Metabolic engineering for high-level flavonoids and stilbenes production in microorganisms
Lim, Chin Giaw
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This dissertation represented an effort in efficient biosynthesis of both natural and unnatural stilbenes and flavonoids in microbial platform utilizing state of the art metabolic engineering strategies. Flavonoids are plant secondary metabolites that possess a variety of nutraceutical and pharmaceutical properties, also the potential use as natural colorants. Therefore, it was of interest to reconstitute the entire flavonoids biosynthetic pathway in heterologous host, specifically microorganisms such as E. coli and S. cerevasiae due to their versatility and short doubling time. The present work focused on the biosynthesis of three high-valued compounds: stilbenes and two flavonoids, namely isoflavonoids and anthocyanins. First, their specific pathways were established in microorganisms. Next, different metabolic engineering strategies were utilized to optimize the production of individual molecule: (i) Identify catalytic protein with highest activity or best substrate specificity from a variety of plant homologs; (ii) Optimize the "construct environment" through promoter, vector, host strain...; (iii) Pinpoint the rate limiting precursor and identify possible metabolic intervention targets; (iv) Improve culture conditions: carbon source selection, inducer concentration, induction time and medium. The optimized platform allowed downstream assessment of their medicinal properties, such as the inhibition against digestive enzyme, α-glucosidase and binding affinity towards human estrogen receptor α and β.