Improving flavonoid and stilbene titers in recombinant microorganisms using metabolic engineering and directed evolution.

摘要

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-biotechnology1. 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.