Year of Publication
Doctor of Philosophy (PhD)
Dr. Joe Chappell
B. braunii is a green alga that has attracted attention as a potential renewable fuel source due to its high oil content and the archeological record of its unique contribution to oil and coal shales. Three extant chemotypes of B. braunii have been described, namely race A, race B, and race L, which accumulate alkadienes and alkatrienes, botryococcene and squalene and their methylated derivatives, and lycopadiene, respectively. The methylated triterpenes, particularly botryococcenes, produced by race B can be efficiently converted to high quality combustible fuels and other petrochemicals; however, botryococcene biosynthesis has remained enigmatic.
It has been suggested that botryococcene biosynthesis could resemble that of squalene, arising from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form pre-squalene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene, or in an alternative reductive rearrangement, botryococcene. Based on the proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence, isolated squalene synthase-like genes from B. braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encode for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether and to a lesser extent squalene, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when SSL-1 is combined with either SSL-2 or SSL-3, in vivo and in vitro, robust squalene or botryococcene biosynthesis was observed, respectively. 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 inter-dependently for specialized triterpene production. Expression of various configurations of the SSL genes in TN-7 yeast demonstrates that botryococcene can be efficiently produced in a heterologous host.
Additionally, three triterpene methyltransferase (TMTs) were isolated which efficiently catalyze the transfer of a methyl group from S-adenosyl methionine (SAM) to either squalene (TMT-1 and TMT-2) or botryococcene (TMT-3) in vivo and in vitro. Co-expression of the various TMT genes with either squalene synthase or botryococcene synthase in TN-7 yeast resulted in the accumulation of C31 and C32 methyl derivatives of squalene or botryococcene, demonstrating their potential for heterologous production. The methylation sites were determined by NMR spectroscopy to be identical to C31 and C32 methyl-derivatives of squalene or botryococcene observed in B. braunii race B.
Expression studies of various heterologous squalene synthase genes in S. cerevisiae corroborated an earlier but surprising observation reported in the literature. While the squalene synthase gene of S. cerevisiae was able to complement an erg9 (squalene synthase) knockout in yeast, squalene synthase genes from plants and animals were not. Chemical profiles revealed that squalene accumulated to significant levels in yeast expressing the squalene synthase of plant, animal, or S. cerevisiae. This suggested that it was not the ability of these heterologous synthase enzymes to produce squalene, but their inability to feed squalene into the native sterol biosynthetic pathway that prevented them from restoring normal ergosterol biosynthesis in S. cerevisiae. By examining the ability of chimera squalene synthase enzymes to complement the erg9 mutation, a discrete sequence of amino acids near the C-terminus of the enzyme was identified which is necessary and sufficient for allowing any squalene synthase to restore normal sterol metabolism.
Niehaus, Thomas Daniel, "ELUCIDATING THE BIOCHEMICAL WIZARDRY OF TRITERPENE METABOLISM IN BOTROYCOCCUS BRAUNII" (2011). Theses and Dissertations--Plant and Soil Sciences. 1.