Tetraterpene Synthase Substrate and Product Specificity in the Green Microalga Botryococcus braunii Race L

Recently, the biosynthetic pathway for lycopadiene, a C₄₀ tetraterpenoid hydrocarbon, was deciphered from the L race of Botryococcus braunii, an alga that produces hydrocarbon oils capable of being converted into combustible fuels. The lycopadiene pathway is initiated by the squalene synthase (SS)-like enzyme lycopaoctaene synthase (LOS), which catalyzes the head-to-head condensation of two C₂₀ geranylgeranyl diphosphate (GGPP) molecules to produce C₄₀ lycopaoctaene. LOS shows unusual substrate promiscuity for SS or SS-like enzymes by utilizing C₁₅ farnesyl diphosphate (FPP) and C₂₀ phytyl diphosphate in addition to GGPP as substrates. These three substrates can be combined by LOS individually or in combinations to produce six different hydrocarbons of C₃₀, C₃₅, and C₄₀ chain lengths. To understand LOS substrate and product specificity, rational mutagenesis experiments were conducted based on sequence alignment with several SS proteins as well as a structural comparison with the human SS (HSS) crystal structure. Characterization of the LOS mutants in vitro identified Ser276 and Ala288 in the LOS active site as key amino acids responsible for controlling substrate binding, and thus the promiscuity of this enzyme. Mutating these residues to those found in HSS largely converted LOS from lycopaoctaene production to C₃₀ squalene production. Furthermore, these studies were confirmed in vivo by expressing LOS in E. coli cells metabolically engineered to produce high FPP and GGPP levels. These studies also offer insights into tetraterpene hydrocarbon metabolism in B. braunii and provide a foundation for engineering LOS for robust production of specific hydrocarbons of a desired chain length.