geranylgeranyl-pyrophosphate and astaxanthine

Topics: Biosynthetic Pathways; Endosperm; Genetic Engineering; Oryza; Plant Proteins; Plants, Genetically Modified; Polyisoprenyl Phosphates; Xanthophylls

The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.

Topics: Amino Acid Sequence; Basidiomycota; Binding Sites; Carbon; Carotenoids; Chromatography, Thin Layer; Escherichia coli; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Geranyltranstransferase; Molecular Sequence Data; Mutation; Plasmids; Polyisoprenyl Phosphates; Protein Engineering; Recombinant Proteins; Sequence Homology, Amino Acid; Sesquiterpenes; Sterols; Xanthophylls

An astaxanthin-overproducing (∼1000 μg g(-1)) strain of Phaffia rhodozyma, termed MK19, was established through 1-methyl-3-nitro-1-nitrosoguanidine and Co60 mutagenesis from wild-type JCM9042 (merely 35-67 μg g(-1)). The total fatty acid content of MK19 was much lower than that of the wild type. Possible causes of the astaxanthin increase were studied at the gene expression level. The expression of the carotenogenic genes crtE, crtI, pbs, and ast, which are responsible for astaxanthin biosynthesis from geranylgeranyl pyrophosphate, was highly induced at the mRNA level, leading to excessive astaxanthin accumulation. In contrast, transcription levels of the genes (hmgs, hmgr, idi, mvk, mpd, fps), responsible for the initial steps in the terpenoid pathway, were essentially the same in wild type and MK19. Although fatty acid and total ergosterol content were reduced by 40-70 mg g(-1) and 760.3 μg g(-1) , respectively, in MK19 as compared with the wild type, but the transcription levels of rate-limiting genes in fatty acid and ergosterol pathways such as acc and sqs were similar. Because fatty acids and ergosterol are two branch pathways of astaxanthin biosynthesis in P. rhodozyma, our findings indicate that enhancement of astaxanthin in MK19 results from decreased fatty acid and ergosterol biosynthesis, leading to precursor accumulation, and transfer to the astaxanthin pathway. Strengthening of the mevalonate pathway is suggested as a promising metabolic engineering approach for further astaxanthin enhancement in MK19.

Topics: Basidiomycota; Ergosterol; Fatty Acids; Gene Expression; Gene Expression Profiling; Mutagenesis; Mutation; Polyisoprenyl Phosphates; RNA, Fungal; RNA, Messenger; Transcription, Genetic; Xanthophylls