Improving flavonoid and stilbene titers in recombinant microorganisms using metabolic engineering and directed evolution
Bhan, Namita Jayant
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The idea behind this thesis is to remove the bottle-neck in the production of phenylpropanoids-naringinine and resveratrol by using biomolecular engineering. Biomolecular engineering is very broad and highly interdisciplinary field; it includes, but is not limited to, protein engineering, metabolic engineering, bioinformatics, bioprocessing, gene therapy, drug design, discovery and delivery, biomaterials, and nano-biotechnology 1 . In particular we used metabolic engineering and protein engineering to improve the titers of flavonoids and stilbenes in bacteria (E.coli) and yeast (Saccharomyces cervisiea). The precursor to phenylpropanoid pathway; malonyl-CoA is very tightly regulated to very low cellular levels in both E.coli and yeast, we improved its availability by over expressing the gene for acetyl-CoA carboxylase which converts acetyl-CoA to malonyl-CoA in yeast. Acetyl-CoA carboxylase is the first step to fatty acid synthesis, which is the major consumer of malonyl-CoA, so we tried down-regulation of the fatty acid synthesis pathway by using anti-sense RNA technique in the yeast Saccharomyces cerevisiea. S. cerevisiea lacks the RNAi pathway; our technique involved a steric inhibition of the RNA encoding for fatty acid synthase I by expressing approximately 500 base pairs of anti-sense RNA, thus leading to transcriptional inhibition. Stilbene synthase, the enzyme responsible for the final step in the production of the phenylpropanoid stilbene, upon sequence comparison of the stilbene synthases from different plant species, shows a lot of sequence dissimilarity around the functionally important residues, thus we tried to use the directed evolution technique to evolve a better stilbene synthase.