Expression of the genes (, , , , and ) in

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 FMNH2 co-factor required for the bioluminescent reaction. Expression of frp with the lux genes not only stabilized but also enhanced bioluminescence to levels approaching 9.0U105 times above background.