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.algae | biofuels | terpene enzymology

Functional Identification of Triterpene Methyltransferases from Botryococcus braunii Race B

“…Manually assembled cDNA sequences were then screened against the National Center for Biotechnology Information (NCBI) tBlastn search function across all available databases, and TMT-3 was identified as exhibiting similarity to C-24-sterol methyltransferase (SMT) genes. All other SMTlike genes were identified in a B. braunii 454 transcriptomic dataset as described previously (17). This dataset was screened computationally using an NCBI BLAST search window with the Chlamydomonas reinhardtii SMT-1 protein sequence (EDP05221) and the Arabidopsis thaliana SMT-1 sequence (AAG28462) as the queries, which led to the identification of six full-length ORFs that were at least 42% identical and 59% similar to C. reinhardtii SMT.…”

“…Cloning SMT-like Genes-The triterpene methyltransferase-3 (TMT-3) was identified through a random sequencing effort of expressed sequence tags using a B. braunii phage cDNA library as described previously (17). Briefly, phages were converted to their plasmid form using the mass excision protocol as described by the manufacturer (Stratagene), and ϳ500 individual colonies were randomly selected for automated DNA sequencing using sequencing primers flanking the cDNA insertion sites.…”

“…Although the unique mechanisms for C 30 botryococcene and squalene biosynthesis in B. braunii have been elucidated (17), the specific mechanism(s) by which these triterpenes are methylated was unknown. Small molecule methylation has been extensively characterized for many diverse compounds and typically consists of a methyltransferase (MT) that utilizes the universal methyl donor S-adenosyl methionine (AdoMet), and exhibits variable degrees of selectivity for a wide range of methyl acceptor molecules (26,27).…”

“…A third enzyme, SSL-2, catalyzes the biosynthesis of squalene from pre-squalene diphosphate produced by SSL-1 but cannot efficiently use farnesyl diphosphate as a substrate. Overall, it was suggested that the squalene and botryococcene produced by the SSL enzymes were channeled into the production of the liquid oils and the biosynthesis of squalene derivatives (17), such as the extracellular matrix, whereas the conventional B. braunii squalene synthase (18) appears to synthesize squalene destined for sterol biosynthesis.…”

Active Hydrocarbon Biosynthesis and Accumulation in a Green Alga, Botryococcus braunii (Race A)

“…Race A produces alkadienes and alkatrienes (14), race B produces mainly triterpenoids known as botrococcenes (15,16), and race L produces a tetraterpenoid known as lycopadiene (17). In both races A and B, the biosynthetic pathways to these hydrocarbons have been clarified, and some of the enzymes involved in the synthetic activity have been characterized (18)(19)(20)(21)(22)(23)(24). In addition, the associated patterns of gene expression have been examined to clarify the biosynthetic pathways to hydrocarbons (25)(26)(27).…”