基于游离质粒的木糖诱导型枯草芽孢杆菌转化体系的建立及其应用

doi:10.19586/j.2095-2341.2021.0044

生物技术进展 ›› 2021, Vol. 11 ›› Issue (6): 741-748.DOI: 10.19586/j.2095-2341.2021.0044

基于游离质粒的木糖诱导型枯草芽孢杆菌转化体系的建立及其应用

田辉(), 王帅, 刘波()

中国农业科学院生物技术研究所，北京 100081

收稿日期:2021-04-08 接受日期:2021-05-12 出版日期:2021-11-25 发布日期:2021-11-26
通讯作者: 刘波
作者简介:田辉 E-mail：tianhuicaas@163.com；
基金资助:
国家自然科学基金项目(31671802)

Construction and Application of Plasmid‑based and Xylose‑induced Transformation System of Bacillus subtilis

Hui TIAN(), Shuai WANG, Bo LIU()

Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2021-04-08 Accepted:2021-05-12 Online:2021-11-25 Published:2021-11-26
Contact: Bo LIU

摘要/Abstract

摘要：

枯草芽孢杆菌（Bacillus subtilis）作为食品级安全菌株，因其具有理化特征清晰、培养发酵方便等特点，广泛应用于异源蛋白质的高效表达以及高附加值物质的合成。传统的B. subtilis遗传转化方法存在操作流程繁琐、效率低等缺点，因此，开发方便高效的遗传转化系统具有重要意义。转录因子ComK被证实能调控B. subtilis感受态的形成，并在B. subtilis高效转化中有重要作用。构建1个含有木糖诱导启动子P_xyl调控comK表达的穿梭质粒pUBC01?P_xyl?comK的菌株B. subtilis K1，经木糖诱导条件优化后，质粒pHY300?p43?egfp的转化效率达到4.8×10³ CFU·μg^-1。此外，质粒pUBC01?P_xyl?comK可在无胁迫条件下连续培养及消除。木糖诱导感受态体系及质粒消除极大地提高了芽孢杆菌基因编辑和菌株改造的便捷性，同时增强了菌株尤其是生产菌株的性状稳定性。

关键词: 枯草芽孢杆菌, ComK, 感受态, 木糖诱导, 质粒消除

Abstract:

Bacillus subtilis is a kind of food?grade safe strain. Due to clear physicochemical characteristics and simple fermentation， it is widely used in the high?efficiency expression of heterologous proteins and the synthesis of high value?added compounds. The traditional genetic transformation methods for B. subtilis have some demerits， such as complicated operation procedures and low efficiency. It is significant important to develop a convenient and highly efficient genetic transformation system. The transcription factor ComK was proved to regulate the formation of competent cells and play an important role in highly efficient transformation of B. subtilis. A shuttle plasmid pUBC01?P_xyl?comK containing a xylose?induced P_xyl?comK expression cassette was constructed and a recombinant strain B. subtilis K1 containing this plasmid was developed. The transformation efficiency of plasmid pHY300?p43?egfp into B. subtilis K1 could reach 4.8×10³ CFU·μg^-1 after optimized xylose induction condition. In addition， pUBC01?P_xyl?comK could be cured via serial subculture under nonselective condition. The system of xylose?induced competent cells and plasmid curing greatly increased the convenience of gene editing and strain improvement of B. subtilis and enhanced character stability of the transformed strains， especially production strains.

Key words: Bacillus subtilis, ComK, competent cells, xylose induction, plasmid curing

中图分类号:

田辉, 王帅, 刘波. 基于游离质粒的木糖诱导型枯草芽孢杆菌转化体系的建立及其应用[J]. 生物技术进展, 2021, 11(6): 741-748.

Hui TIAN, Shuai WANG, Bo LIU. Construction and Application of Plasmid‑based and Xylose‑induced Transformation System of Bacillus subtilis[J]. Current Biotechnology, 2021, 11(6): 741-748.

