Biosynthesis of Unnatural Aromatic Polyketides

目的是工程化细菌芳香族合成酶（PKS），用于生物合成含有独特官能团（例如C1处的烯烃、羟基、胺、氯或腈）的DMAC（3，8-二羟基-1-甲基蒽醌-2-羧酸）类似物。反应柄可以很容易地转化为醛，这是一个有吸引力的起点，在合成有效的抗糖尿病药物，mumbaistatin。新的结构单元将通过非天然PKS起始单元而不是天然乙酸酯单元引入。我们将确定在引发“最小PKS”中最有效的非天然底物，其催化聚酮化合物生物合成的起始和延长。将使用不同的最小PKS在体外评价ACP（酰基载体蛋白）形式的每个起始单元支持DMAC合成的能力，并将其与乙酰基-ACP的引发速率进行定量比较。来自R1128 PKS的加载酰基-ACP生物合成途径将被用于非天然酰基-ACP的体内积累。非天然酰基-CoA及其膜可渗透的N-乙酰半胱胺衍生物将用于测定ZhuH识别和随后转化为酰基-ACP。基于结构数据对ZhuH进行定点突变和组合突变，以提高ZhuH对非天然底物的识别能力。针对引发级联的遗传改变将被引入宿主生物体S。用于我们靶分子生物合成的有效体内途径。

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.