Synthetic Biology Approach to Regioselectively Modified Polyketides

区域选择性修饰聚酮化合物的合成生物学方法

• 批准号: 8843895
• 负责人: Gavin J Williams
• 金额: $ 27.32万
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8843895
• 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; Health; 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; 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）：聚酮化合物的广泛和有效的生物活性由其化学结构决定，其由聚酮化合物脱氢酶（PKS）构建。由于需要提供定制的生物合成途径用于产生非天然延伸物单元并将其安装到聚酮化合物中，因此旨在产生用于产生聚酮化合物类似物的设计者PKS的组合生物合成方法在范围和效率方面受到限制。作为我们重新编程天然产物的生物合成以合成潜在药物的长期目标的一部分，这里的总体目标是使用定制的酶来构建人工生物合成途径，用于将不同的扩展单元合成和安装到聚酮化合物中。我们的假设是（1）可以产生混杂的酰基辅酶A合成酶并用于合成不同的延伸单元，（2）可以通过探测具有不同延伸单元的这些酶的特异性来鉴定PKS和trans-AT的新的非天然底物，和（3）PKS的固有混杂性和/或反式-AT可以被利用来构建原型细菌菌株，用于将非天然延伸单元区域选择性地安装到聚酮化合物中。这些假设得到以下方面的支持：（1）显示丙二酰辅酶A合成酶的底物特异性可以扩展的强初步数据，（2）暗示PKS和trans-AT潜在混杂性的初步数据和文献先例，以及（3）在异源宿主中产生天然聚酮化合物的能力和所需小分子前体的已知细胞渗透性。所提出的研究的基本原理是，我们提出的酰基辅酶A合成酶/反式-AT路线提供了在体内和体外产生各种各样的扩展单元的能力，从而能够鉴定新的PKS特异性，并最终合成区域选择性修饰的聚酮化合物。将完成以下具体目标：（1）创建用于扩展单元生成的混杂酰基辅酶A合成酶，（2）表征和改变PKS和相关生物合成机制的特异性，以及（3）构建用于扩展单元生成和安装到聚酮化合物中的原型细菌菌株。拟议研究的预期成果包括（1）PKS底物合成的突变酶，（2）PKS和相关机制的新特异性，（3）异源生产聚酮类似物的细菌菌株，以及（4）提高对PKS底物特异性和催化作用的理解。这些结果预计将产生广泛的积极影响，并导致天然产物合成，合成生物学和药物发现的纵向进展，（1）为天然产物多样化提供新的策略，（2）扩展我们对PKS催化反应的理解，（3）为工程PKS和其他酶提供新的方法，以及（4）获得通过常规有机合成或遗传操作不容易获得的生物活性天然产物。