图/表 8

表1 研究所用引物列表

Table 1 List of primers used in research

引物名称	引物序列（5'→3'）	引物用途
PxylF	5'‑TTGAGGGGTTTTTTGGGCGCGCCTTTCAAGAGGCTCAACTCCAAA‑3'	P_xyl‑comK表达盒的扩增
TTRR	5'‑AACAAACGGGCCAGTTTAATTAATTGGTGTATCATTTCGTTTTTC‑3'	P_xyl‑comK表达盒的扩增
01seqF	5'‑TCGGGAAGAGGTTAAAATTTATTGT‑3'	pUBC01质粒上验证引物
01seqR	5'‑GAAGAAGGCGATTTGATTCATACA‑3'	pUBC01质粒上验证引物
pHseqF	5'‑CTTTTTTCAGGAATCATTGTCATTAGTTGGC‑3'	pHY3000‑PLK质粒上验证引物
pHseqR	5'‑GCCAGGGGGAAACGCCTGGTATCTTTATAG‑3'	pHY3000‑PLK质粒上验证引物
amyE‑LF	5'‑ATGTTTGCAAAACGATTCAAAACCTCTTTAC‑3'	amyE左边同源臂的扩增
amyE‑LR	5'‑TGTGTGTTTCCATGTGTCCAGTTTGGAATA‑3'	amyE左边同源臂的扩增
Tet‑F	5'‑TGGACACATGGAAACACACATTCAACAAACGGGCCATATTGTTGT‑3'	pHY300‑PLK上Tet表达盒的扩增
Tet‑R	5'‑ATTCGCATCTGCACGCAAGGGAATTCCTGTTATAAAAAAAGGATCTG‑3'	pHY300‑PLK上Tet表达盒的扩增
amyE‑RF	5'‑CCTTGCGTGCAGATGCGAATACAACAAAAG‑3'	amyE右边同源臂的扩增
amyE‑RR	5'‑TCAATGGGGAAGAGAACCGCTTAAGCCCG‑3'	amyE右边同源臂的扩增
amyE‑VF	5'‑CGGTTTGAAAGGAGGAAGCGGAAGAATGAAG‑3'	amyE敲除验证
amyE‑VR	5'‑CTTTTGCAAGAGAAAAGTTTTGTCTGATTTATG‑3'	amyE敲除验证

图1 Pxyl?comK表达盒的扩增以及pUBC01?Pxyl?comK的PCR鉴定A：Pxyl?comK表达盒的扩增。M—8 kb DNA maker；1—Pxyl?comK。B：pUBC01?Pxyl?comK的PCR鉴定。M—8 kb DNA maker；1~4—转化子菌落PCR产物；5—质粒pUBC01的PCR产物。

Fig.1 Amplification of Pxyl?comK cassette and verification of plasmid pUBC01?Pxyl?comK byPCR

图2 B. subtilis K1菌株不同诱导时间的单位转化效率注：xylose + —木糖添加；xylose - —无木糖添加。

Fig.2 The transformation efficiency of strain B. subtilis K1 under different induced times

图3 转化结果以及荧光的观察注：A：转化结果。左图—pHY300?P43?egfp转化WB600?01；右图—pHY300?P43?egfp转化B. subtilis K1。B：荧光观察。CK—发菌株WB600；1～8—pHY300?P43?egfp转化B. subtilis K1的转化子。

Fig.3 The result of transformation and fluorescence visualization

图4 抗生素抗性验证质粒pUBC01?Pxyl?comK的消除A：双抗培养，点接于四环素LB平板；B：双抗培养，点接于卡那霉素LB平板；C：单抗培养，点接于四环素LB平板；D：单抗培养，点接于卡那霉素LB平板

Fig.4 Identification of plasmid pUBC01?Pxyl?comK eliminationby antibiotic resistance assay

图5 PCR验证pHY300?p43?egfp以及pUBC01?Pxyl?comK质粒A：PCR验证pHY300?p43?egfp。M—8 kb DNA maker；1~5—挑取的1~5号菌；6—H20；7—pHY300?p43?egfp质粒对照。B：pUBC01?Pxyl?comK质粒。M—8 kb DNA maker；1～5—挑取的1～5号菌；6—H20；7—pUBC01?Pxyl?comK质粒对照。

Fig.5 PCR verification of pHY300?p43?egfp andpUBC01?Pxyl?comK plasmids

图6 整合片段的扩增及amyE敲除菌株的PCR验证A：整合片段的扩增。M—8 kb DNA maker；1—amyE?L片段；2—amyE?R片段；3—Tet片段；4—LTR片段。B： amyE敲除菌株的PCR验证。M—8 kb DNA maker；1~4—挑取的1～4号菌；5—H20；6—WB600菌株。

Fig.6 Amplification of integration fragments and PCR vertification of strains with amyE knocked out

图7 淀粉水解实验A：含有1%淀粉的LB平板。B：添加碘液的1%淀粉的LB平板。WT—WB600野生型；ΔamyE—淀粉酶敲除的WB600菌株。

Fig.7 Starch hydrolysis experiment

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基于游离质粒的木糖诱导型枯草芽孢杆菌转化体系的建立及其应用

Construction and Application of Plasmid‑based and Xylose‑induced Transformation System of Bacillus subtilis

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