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, PF[1]

机构：[1]Beijing Univ Chem Technol, Beijing Key Lab Bioproc, Coll Lire Sci & Technol, Beijing 100029, Peoples R China;[2]Beijing Union Univ, Coll Biochem Engn, Beijing, Peoples R China

第一机构：Beijing Univ Chem Technol, Beijing Key Lab Bioproc, Coll Lire Sci & Technol, Beijing 100029, Peoples R China

通讯机构：[1]corresponding author), Beijing Univ Chem Technol, Beijing Key Lab Bioproc, Coll Lire Sci & Technol, Beijing 100029, Peoples R 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：;WOS:【SCI-EXPANDED(收录号:WOS:000537303600001)】；

基金：National Key Research and Development Program of China, Grant/Award Number: 2018YFA0901800; National Natural Science Foundation of China, Grant/Award Number: 21476011; National High Technology Research and Development Program, Grant/Award Number: 2015AA021003

语种：英文

外文关键词：3-hydroxypropionic acid; Klebsiella pneumoniae; orthogonal expression; T7 RNA polymerase

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.