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

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.

The Colonial Microalgae Botryococcus braunii as Biorefinery

“…With NADPH, the PSPP has two options; one forms the botryococcene with a C3-C1 connection between the FPP molecules ( Figure 3d). The other option forms a C1-C1 0 between two FPP molecules producing squalene [15].…”

“…One encodes a squalene synthase (LSS) and the other for a lycopaoctaene synthase (LOS). LOS uses preferentially in vivo GGPP, and C 15 and C 20 prenyl diphosphates as substrates [15] (Figure 4).…”

“…Race L produces a tetraterpene C 40 known as lycopadiene and constitutes from 0.1 to 8% of the dry weight [13,14]. This race contains 5% of lycopatriene, lycopatetraene, lycopapentaene, and lycopahexaene [15]. In addition, a race S is proposed, which synthesizes saturated n-alkanes C 18 and C 20 , and epoxy-alkanes; however, its existence is not yet fully accepted [6].…”

Raman spectra and DFT calculations for tetraterpene hydrocarbons from the L race of the green microalga Botryococcus braunii

“…1) [14,15]. We recently deciphered the biosynthetic pathway for lycopadiene [16], which begins with the condensation of two molecules of the C 20 isoprenoid intermediate geranylgeranyl diphosphate (GGPP) to produce the eight carbon-carbon double-bond (C=C)containing lycopaoctaene (Fig. 1).…”

“…1). Lycopadiene constitutes 95% of all L race hydrocarbons with the intermediates accounting for the remaining 5% [16].…”