Construction and application of an expression vector from the new plasmid pLAtc1 of Acidithiobacillus caldus----中国科学院微生物研究所

科研成果

论文题目:	Construction and application of an expression vector from the new plasmid pLAtc1 of Acidithiobacillus caldus
作者:	Zhang Ming-Jiang, Jiang Cheng-Ying, You Xiao-Yan, Liu Shuang-Jiang
联系作者:	Liu Shuang-Jiang*
刊物名称:	Applied Microbiology and Biotechnology
期:	98
卷:	9
页:	4083-4094
年份:	2014
影响因子:	3.762
论文下载:	http://link.springer.com/article/10.1007%2Fs00253-014-5507-z
摘要:	In this study, a recently sequenced 9.8-kb plasmid, pLAtc1, from Acidithiobacillus caldus strain SM-1 was characterized and developed into an expression vector. The pLAtc1 backbone carried an oriV, three rep genes, five mob genes, a Nic site, and an addiction system. Multilocus sequence analysis indicated that pLAtc1 was phylogenetically more related to the IncQ-like broad host range plasmids than to other IncQ plasmids. pLAtc1 was able to replicate and reside in Gram-negative Escherichia coli, Comamonas testosteroni, but not in Gram-positive Corynebacterium glutamicum. pLAtc1 was mobilized via conjugation into E. coli BL21 and A. caldus SM-1 from E. coli S17-1. Quantitative PCR revealed seven and four copies of plasmid in A. caldus and E. coli cells, respectively. The expression vector pLAtcE was constructed from pLAtc1 by introducing a regulatable promoter (P (tetH) ), a transcriptional terminator, a multiple cloning site, a kanamycin resistance gene, and a streptomycin resistance gene. The functionality of pLAtcE was demonstrated by expressing a gene encoding enhanced green fluorescence protein in E. coli and in A. caldus. pLAtcE was used to express alpha-ketoglutarate dehydrogenase (sucAB) and succinate dehydrogenase (sdhA) genes in A. caldus. The newly engineered strain that harbored sucAB and sdhA on a plasmid pLAtcE-sucA-sucB-sdhA grew better than the parent strain SM-1/pLAtcE in tetrathionate and glucose-supplemented medium and produced more acidity and resulted in a more oxidative environment. This study created a useful molecular tool for genetic manipulation of the thermoacidophilic and autotrophic A. caldus.