Co-overexpression of RspAB Improves Recombinant Protein Production in Escherichia coli

• Published in: Metabolic engineering 2000-10, Vol.2 (4), p.293-299
• Main Authors: Weikert, Christian, Canonaco, Fabrizio, Sauer, Uwe, Bailey, James E.
• Format: Article
• Language: English
• Subjects: beta-Galactosidase - biosynthesis; beta-Galactosidase - genetics; Blotting, Western; Cell Division; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli - physiology; Flagella - physiology; Gene Expression Regulation, Bacterial; Genes, Reporter; Guanosine Tetraphosphate - metabolism; homoserine lactone; Mutation - genetics; Operon - genetics; Phenotype; Promoter Regions, Genetic - genetics; protein production; quorum-sensing; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; RspA; Sigma Factor - genetics; Sigma Factor - metabolism; Transformation, Bacterial
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1006/mben.2000.0163

The Escherichia coli mutant CWML2 was previously reported to exhibit improved physiological characteristics, including recombinant protein production. Here we investigate the molecular basis of this phenotype by comparing the cellular level of three RNA polymerase sigma subunits by immunoblot analysis. While the level of housekeeping σD was similar in parent and mutant, the levels of the flagella synthesis regulator σF and the stationary phase regulator σS were higher in the mutant strain, indicating a different motility and stationary phase phenotype. Evidence for this conclusion was provided by the significantly higher motility of CWML2, compared to its parent. Based on these results, we hypothesized that alterations in ppGpp regulation via a homoserine lactone-dependent mechanism may be relevant for the mutant phenotype. Indeed, transcription of the rspAB operon, which was previously described to be involved in the degradation of homoserine lactone, was found to be deregulated in CWML2 in a plasmid-based reporter protein assay. By overexpression of the E. coli rspAB operon, we could partly mimic the mutant phenotype and demonstrate that co-overexpression of RspAB is a pertinent metabolic engineering strategy to improve recombinant protein production.