title: Functional characterization of the Xanthophyllomyces dendrorhous farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoding genes that are involved in the synthesis of isoprenoid precursors.
abstract: 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.
authors.affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Alcaíno
authors.affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Romero
authors.affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Niklitschek
authors.affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Dionisia
authors.initials: D
authors.lastname: Sepúlveda
authors.affiliation: Departamento de Química, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: María Cecilia
authors.initials: MC
authors.lastname: Rojas
authors.affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Baeza
authors.affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Víctor
authors.initials: V
authors.lastname: Cifuentes
mesh_terms: D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology*; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D002338:Carotenoids / Q000096:biosynthesis; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000378:metabolism; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase / Q000737:chemistry / Q000235:genetics*; D051228:Geranyltranstransferase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes; D013261:Sterols / Q000737:chemistry; D024341:Xanthophylls / Q000737:chemistry
chemical_list: D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D013261:Sterols; D024341:Xanthophylls; D002338:Carotenoids; D002244:Carbon; C004808:farnesyl pyrophosphate; C005948:astaxanthine; D051228:Geranyltranstransferase; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; C002963:geranylgeranyl pyrophosphate