Biosynthesis of Unnatural Aromatic Polyketides

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The objective is the engineering of bacterial aromatic synthetases (PKS) for the biosynthesis of DMAC (3,8-dihydroxy-1-methylanthraquinone-2- carboxylic acid) analogs that contain unique functionalities, such as alkene, hydroxyl, amine, chloride or nitrile at C1. The reactive handles can be readily transformed to an aldehyde, which is an attractive starting point in the synthesis of the potent anti-diabetic drug, mumbaistatin. The novel building blocks will be introduced via an unnatural PKS starter unit instead of the natural acetate unit. We will determine the unnatural substrate that is most efficient in priming a "minimal PKS), which catalyzes the initiation and elongation of polyketide biosynthesis. The abilities of each starter unit in its ACP (acyl-carrier protein) form to support DMAC synthesis will be evaluated using different minimal PKS in vitro and will be quantitatively compared to the priming rate of acetyl- ACPs. The loading acyl-ACP biosynthesis pathway from R1128PKS will be exploited for in vivo accumulation of unnatural acyl-ACP. Unnatural acyl-CoAs and their membrane-permeable, N- acetylcysteamine derivatives will e assayed for ZhuH recognition and the subsequent conversion to acyl-ACPs. Site-directed and combinatorial mutagenesis of ZhuH based on s structural data will be performed to improve ZhuH recognition of unnatural substrates. Genetic alternations targeted at the priming cascade will be introduced to the host organism S. effective in vivo pathway for biosynthesis of our target molecules.