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Genes and enzymes affecting carotenoid biosynthesis in Xhanthophyllomyces

Two prominent genes of the carotenogenic pathway in Xanthophyllomyces encoding for pytoene synthase / lycopene cyclase and astaxanthin synthase were expressed and functionally characterized. Furthermore, an improvement of the catalytic efficiency of phytoene synthase by E.coli-mediated random mutagenesis was attempted.

A promising candidate for the astaxanthin synthase gene is a novel monooxygenase of the cytochrome P450 3A subfamily type since its expression in a beta-carotene producing Xanthophyllomyces mutant led to the formation of astaxanthin. Therefore, the function of this monooxygenase to catalyse the introduction of 3-hydroxy groups and 4-keto groups into beta-carotene as analysed. First, the cDNA of this gene was expressed in E. coli with an established ß-carotene background together with a cDNA for a NADPH cytochrome P450 reductase from yeast. After supplementation of hemin ore aminolevulinic acid, ketolase activitity could be established but no hydroxylation observed. Since only small amounts of canthaxanthin are formed in E. coli, a possible subsequent hydroxylation step may be prevented by these low substrate level. In a next step, defined Xanthophyllomyces mutants were generated. Into a the beta-carotene accumulating mutant that produced astaxanthin upon transformation with the P450 gene, either a hydroxylase or ketolase gene was introduced. The ketolase transformant yielded high amounts of canthaxanthin as final product but no astaxanthin. In the hydroxylase mutant large amounts of zeaxanthin were found but again no astaxanthin. The conclusion drawn from these results indicate that the P450 gene encodes an enzyme which catalyzes two reactions: first ketolation of beta-carotene and then a hydroxylation step forming astaxanthin as the final product.

Phytoene synthase and lycopene cyclase are encoded by a fusion gene. In this gene, the N-terminal lycopene cyclase region and the C-terminal phytoene synthase region is separated by a putative protease cleavage motif. In order to test the hypothesis that that the protein is post-translationally cleaved into two independent functional enzymes, truncated gene constructs were made and assayed in the E. coli complementation system. One protein expressed from amino acid 249 to 674 was active exclusively as a phytoene synthase. With another expressed enzyme starting from amino acid 1 to exactly 254, it was possible to obtain a protein capable of lycopene cyclization to beta-carotene. This result demonstrated that an individual lycopene cyclase cleaved off the phytoene synthase moity is enzymtically active. For a further biochemical characterization of the phytoene synthase/lycopene cyclase heterologous expression in substantial quantity is important. Expression of the full-length cDNA with a 6-His moiety for metal chelate affinity purification in E. coli was quite low due to 54 rare codons. Therefor, plasmids with t-RNAs for some of these codons were co-expressed. Finally, substantial expression of phytoene / -carotene synthase was achieved in the E. coli strain Rosetta which is capable to synthesize all the rare t-RNAs. However, the yield was comparably low not exceeding 3 to 4% of total protein.

Since phytoene synthesis is the bottle-neck in the carotenogenic pathway in Xanthophyllomyces we mutagenized the phytoene synthase / lycopene cyclase gene by error prone PCR or with the mutator E. coli strain XL-1 red and screened for higher phytoene synthase activity or modified cyclization reaction. In several mutants higher phytoene synthesis capacity or a more pronounced cyclization reaction to bi-cyclic products than in the original transformant was obtained. All new phenotypes could be attributed to mutations in the promoter resulting in higher enzyme expression. Thus, no genetically improved phytoene synthase or lycopene cyclase was obtained. However, our results indicate that over-expression of both enzymes will increase carotenoid production and support formation and of bi-cyclic astaxanthin versus 3-HO-4-keto-torulene. Our finding that higher phytoene synthase activities leads to higher astaxanthin production let us look for physiological conditions that up-regulate carotenogenesis in Xanthophyllomyces.

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