GENETIC AND BIOCHEMICAL ANALYSES OF THE BIOSYNTHESIS OF THE YELLOW CAROTENOID 4,4'-DIAPONEUROSPORENE OF STAPHYLOCOCCUS-AUREUS

The major pigment produced by Staphylococcus aureus Newman is the deep-yellow carotenoid 4,4'-diaponeurosporene; after prolonged cultivation, this pigment is in part converted to the orange end product staphyloxanthin. From this strain a 3.5-kb DNA fragment was identified which after being cloned into Escherichia coli and Staphylococcus carnosus conferred the ability to produce 4,4'-diaponeurosporene. DNA sequencing of this fragment revealed two open reading frames (ORFs) which are very likely cotranscribed. ORF1 encodes a 254-amino acid hydrophobic protein, CrtM (M(r), 30,121). The deduced sequence of CrtM exhibits in three domains similarities to the sequences of Saccharomyces cerevisiae and human squalene synthases and phytoene synthases of various bacteria. ORF2 encodes a 448-amino-acid hydrophobic protein, CrtN, with an M(r) of 50,853 whose deduced sequence is similar to those of phytoene desaturases of other bacteria. At the N terminus of CrtN a classical FAD-, NAD(P)-binding domain is found. Spectrophotometry and gas chromatography-mass spectrometry analyses of the carotenoid production of E. coli and S. carnosus clones containing either ORF1 or both ORFs together suggest that ORF1 and ORF2 represent the dehydrosqualene synthase gene (crtM) and the dehydrosqualene desaturase gene (crtN, respectively. The results furthermore suggest that the biosynthesis of 4,4'-diaponeurosporene starts with the condensation of two molecules of farnesyl diphosphate by dehydrosqualene synthase (CrtM); it is shown that the reaction product of this enzyme is dehydrosqualene and not squalene. Dehydrosqualene (4,4'-diapophytoene) is successively dehydrogenated by a desaturase (CrtN) to form the yellow main intermediate 4,4'-diaponeurosporene.