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

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    THAPA-DISSERTATION-2017.pdf (47.92Mb)
    Date
    2017-06-28
    Author
    Thapa, Hem R
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    Abstract
    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.
    URI
    http://hdl.handle.net/1969.1/165827
    Subject
    Microalgae
    biofuels
    Botryococcus braunii
    squalene synthase
    isoprenoids
    hydrocarbons
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    • Electronic Theses, Dissertations, and Records of Study (2002– )
    Citation
    Thapa, Hem R (2017). Elucidation of the Tetraterpene Hydrocarbon Biosynthetic Pathway in the Green Microalga Botryococcus braunii Race L. Doctoral dissertation, Texas A & M University. Available electronically from http : / /hdl .handle .net /1969 .1 /165827.

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