文献类型：期刊文献

英文题名：Development of orthogonal T7 expression system in Klebsiella pneumoniae

作者：Zhao, Peng[1]; Ren, Minrui[1]; Ge, Xizhen[2]; Tian, Pingfang[1]; Tan, Tianwei[1]

第一作者：Zhao, Peng

通讯作者：Tian, Pingfang

机构：[1] Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China; [2] College of Biochemical Engineering, Beijing Union University, Beijing, China

第一机构：Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China

通讯机构：[1]Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China

年份：2020

卷号：117

期号：8

起止页码：2446-2459

外文期刊名：Biotechnology and Bioengineering

收录：EI(收录号：20202308793008);Scopus(收录号：2-s2.0-85085874087)

语种：英文

外文关键词：Gene expression - Bottles - Escherichia coli - RNA - Fluorescence - Biochemistry

摘要：Most expression systems are tailored for model organisms rather than nonmodel organisms. However, heterologous gene expression in model organisms constrains the innate advantages of original strain carrying gene of interest. In this study, T7 expression system was developed in nonmodel bacterium Klebsiella pneumoniae for production of chemicals. First, we engineered a recombinant K. pneumoniae strain harboring two vectors. One vector was used to express T7 RNA polymerase (T7 RNAP) which would drive the expression of egfp in the other vector. This recombinant strain demonstrated 15.73-fold of fluorescence relative to wild-type K. pneumoniae and showed similar level of fluorescence to recombinant Escherichia coli overexpressing egfp. When egfp was replaced by puuC, an endogenous aldehyde dehydrogenase catalyzing 3-hydroxypropionic acid (3-HP) biosynthesis in K. pneumoniae, the recombinant strain coexpressing T7 RNAP and PuuC showed high-level PuuC expression. In shake-flask cultivation, this recombinant strain produced 1.72 g/L 3-HP in 24 hr, which was 3.24 times that of wild-type K. pneumoniae (0.53 g/L). To mitigate plasmid burden, the vector expressing T7 RNAP was eliminated, but the T7 RNAP expression cassette was integrated into K. pneumoniae genome. The resulting strain harboring only PuuC expression vector produced 2.44 g/L 3-HP in 24 hr under shake-flask conditions, which was 1.46 times that of the strain harboring both T7 RNAP and PuuC expression vectors. In bioreactor cultivation, this strain generated 67.59 g/L 3-HP and did not show significantly halted growth. Overall, these results indicate that the engineered T7 expression system functioned efficiently in K. pneumoniae. This study provides a paradigm for the development of T7 expression system in prokaryotes. ? 2020 Wiley Periodicals LLC