Elucidation of the Tetraterpene Hydrocarbon Biosynthetic Pathway in the Green Microalga Botryococcus braunii Race L

The colony-forming green microalga Botryococcus braunii is a potential source of biofuel feedstocks as it produces large amount of liquid hydrocarbon oils that can be converted into combustion engine fuels. There are three different races of B. braunii based on the hydrocarbons it synthesizes. Race A produces fatty acid derived alkadienes and alkatrienes, race B produces the triterpenoid The colony-forming green microalga Botryococcus braunii is a potential source of biofuel feedstocks as it produces large amount of liquid hydrocarbon oils that can be converted into combustion engine fuels. There are three different races of B. braunii based on the hydrocarbons it synthesizes. Race A produces fatty acid derived alkadienes and alkatrienes, race B produces the triterpenoid hydrocarbons tetramethylsqualene and botryococcenes, and race L, the focus of this study, produces the C₄₀ tetraterpenoid hydrocarbon lycopadiene via a previously uncharacterized biosynthetic pathway. Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C₃₀ squalene. Confirming this hypothesis, the studies presented here identified C₂₀ geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. Two squalene synthase (SS)-like cDNAs were identified in race L with one encoding a true SS, and the other an enzyme with lycopaoctaene synthase (LOS) activity. Interestingly, LOS utilizes alternative C₁₅ and C₂₀ prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively utilizes GGPP in vivo. This discovery highlights how SS enzyme diversification resulted in the production of specialized tetraterpenoid oils in race L of B. braunii. To understand LOS substrate and product specificity, rational mutagenesis experiments were conducted based on sequence alignments 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 tetraterpenoid hydrocarbon metabolism in B. braunii and provide a foundation for engineering LOS for robust production of specific hydrocarbons of a desired chain length

Elucidation of the Tetraterpene Hydrocarbon Biosynthetic Pathway in the Green Microalga Botryococcus braunii Race L

The colony-forming green microalga Botryococcus braunii is a potential source of biofuel feedstocks as it produces large amount of liquid hydrocarbon oils that can be converted into combustion engine fuels. There are three different races of B. braunii based on the hydrocarbons it synthesizes. Race A produces fatty acid derived alkadienes and alkatrienes, race B produces the triterpenoid The colony-forming green microalga Botryococcus braunii is a potential source of biofuel feedstocks as it produces large amount of liquid hydrocarbon oils that can be converted into combustion engine fuels. There are three different races of B. braunii based on the hydrocarbons it synthesizes. Race A produces fatty acid derived alkadienes and alkatrienes, race B produces the triterpenoid hydrocarbons tetramethylsqualene and botryococcenes, and race L, the focus of this study, produces the C₄₀ tetraterpenoid hydrocarbon lycopadiene via a previously uncharacterized biosynthetic pathway. Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C₃₀ squalene. Confirming this hypothesis, the studies presented here identified C₂₀ geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. Two squalene synthase (SS)-like cDNAs were identified in race L with one encoding a true SS, and the other an enzyme with lycopaoctaene synthase (LOS) activity. Interestingly, LOS utilizes alternative C₁₅ and C₂₀ prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively utilizes GGPP in vivo. This discovery highlights how SS enzyme diversification resulted in the production of specialized tetraterpenoid oils in race L of B. braunii. To understand LOS substrate and product specificity, rational mutagenesis experiments were conducted based on sequence alignments 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 tetraterpenoid hydrocarbon metabolism in B. braunii and provide a foundation for engineering LOS for robust production of specific hydrocarbons of a desired chain length

Genetic and Biochemical Reconstitution of Bromoform Biosynthesis in Asparagopsis Lends Insights into Seaweed Reactive Oxygen Species Enzymology

Marine macroalgae, seaweeds, are exceptionally prolific producers of halogenated natural products. Biosynthesis of halogenated molecules in seaweeds is inextricably linked to reactive oxygen species (ROS) signaling as hydrogen peroxide serves as a substrate for haloperoxidase enzymes that participate in the construction these halogenated molecules. Here, using red macroalga Asparagopsis taxiformis, a prolific producer of the ozone depleting molecule bromoform, we provide the discovery and biochemical characterization of a ROS-producing NAD(P)H oxidase from seaweeds. This discovery was enabled by our sequencing of Asparagopsis genomes, in which we find the gene encoding the ROS-producing enzyme to be clustered with genes encoding bromoform-producing haloperoxidases. Biochemical reconstitution of haloperoxidase activities establishes that fatty acid biosynthesis can provide viable hydrocarbon substrates for bromoform production. The ROS production haloperoxidase enzymology that we describe here advances seaweed biology and biochemistry by providing the molecular basis for decades worth of physiological observations in ROS and halogenated natural product biosyntheses

Tetraterpene Synthase Substrate and Product Specificity in the Green Microalga <i>Botryococcus braunii</i> Race L

Recently, the biosynthetic pathway for lycopadiene, a C₄₀ tetraterpenoid hydrocarbon, was deciphered from the L race of <i>Botryococcus braunii</i>, 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 <i>in vitro</i> 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 <i>in vivo</i> by expressing LOS in <i>E. coli</i> cells metabolically engineered to produce high FPP and GGPP levels. These studies also offer insights into tetraterpene hydrocarbon metabolism in <i>B. braunii</i> and provide a foundation for engineering LOS for robust production of specific hydrocarbons of a desired chain length