engleski

Engineering the product outcome of α-eudesmol synthase by active site redesign

The sesquiterpenoid 10-epi-γ-eudesmol is a highly demanded anti-insect compound, naturally found in ginger, rosemary or ginko nuts. Extraction of desired isomer from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods. Therefore, major attention has to be paid to biotechnological approaches. Unfortunately, the gene of the synthase to specifically produce this compound has not been identified yet. Recently, α-eudesmol synthase was isolated from Streptomyces chartreusis (unpublished data), which produces 10-epi-γ-eudesmol as a site-product. Herein, site-directed mutagenesis was employed to alter critical residues in the protein which are responsible to product specificity. Based on possible reaction pathways for αES, it is assumed that the formation of the different α-, β- and γ-isomers is based on the first, cyclisation step. Therefore, a variation of amino acids in close proximity to the atoms C1-C5 of the substrate farnesyl pyrophosphate is expected to influence the product formation. The 17 mutant enzymes were expressed in metabolically engineered Escherichia coli and were found to vary widely in their product selectivity. Mutants containing aromatic amino acids (F, H, W and Y) showed increasing effect on production of desired product, especially those with changes at position R179. This master thesis demonstrates the feasibility of improving product specificity in protein, for synthesis of γ-isomer by rational protein design and site-directed mutagenesis.

α-eudesmol synthase, 10-epi-γ-eudesmol, Keasling strain, GC/MS, product specificity, Streptomyces chartreusis

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