A squalene synthase-like enzyme initiates production of tetraterpenoid hydrocarbons in Botryococcus braunii Race L.

Hem R Thapa,Kentaro Takada,Yuquan Xu,Timothy P Devarenne,Mandar T Naik,István Molnár,Shigeru Okada

doi:10.1038/ncomms11198

Abstract

The green microalga Botryococcus braunii is considered a promising biofuel feedstock producer due to its prodigious accumulation of hydrocarbon oils that can be converted into fuels. B. braunii Race L produces the C40 tetraterpenoid hydrocarbon lycopadiene via an uncharacterized biosynthetic pathway. Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C30 squalene. Confirming this hypothesis, the current study identifies C20 geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. Two squalene synthase (SS)-like complementary DNAs are identified in race L with one encoding a true SS and the other encoding an enzyme with lycopaoctaene synthase (LOS) activity. Interestingly, LOS uses alternative C15 and C20 prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively uses GGPP in vivo. This discovery highlights how SS enzyme diversification results in the production of specialized tetraterpenoid oils in race L of B. braunii.

Highlights

The green microalga Botryococcus braunii is considered a promising biofuel feedstock producer due to its prodigious accumulation of hydrocarbon oils that can be converted into fuels
Nuclear magnetic resonance (NMR) spectroscopy was used to confirm identity and structure, including double bond positions, of each molecule (Fig. 2a, Supplementary Table 1 and Supplementary Figs 8–15). This is the first report of lycopatetraene, lycopapentaene and lycopahexaene hydrocarbons from B. braunii race L
As lycopaoctaene synthase (LOS) may have arisen from an SS paralogue that evolved to accept geranylgeranyl diphosphate (GGPP) as substrate for lycopaoctaene production, we considered that LOS may have retained the ability to use farnesyl diphosphate (FPP) to produce squalene

Summary

Introduction

The green microalga Botryococcus braunii is considered a promising biofuel feedstock producer due to its prodigious accumulation of hydrocarbon oils that can be converted into fuels. B. braunii Race L produces the C40 tetraterpenoid hydrocarbon lycopadiene via an uncharacterized biosynthetic pathway Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C30 squalene. Confirming this hypothesis, the current study identifies C20 geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. LOS uses alternative C15 and C20 prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively uses GGPP in vivo This discovery highlights how SS enzyme diversification results in the production of specialized tetraterpenoid oils in race L of B. braunii. The colony-forming green microalga B. braunii is an exciting candidate for biofuel feedstock production, as it produces up to 61% of its dry weight as liquid hydrocarbon oils[6]. We report the elucidation of the first committed step in the lycopadiene hydrocarbon biosynthetic pathway in B. braunii Race L, which is catalysed by a new SS-like (SSL) enzyme

Methods

Results

Conclusion