Improvement of pyruvate production by Escherichia coli via pathway engineering and Tn5 transposon mediated mutagenesis.

Xiaorong Shi; Jun Liu; Yanfeng Peng; Lin LI; Wenke Wang; Qinhong Wang

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Improvement of pyruvate production by Escherichia coli via pathway engineering and Tn5 transposon mediated mutagenesis.

Author: Xiaorong SHI ¹ ; Jun LIU ¹ ; Yanfeng PENG ² ; Lin LI ² ; Wenke WANG ¹ ; Qinhong WANG ²
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1. School of Life Sciences, Shanxi Normal University, Linfen 041004, Shanxi, China.
2. Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Publication Type:Journal Article
Keywords: Escherichia coli; Tn5 transposon; genome; high throughput screening; pathway engineering; pyruvate
From: Chinese Journal of Biotechnology 2017;33(12):1913-1922
CountryChina
Language:Chinese
Abstract: To develop a high-yield pyruvate strain, we first engineered a pyruvate-producing Escherichia coli KLPP from wild-type E. coli MG1655 by blocking the pathways for byproduct formation via gene knockout. Then, we built a library of mutant containing 7 197 monoclones by using the pUT Mini-Tn5 transposon vector for random mutagenesis with E. coli KLPP. We developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader. After two-round screening we successfully obtained six mutants with increased pyruvate titer using this method, the titer of pyruvate was increased by 38%, 31%, 19%, 28%, 44% and 14%, respectively. The position of transposon insertion was determined by whole genome re-sequencing, and the gene locus possibly influencing pyruvate production was analyzed, which laid the foundation for subsequent strain improvement by metabolic engineering.