Abstract
Most Staphylococcus aureus strains produce the orange carotenoid staphyloxanthin. The staphyloxanthin biosynthesis genes are organized in an operon, crtOPQMN, with a sigma(B)-dependent promoter upstream of crtO and a termination region downstream of crtN. The functions of the five encoded enzymes were predicted on the basis of their sequence similarity to known enzymes and by product analysis of gene deletion mutants. The first step in staphyloxanthin biosynthesis is the head-to-head condensation of two molecules of farnesyl diphosphate to form dehydrosqualene (4,4'-diapophytoene), catalyzed by the dehydrosqualene synthase CrtM. The dehydrosqualene desaturase CrtN dehydrogenates dehydrosqualene to form the yellow, main intermediate 4,4'-diaponeurosporene. CrtP, very likely a mixed function oxidase, oxidizes the terminal methyl group of 4,4'-diaponeurosporene to form 4,4'-diaponeurosporenic acid. CrtQ, a glycosyltransferase, esterifies glucose at the C(1)'' position with the carboxyl group of 4,4'-diaponeurosporenic acid to yield glycosyl 4,4'-diaponeurosporenoate; this compound was the major product in the clone expressing crtPQMN. In the final step, the acyltransferase CrtO esterifies glucose at the C(6)'' position with the carboxyl group of 12-methyltetradecanoic acid to yield staphyloxanthin. Staphyloxanthin overexpressed in Staphylococcus carnosus (pTX-crtOPQMN) and purified was analyzed by high pressure liquid chromatography-mass spectroscopy and NMR spectroscopy. Staphyloxanthin was identified as beta-D-glucopyranosyl 1-O-(4,4'-diaponeurosporen-4-oate)-6-O-(12-methyltetradecanoate).
Highlights
In previous work, we cloned the genes for staphyloxanthin biosynthesis from S. aureus and analyzed the function of two enzymes involved in the pathway [5]
When unpigmented S. carnosus was transformed with pOC1, the colonies of the transformants produced staphyloxanthin and were orange, which suggested that all genes necessary for the synthesis of staphyloxanthin were present on pOC1
Staphyloxanthin expression studies showed that all five genes are necessary for staphyloxanthin biosynthesis
Summary
We cloned the genes for staphyloxanthin biosynthesis from S. aureus and analyzed the function of two enzymes involved in the pathway [5]. The biosynthesis of the pigment starts with the headto-head condensation of two farnesyl diphosphate molecules, catalyzed by the dehydrosqualene synthase CrtM, to yield 4,4Ј-diapophytoene (dehydrosqualene). Dehydrosqualene desaturase, CrtN, catalyzes the formation of the first deep yellow-colored carotenoid intermediate product, 4,4Ј-diaponeurosporene, which is formed via successive dehydrogenation reactions [5]. We analyzed the complete staphyloxanthin biosynthesis operon crtOPQMN. We postulated the function of the encoded proteins based on product analysis of crt mutants and sequence similarity comparisons. Staphyloxanthin was purified, and its structure was determined by NMR spectroscopy
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