Construction and Efficiency Evaluation of the Super-competence of Wild- type Bacillus subtilis
LIU Gong-Wei1, WU Jing-Yun1, JIN Miao-Han1, WANG Xiao-Yu1,2, YANG Yu-Xin1*, CHEN Yu-Lin1*
1 College of Animal Science and Technology, Northwest A & F University, Yangling 712100, China;
2 Qinling Giant Panda Breeding Research Center, Shaanxi Academy of Forestry Sciences, Zhouzhi 710402, China
AbstractBacillus 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.
LIU Gong-Wei,WU Jing-Yun,JIN Miao-Han, et al. Construction and Efficiency Evaluation of the Super-competence of Wild- type Bacillus subtilis[J]. 农业生物技术学报, 2022, 30(7): 1432-1442.
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