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UBC Theses and Dissertations

Characterization and heterologous expression of cis-abienol synthase from balsam fir (Abies balsamea) towards high-end fragrance components Redfern, Baillie


Background The cis-abienol synthase (AbCAS) gene of balsam fir (Abies balsamea) encodes a diterpene synthase (diTPS), which produces a compound, cis-abienol, of value for the fragrance industry. The same compound is also produced in Abies lasiocarpa. cis-Abienol can be used as a starting material for semisynthesis of the commercial substance Ambrox® which is used in the formulation of high end perfumes. AbCAS is a bifuncational diTPS which produces cis-abienol in a two-step process involving two active sites responsible for the class I and class II activities. Class-II activity generates the hydroxylated labda-13-en-8-ol diphosphate (LPP) intermediate, followed by the class-I activity which produces the cis-abienol product. Results We investigated the effects that amino acid residue changes have on catalytic activity of AbCAS and related diTPSs and the product profiles of these protein variants. We identified critical amino acid residues (D348H and S345S) that are responsible for product specificity in AbCAS. In addition, in order to determine if the mutations in AbCAS were also relevant to other diTPSs, this study extended investigation into the AbLAS enzyme, a multiproduct levopimaradiene/abietadiene synthase. The AbLAS:C345S:H346D protein variant showed a new activity and produced the oxygenated diterpene manoyl oxide (100% product profile), where as AbLAS (WT) produces exclusively olefins. To develop a metabolic engineering platform for cis-abienol production we investigated yeast (Saccharomyces cerevisiae) and in the bryophyte Physomitrella patens as hosts for expression of AbCAS. We examined expression of AbCAS with three different S. cerevisiae strains (Am94, BY4741 and KE Strain) and successfully produced cis-abienol in all three strains with up to 12.8 mg x L-¹ in the AM94 strain. Physcomitrella transformed with AbCAS did not produce detectable levels of cis-abienol. Conclusions Product specificity of the diTPS enzymes AbCAS and AbLAS can be altered by changing as few as one or two amino acid residues. Expression of AbCAS in yeast resulted in the formation of cis-abienol indicating opportunities for metabolic engineering of a recombinant production system for this valuable diterpene compound.

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