Synthetic Biology Approach to Regioselectively Modified Polyketides

区域选择性改性聚酮化合物的合成生物学方法

• 批准号: 8421112
• 负责人: Gavin J Williams
• 金额: $ 25.98万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-15 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8421112
• 关键词: Acyl Carrier Protein; Acyl Coenzyme A; Acyltransferase; Address; Anabolism; Biochemical; Biological; Biological Factors; Carbon; Catalysis; Cells; Chemical Structure; Chemicals; Coenzyme A; Coenzyme A Ligases; Complex; Coupled; Data; Engineering; Enzymes; Generations; Goals; In Vitro; Lead; Ligase; Literature; Malonates; Malonyl Coenzyme A; Medicine; Molecular; Organic Synthesis; Outcome; Pathway interactions; Permeability; Pharmaceutical Preparations; Physical condensation; Positioning Attribute; Production; Public Health; Reaction; Research; Role; Route; Specificity; Staging; Structure; Substrate Specificity; System; Therapeutic; Vertebral column; analog; base; carboxylation; combinatorial; design; drug discovery; genetic manipulation; improved; in vivo; mutant; novel; novel strategies; phosphopantetheinyl transferase; polyketide synthase; prototype; public health relevance; small molecule; synthetic biology; thioester

DESCRIPTION (provided by applicant): The broad and potent biological activities of polyketides are determined by their chemical structures, which are constructed by polyketide synthases (PKSs). Combinatorial biosynthesis approaches aimed at creating designer PKSs for the generation of polyketide analogues have been limited in terms of scope and efficiency due to the requirement to provide tailored biosynthetic pathways for the generation and installation of non-natural extender units into polyketides. As part of our long term goal of reprogramming the biosynthesis of natural products for the synthesis of potential drugs, the overall objective here is to use tailor-made enzymes to build an artificial biosynthetic pathway for the synthesis and installation of diverse extender units into polyketides. Our hypotheses are (1) a promiscuous acyl-CoA synthetase can be created and used to synthesize diverse extender units, (2) novel non-natural substrates for PKSs and trans-ATs can be identified by probing the specificity of these enzymes with diverse extender units, and (3) inherent promiscuity of PKSs and/or inherent/engineered promiscuity of trans-ATs can be harnessed to construct prototype bacterial strains for regioselective installation of non-natural extender units into polyketides. These hypotheses are supported by (1) strong preliminary data that shows the substrate specificity of a malonyl-CoA synthetase can be expanded, (2) preliminary data and literature precedent that hint at potential promiscuity of PKSs and trans-ATs, and (3) the ability to produce natural polyketides in heterologous hosts and the known cell permeability of required small molecule precursors. The rationale for the proposed research is that our proposed acyl-CoA synthetase/trans-AT route offers the ability to produce a broad variety of extender units in vivo and in vitro, enabling identification of new PKS specificities, and ultimately the synthesis of regioselectively modified polyketides. The following specific aims will be completed (1) create promiscuous acyl-CoA synthetases for extender unit generation, (2) characterize and alter the specificity of PKSs and related biosynthetic machinery, and (3) construct prototype bacterial strains for extender unit generation and installation into polyketides. The expected outcomes of the proposed research include (1) mutant enzymes for PKS substrate synthesis, (2) novel specificities for PKSs and related machinery, (3) bacterial strains for heterologous production of polyketide analogues, and (4) improved understanding of substrate specificity and catalysis in PKSs. The results are expected to have broad positive impact and lead to vertical advances in natural product synthesis, synthetic biology, and drug discovery by (1) providing new strategies for natural product diversification, (2) extending our understanding of PKS catalyzed reactions, (3) providing new approaches for engineering PKSs and other enzymes, and (4) access to biologically active natural products not readily accessible by conventional organic synthesis or genetic manipulation.

描述（由申请人提供）：聚酮化合物的广泛而有效的生物学活性取决于其化学结构，这些结构由聚酮化合物合酶（PKS）构成。旨在创建用于生成聚酮化合物类似物的设计器PKS的组合生物合成方法在范围和效率方面受到限制，因为要求提供定制的生物合成途径，以生成和安装非天然扩展单元到聚酮化合物中。作为我们对潜在药物合成天然产物的生物合成的长期目标的一部分，这里的总体目标是使用量身定制的酶来构建人工生物合成途径，以合成和安装多种扩展单元到聚酮化合物中。 Our hypotheses are (1) a promiscuous acyl-CoA synthetase can be created and used to synthesize diverse extender units, (2) novel non-natural substrates for PKSs and trans-ATs can be identified by probing the specificity of these enzymes with diverse extender units, and (3) inherent promiscuity of PKSs and/or inherent/engineered promiscuity of trans-ATs can be harnessed to construct用于将非天然扩展单元的区域选择性安装到聚酮化合物中的原型细菌菌株。这些假设得到了（1）强大的初步数据的支持，这些数据表明，可以扩展丙二酰辅酶A合成酶的底物特异性，（2）初步数据和文献，表明PKS和Trans-Ats的潜在混杂性，以及（3）在众所周知的细胞中产生天然多吉比的能力，并允许较小的寄生类群体中的多吉比。拟议的研究的基本原理是，我们提出的酰基-COA合成酶/Trans-AT途径具有在体内和体外生产各种扩展单元的能力，从而可以鉴定出新的PKS特异性，并最终合成区域选择性修饰的聚酮化合物。将完成以下特定目标（1）为扩展单位生成创建混杂的酰基-COA合成酶，（2）表征和改变了PKS和相关生物合成机械的特异性，以及（3）构造用于扩展单位生成和安装在聚酮化合物中的构造原型细菌量。拟议研究的预期结果包括（1）PKS底物合成的突变酶，（2）PKS和相关机械的新型特异性，（3）用于聚酮化合物类似物的细菌毒性，以及（4）（4）对PKS中底物的特异性和催化的理解提高了。 The results are expected to have broad positive impact and lead to vertical advances in natural product synthesis, synthetic biology, and drug discovery by (1) providing new strategies for natural product diversification, (2) extending our understanding of PKS catalyzed reactions, (3) providing new approaches for engineering PKSs and other enzymes, and (4) access to biologically active natural products not readily accessible by conventional organic synthesis or genetic manipulation.