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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During the fermentation of 2-keto-L-gulonic acid (2KGA) from L-[U-14C]sorbose by the 2KGA-producing mutant UV10, derived from G. melanogenus IFO 3293, 40% of the metabolized substrate was converted to l4CO2. The CO2 evolved was mainly via the pentose phosphate pathway and partly via the intermediates such as L-sorbosone or the by-products of 2KGAformation. CO2 evolution from 2KGAwas not observed. 2KGA is an important intermediate in the synthesis of L-ascorbic acid (vitamin C). Various approaches to produce 2KGA by fermentation have been tried by using strains of the genera Gluconobacter,1} Erwinia and Corynebacterium,2) and a mixture of Gluconobacter and Pseudomonas.3~5) As previously reported, we have developed a fermentation process6) that produces 2KGAfrom L-sorbose via L-sorbosone by a mutant derived from G. melanogenus IFO 3293. G. melanogenus IFO 3293 produced 1 g of2KGA per liter from 70g of L-sorbose per liter. Mutant UV10, which is one of the 2KGAproducing mutants obtained on the way to establish the process described above, produced 30g of 2KGAper liter from 70 g ofL-sorbose per liter. Besides 2KGA,only a few grams ofL-idonic acid per liter have been detected and most of the remaining carbon was not recovered. We also obtained the

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