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 staphylo xanthin. From this strain a 3.5-kb DNA fragment was identified which a fter being cloned into Escherichia coli and Staphylococcus carnosus co nferred the ability to produce 4,4'-diaponeurosporene. DNA sequencing of this fragment revealed two open reading frames (ORFs) which are ver y likely cotranscribed. ORF1 encodes a 254-amino acid hydrophobic prot ein, CrtM (M(r), 30,121). The deduced sequence of CrtM exhibits in thr ee domains similarities to the sequences of Saccharomyces cerevisiae a nd 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 des aturases of other bacteria. At the N terminus of CrtN a classical FAD- , NAD(P)-binding domain is found. Spectrophotometry and gas chromatogr aphy-mass spectrometry analyses of the carotenoid production of E. col i 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. T he results furthermore suggest that the biosynthesis of 4,4'-diaponeur osporene starts with the condensation of two molecules of farnesyl dip hosphate by dehydrosqualene synthase (CrtM); it is shown that the reac tion product of this enzyme is dehydrosqualene and not squalene. Dehyd rosqualene (4,4'-diapophytoene) is successively dehydrogenated by a de saturase (CrtN) to form the yellow main intermediate 4,4'-diaponeurosp orene.