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.

目的是工程的细菌芳香合成酶（PKS）的生物合成DMAC（3,8-二羟基-1-甲基蒽醌-2-羧酸）类似物，含有独特的功能，如烯烃，羟基，胺，氯或腈在C1。反应柄可以很容易地转化为醛，这是合成有效的抗糖尿病药物孟买抑素的一个有吸引力的起点。新型构建模块将通过非天然PKS启动单元而不是天然醋酸酯单元引入。我们将确定在启动“最小PKS”中最有效的非自然底物，它催化聚酮生物合成的起始和延伸。每个ACP（酰基载体蛋白）形式的起始单元支持DMAC合成的能力将在体外使用不同的最小PKS进行评估，并将定量地与乙酰基ACP的引发率进行比较。R1128PKS的装载酰基- acp生物合成途径将被用于非天然酰基- acp的体内积累。非天然酰基辅酶a及其膜渗透性N-乙酰半胱胺衍生物将被测定以进行ZhuH识别并随后转化为酰基acp。为了提高ZhuH对非天然底物的识别能力，将基于s结构数据对ZhuH进行位点定向和组合诱变。针对启动级联的遗传改变将被引入宿主生物体内，从而有效地合成我们的目标分子。