Enhancement of protein secretion by optimizing protein synthesis: isolation and characterization of Escherichia coli mutants with increased secretion ability of alkaline phosphatase

H. Kadokura, K. Yoda, S. Watanabe, Y. Kikuchi, G. Tamura, M. Yamasaki

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase, on multicopy plasmids caused the accumulation of the precursor form of alkaline phosphatase. The cells lost their viability by a half-life of 60 min and exhibited high sensitivity to 1% sodium dodecyl sulfate (SDS), suggesting that the assembly of the surface proteins were affected by overexpression of the phoA gene. From the cells exhibiting resistance to 1% SDS, we obtained 20 mutants that secrete more alkaline phosphatase into the periplasmic space. Three representatives of the mutants accumulated no precursor molecules and secreted alkaline phosphatase by five- to six-fold that of the wild-type cells carrying multicopy phoA. In all of the three mutants, the amount of phoA transcripts were two to four times less than those in the wild-type cells, indicating that the ability to secrete a large amount of alkaline phosphatase was conferred by decreasing the synthetic rates of the phoA gene product. When the promoter of phoA was replaced with the tacI promoter and the expression level of the phoA gene was regulated with isopropyl-1-thio-β-d-galactoside, the secretion of alkaline phosphatase into the periplasm decreased as the synthetic rate of the phoA gene product increased over a threshold. All these results indicate that overproduction of the phoA gene product causes defects in the secretion of alkaline phosphatase and that the regulation of the expression level is essential for efficient translocation.

Original languageEnglish
Pages (from-to)163-169
Number of pages7
JournalApplied Microbiology and Biotechnology
Volume41
Issue number2
DOIs
Publication statusPublished - 1994 Apr 1
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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