Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae.
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.
Gupta
Rakesh K
RK
The Center for Environmental Biotechnology and Department of Microbiology, University of Tennessee, 676 Dabney Hall, Knoxville, TN 37996-1605, USA.
Patterson
Stacey S
SS
Ripp
Steven
S
Simpson
Michael L
ML
Sayler
Gary S
GS
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D020637:Photorhabdus / Q000201:enzymology / Q000235:genetics*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D010088:Oxidoreductases; C055198:LuxC protein, Bacteria; D000217:Acyltransferases; C065928:luxD protein, Bacteria