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- Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from Photobacterium phosphoreum
- Chan Yong Lee , Edward A. Meighen
- J. Microbiol. 2000;38(2):80-87.
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Abstract
- The nucleotide sequence of the luxC gene coding for lux-specific fatty acyl-CoA reductase and the upstream DNA (325 bp) of the structural gene from bioluminescent bacterium, Photobacterium phosphoreum, has been determined. An open reading frame extending for more than 20 codons in 325 bp DNA upstream of luxC was not present in both directions. The lux gene can be translated into a polypeptide of 54 kDa and the amino acid sequences of lux specific reductases of P. phosphoreum shares 80, 65, 58, and 62% identity with those of the Photobacterium leiognathi, Vibrio fischeri, Vibrio harveyi, and Xehnorhabdus luminescens reductases, respectively. Analyses of codon usage, showing that a high frequency (2.3%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA and B genes, suggested that the AUA codon may play a modulator role in the expression of lux gene in E. coli. The structural genes (luxC, D, A, B, E) of the P. phosphoreum coding for luciferase ([alpha], [beta]) and fatty acid reductase (r, s, t) polypeptides can be expressed exclusively in E. coli under the T7 phage RNA polymerase/promoter system and identification of the [^35 S]methionine labelled polypeptide products. The degree of expression of lux genes in this system, high level of luxA, B genes whereas low level of luxC, D genes, were consistent with the analyses of codon usage. High expression of the luxC gene could only be accomplished in a mutant E. coli 43R. Even in crude extracts, the acylated acyl-CoA reductase intermediate as well as acyl-CoA reductase activities could be readily detected.
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