野生型枯草芽胞杆菌超级感受态的构建及效果评价

doi:10.3969/j.issn.1674-7968.2022.07.019

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摘要枯草芽胞杆菌(Bacillus subtilis)因其安全性和良好的异源蛋白表达特性 ,被广泛应用于食品工业、饲料发酵及生物工程等行业 ,尤其以模式菌枯草芽胞杆菌 168 为宿主菌进行基因工程改造的研究越来越多。但是,来自自然环境的野生型枯草芽胞杆菌由于转化效率极低,其基因工程改造受到了很大程度的限制。本研究通过双交叉同源重组方法 ,将源自枯草芽胞杆菌 168 的感受态转录因子(competence transcription factor, comK)基因融合木糖启动子 PxylA,并插入至野生型枯草芽胞杆菌 C6 (BS-C6)的胞外丝氨酸蛋白酶(extracellular serine protease, epr)基因位点,成功获得野生型枯草芽胞杆菌超级感受态菌株 C6-comks。结果表明 ,超级感受态 C6-comks 的质粒转化效率可达到(4117±363) CFU/µg,相对 BS-C6 提高了 8 倍(P<0.01),转化 PCR 纯化产物效率可达到(442±52) CFU/µg,超过 BS-C6 转化效率的 73.7 倍(P<0.01)。同时,荧光定量结果表明,C6-comks 中感受态形成关键基因 comK、comGB、comGF、comFA 和 comFC的基因表达量相对 BS-C6 分别极显著升高了 77、1 654、1 180、885 和 108 倍(P<0.01)。本研究成功构建了野生型枯草芽胞杆菌超级感受态菌株,并分析了感受态转化效率产生差异的原因,为基于野生型枯草芽胞杆菌作为表达宿主的基因工程应用提供了良好的借鉴。

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	刘功炜
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	王晓宇
	杨雨鑫
	陈玉林

关键词 ：枯草芽胞杆菌, 超级感受态, 感受态转录因子(comK), 基因工程

Abstract：Bacillus subtilis is a bacterial species wildly used in food industry, feed fermentation and bioengineering. This is owing to its safety and excellent ability of heterologous protein expression. In particular, the expansion of studies proved the use of B. subtilis 168 as the host strain for genetic engineering. However, the transformation efficiency of B. subtilis from the natural environment was extremely low, which greatly limited the application and modification of genetic engineering. In this study, wild-type B. subtilis C6 (BS-C6) was selected as the original bacteria for genetic engineering, which was isolated from the intestinal tract of Reticulitermes labralis and stored in our laboratory. The engineering bacteria B. subtilis C6-comk (C6- comk) and B. subtilis C6-comks (C6-comks) were constructed via double-crossover homologous recombination. Competence transcription factor (comK) is a key regulatory protein that affects genetic competence and DNA uptake in B. subtilis. The C6-comk strain was obtained by replacing the original promoter of the comK gene with xylose-inducible promoter (PxylA). On the other hand, the comK gene was amplified from the genome of B. subtilis 168, and then fused with the xylose-inducible promoter (PxylA). The overlap product was inserted into the extracellular serine protease (epr) site of wild-type B. subtilis C6 by using the homologous recombination approach. The super-competent C6-comks strain was successfully obtained. The results showed that the C6-comk strain transformed with plasmids and PCR products could not obtain positive clones, indicated that the method needs further improvement. Fortunately, the desirable phenotypes of C6-comks strain were observed. The results showed that the plasmid transformation efficiency of C6-comks strain was (4117±363) CFU/µg, the efficiency was improved by about 8 folds (P<0.01) compared to BS-C6 (wild type). Notably, the transformation efficiency of PCR products was (442±52) CFU/µg, transcending BS- C6 by about 73.7 folds (P<0.01). Furthermore, the qRT-PCR results showed that the expression of key genes for competence formation were significantly increased compared to BS-C6. The comK, comGB, comGF, comFA and comFC was improved 77, 1 654, 1 180, 885 and 108 folds (P<0.01), respectively. In contrast, the gene expression levels of flagellar basal-body rod protein (flgB), xylose isomerase (xylA) and xylulokinase (xylB) were only 64% (P<0.05), 12% (P<0.01) and 11% (P<0.01) compared to BS-C6. The gene expression levels of C6-comks strain and C6-comk strain were also compared. The results showed that the gene expression levels of comK, comGB, comGF, comFA and comFC were significantly increased by 1.5, 451, 403, 797 and 100 folds (P<0.01) in C6-comks, respectively; while xylA and xylB were significantly reduced by 5.2 and 6.6 folds (P<0.01). There was no treatment effect on flgB gene expression between C6-comks and C6- comk strains (P>0.05). Taken together, this study successfully generated super-competence B. subtilis from the natural environment and analyzed the reasons for the difference of transformation efficiency, which provided a valuable reference for genetic engineering application of wild-type B. subtilis as a host cell.

Key words： Bacillus subtilis Super-competence Competence transcription factor (comK) Genetic engineering

收稿日期: 2021-10-26

ZTFLH:	S182
	S188

通讯作者: *yangyuxin2002@126.com;chenyulin@nwafu.edu.cn

引用本文:

刘功炜, 伍静昀, 金妙函, 王晓宇, 杨雨鑫, 陈玉林. 野生型枯草芽胞杆菌超级感受态的构建及效果评价[J]. 农业生物技术学报, 2022, 30(7): 1432-1442.
LIU Gong-Wei, WU Jing-Yun, JIN Miao-Han, WANG Xiao-Yu, YANG Yu-Xin, CHEN Yu-Lin. Construction and Efficiency Evaluation of the Super-competence of Wild- type Bacillus subtilis. 农业生物技术学报, 2022, 30(7): 1432-1442.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.07.019 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I7/1432

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