A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this beta-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from beta-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of beta-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both beta-ionone ends.

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The physical map of the chromosome of T/2evrnus therrnophilus HB27 was constructed using three restriction enzymes: EcoRI, SspI was detected m HB27, and its physical map was also constructed. In addition, several genes were located on the chromosomal physical map.

We have constructed refined physical maps of the chromosome (1.82 Mb) and the large plasmid pTT27 (250 kb) of Thennus thennophilus HB27. A total of 49 cleavage sites with five restriction enzymes, EcoRI, Sspl, MunI, EcoRV and Clal, were determined on the maps. The location of 61 genes was determined by using as probes 64 genes cloned from T. themphilus or other Thermus strains. Comparison of the genomic organization of the chromosomes of T. thermophilus HB27 and HB8 revealed that they were basically identical, but some genes were located in different regions. Among 32 genes whose locations were determined on both the HB27 and the HB8 chromosomes, the copy number of rpsl-rpsG-fus-tufA, the locations of glyS, pol, and one copy of nusG+p/K+p/A were different. The lSlOO0 sequence was located only in one region on the HB27 chromosome. In contrast, ISlOOO sequences were scattered over four regions on the chromosome of HB8. As each region in which glyS, pol, or one copy of nusG-rplK-rplA are present also contained ISlOOO in HB8, it is suggested that IS1000 may play an important role in genomic rearrangements in 7hermus strains.