Biosynthesis of Unnatural Aromatic Polyketides

非天然芳香族聚酮化合物的生物合成

• 批准号: 6640485
• 负责人: YI TANG
• 金额: $ 4.16万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6640485
• 关键词: Streptomyces; acyl carrier protein; biological products; bioreactors; biosynthesis; biotechnology; chimeric proteins; gene targeting; genetic library; high throughput technology; polyketide synthase; postdoctoral investigator; protein engineering; quinones; site directed mutagenesis; thin layer chromatography; tissue /cell culture

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.

目标是对细菌芳香合成酶 (PKS) 进行工程设计，用于生物合成 DMAC（3,8-二羟基-1-甲基蒽醌-2-羧酸）类似物，这些类似物包含独特的功能，例如 C1 处的烯烃、羟基、胺、氯或腈。反应性手柄可以很容易地转化为醛，这是合成强效抗糖尿病药物孟巴他汀的一个有吸引力的起点。新的构建模块将通过非天然 PKS 起始单元而不是天然醋酸盐单元引入。我们将确定最有效引发“最小 PKS”的非天然底物，该“最小 PKS”催化聚酮化合物生物合成的起始和延伸。将使用不同的体外最小 PKS 评估其 ACP（酰基载体蛋白）形式的每个起始单元支持 DMAC 合成的能力，并将与乙酰基 ACP 的引发速率进行定量比较。 R1128PKS 的酰基-ACP 生物合成途径将用于非天然酰基-ACP 的体内积累。将分析非天然酰基辅酶A及其可渗透膜的N-乙酰半胱胺衍生物的ZhuH识别以及随后转化为酰基ACP的情况。 ZhuH 的定点组合诱变 将进行基于结构数据的分析，以提高 ZhuH 对非天然基质的识别。针对引发级联的遗传改变将被引入宿主生物体中，从而为我们的目标分子的生物合成提供有效的体内途径。