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Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production

Paper ID Volume ID Publish Year Pages File Format Full-Text
23725 43467 2011 8 PDF Available
Title
Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production
Abstract

In this study, the expression level of the pyc gene from Lactococcus lactis was fine tuned to improve succinate production in Escherichia coli SBS550MG. IPTG induction in the cultures of SBS550MG with pHL413, a positive control plasmid previously constructed ( Sanchez et al., 2005), gave drastically decreased PYC activity and succinate yield. We constructed several plasmids for the expression of pyc to change copy number and variant promoters. Among the constructs, as compared to pHL413, the PYC activity dropped significantly with the Plac, Ptac, Ptrc or native Ppyc promoters in medium or high copy vectors, which resulted in a decrease in succinate yield. Three constructs pThio12, pHL413-Km, and pHL413-Km(lacIq-)N showed considerable PYC activity and improved succinate production in E. coli SBS550MG. The native Ppyc promoter was also modified in order to vary pyc expression levels by site-directed mutagenesis of the −10, −35, −44 regions, and the spacer regions between −10 to −35 and −35 to −44 regions. Out of 9 native promoter variants, the MIII variant resulted in a 20% increase in PYC activity, and improved succinate yield in SBS550MG. We also determined the copy number and stability of pHL413 and pHL413-Km. The two plasmids showed roughly the same copy number, but the pHL413-Km plasmid was relatively more stable. This study provides more understanding of the plasmid characteristics and fine tuning of the expression level of pyc for optimization of the succinate production processes.

Keywords
Succinate; Escherichia coli; Pyruvate carboxylase; Promoter engineering; Fine-tuning gene expression
First Page Preview
Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 155, Issue 2, 10 September 2011, Pages 236–243
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering