Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae

FEMS Yeast Res. 2003 Dec;4(3):305-13. doi: 10.1016/S1567-1356(03)00174-0.

Abstract

The luxA, B, C, D, and E genes from Photorhabdus luminescens were cloned and functionally expressed in Saccharomyces cerevisiae to construct a bacterial lux-based yeast bioreporter capable of autonomous bioluminescence emission. The bioreporter was engineered using a series of pBEVY yeast expression vectors that allowed for bi-directional constitutive or inducible expression of the individual luxA, B, C, and E genes. The luxD gene, encoding the acyl-ACP transferase that ultimately supplies the requisite aldehyde substrate for the bioluminescent reaction, was fused to a yeast internal ribosomal entry site (IRES) sequence to ensure high bi-cistronic expression. Although self-generation of bioluminescence was achieved by the bioreporter, the signal was relatively weak and decayed rapidly. To overcome this instability, a flavin oxidoreductase gene (frp) from Vibrio harveyi was co-expressed to provide sufficient concentrations of the FMNH(2) co-factor required for the bioluminescent reaction. Expression of frp with the lux genes not only stabilized but also enhanced bioluminescence to levels approaching 9.0x10(5) times above background.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acyltransferases / genetics
  • Acyltransferases / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Genes, Reporter
  • Operon
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism*
  • Photorhabdus / enzymology
  • Photorhabdus / genetics*
  • Recombination, Genetic
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Vibrio / enzymology
  • Vibrio / genetics*

Substances

  • Bacterial Proteins
  • Oxidoreductases
  • LuxC protein, Bacteria
  • Acyltransferases
  • luxD protein, Bacteria