title: Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii.
abstract: Botryococcene biosynthesis is thought to resemble that of squalene, a metabolite essential for sterol metabolism in all eukaryotes. Squalene arises from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene. In principle, botryococcene could arise from an alternative rearrangement of the presqualene intermediate. Because of these proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence isolated squalene synthase-like genes from Botryococcus braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encodes for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when combinations of the synthase-like enzymes were mixed together, in vivo and in vitro, robust botryococcene (SSL-1+SSL-3) or squalene biosynthesis (SSL1+SSL-2) was observed. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved interdependently for specialized triterpene oil production greater than 500 MYA. Coexpression of the SSL-1 and SSL-3 genes in different configurations, as independent genes, as gene fusions, or targeted to intracellular membranes, also demonstrate the potential for engineering even greater efficiencies of botryococcene biosynthesis.
authors: University of Kentucky, Lexington, KY 40546-0312, USA.
authors: Tom D
authors: TD
authors: Niehaus
authors: Shigeru
authors: S
authors: Okada
authors: Timothy P
authors: TP
authors: Devarenne
authors: David S
authors: DS
authors: Watt
authors: Vitaliy
authors: V
authors: Sviripa
authors: Joe
authors: J
authors: Chappell
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D000460:Chlorophyta / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics / Q000378:metabolism; D017343:Genes, Plant; D007700:Kinetics; D008969:Molecular Sequence Data; D010938:Plant Oils / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013185:Squalene / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014315:Triterpenes / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D018744:DNA, Plant; D010938:Plant Oils; D010940:Plant Proteins; D011994:Recombinant Proteins; D014315:Triterpenes; D013185:Squalene; D013186:Farnesyl-Diphosphate Farnesyltransferase