杆菌碱性蛋白酶信号肽，枯草芽孢杆菌中性蛋白酶信号肽，枯草芽孢杆菌中温α-淀粉酶信号肽，解淀粉芽孢杆菌中温α-淀粉酶信号肽，和/或地衣芽孢杆菌几丁质酶信号肽中的一种或多种。
  2. 根据权利要求1所述的在枯草芽孢杆菌中高效表达普鲁兰酶的方法，其特征在于，所述启动子为枯草芽孢杆菌碱性蛋白酶启动子，所述信号肽为枯草芽孢杆菌中温α-淀粉酶信号肽。
  3. 一种在枯草芽孢杆菌中高效表达普鲁兰酶的方法，其特征在于，所述方法包括步骤：
    构建碱性蛋白酶和中性蛋白酶缺失的枯草芽孢杆菌菌种；
    构建包含启动子、信号肽和普鲁兰酶基因的表达载体；
    将上述构建的表达载体转入碱性蛋白酶和中性蛋白酶缺失的枯草芽孢杆菌菌种，培养获得普鲁兰酶，
    其中，所述启动子为选自核苷酸序列如SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16和/或SEQ ID NO.17所示杂合启动子中的一种或多种，
    所述信号肽为选自：地衣芽孢杆菌高温α-淀粉酶信号肽，枯草芽孢杆菌碱性蛋白酶信号肽，枯草芽孢杆菌中性蛋白酶信号肽，枯草芽孢杆菌中温α-淀粉酶信号肽，解淀粉芽孢杆菌中温α-淀粉酶信号肽，和/或地衣芽孢杆菌几丁质酶信号肽中的一种或多种。
  4. 能够高效表达普鲁兰酶的重组枯草芽孢杆菌，其特征在于，所述重组枯草芽 孢杆菌的蛋白酶和中性蛋白酶缺失，并且包普鲁兰酶基因、以及适于普鲁兰酶基因表达的启动子和信号肽，
    其中，所述启动子为选自：地衣芽孢杆菌高温α-淀粉酶启动子，枯草芽孢杆菌碱性蛋白酶启动子，枯草芽孢杆菌中性蛋白酶启动子，枯草芽孢杆菌中温α-淀粉酶启动子，解淀粉芽孢杆菌中温α-淀粉酶启动子，和/或来源于苏云金芽孢杆菌的启动子中的一种或多种，
    所述信号肽为选自：地衣芽孢杆菌高温α-淀粉酶信号肽，枯草芽孢杆菌碱性蛋白酶信号肽，枯草芽孢杆菌中性蛋白酶信号肽，枯草芽孢杆菌中温α-淀粉酶信号肽，解淀粉芽孢杆菌中温α-淀粉酶信号肽，和/或地衣芽孢杆菌几丁质酶信号肽中的一种或多种。
  5. 根据权利要求4所述的能够高效表达普鲁兰酶的重组枯草芽孢杆菌，其特征在于，所述启动子为枯草芽孢杆菌碱性蛋白酶启动子，所述信号肽为枯草芽孢杆菌中温α-淀粉酶信号肽。
  6. 能够高效表达普鲁兰酶的重组枯草芽孢杆菌，其特征在于，所述重组枯草芽孢杆菌的蛋白酶和中性蛋白酶缺失，并且包普鲁兰酶基因、以及适于普鲁兰酶基因表达的启动子和信号肽，
    其中，所述启动子为选自核苷酸序列如SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16和/或SEQ ID NO.17所示杂合启动子中的一种或多种，
    所述信号肽为选自：地衣芽孢杆菌高温α-淀粉酶信号肽，枯草芽孢杆菌碱性蛋白酶信号肽，枯草芽孢杆菌中性蛋白酶信号肽，枯草芽孢杆菌中温α-淀粉酶信号肽，解淀粉芽孢杆菌中温α-淀粉酶信号肽，和/或地衣芽孢杆菌几丁质酶信号肽中的一种或多种。
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