Biosynthesis of Unnatural Aromatic Polyketides

非天然芳香族聚酮化合物的生物合成

• 批准号: 6550933
• 负责人: YI TANG
• 金额: $ 3.66万
• 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/6550933
• 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) 进行工程改造，用于生物合成含有独特功能（如烯烃、羟基、胺、氯化物或腈）的 DMAC（3,8-二羟基-1-甲基蒽醌-2-羧酸）类似物在C1。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”可催化聚酮化合物生物合成的起始和延长。ACP（酰基载体蛋白）形式的每个起始单元支持 DMAC 合成的能力将是使用不同的最小 PKS 进行体外评估，并将与乙酰 ACP 的加载速率生物合成途径进行定量比较。 R1128PKS 将用于非天然酰基-ACP 的体内积累，并对其可渗透膜的 N-乙酰半胱胺衍生物进行 ZhuH 识别以及随后转化为酰基-ACP 的定点和组合诱变。将进行基于 s 结构数据的 ZhuH，以提高 ZhuH 对非天然底物的识别。针对引发级联的遗传改变将被引入宿主。生物体S.我们的目标分子生物合成的有效体内途径。