Synthetic Biology Approach to Regioselectively Modified Polyketides

描述(申请人提供)：由聚酮合成酶(PKS)构建的聚酮化合物的化学结构决定了其广泛而有效的生物活性。为了产生聚酮类似物而设计的PKS的组合生物合成方法在范围和效率方面受到限制，这是因为需要提供定制的生物合成途径来产生和安装非天然延伸单元到聚酮中。作为我们重新编程天然产物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，用于合成和安装不同的延伸单元到聚酮中。我们的假设是(1)可以创建一个混杂的酰基-CoA合成酶并用于合成不同的延伸单元，(2)可以通过探测这些酶与不同的延伸单元的专一性来鉴定这些酶的新的非自然底物，以及(3)可以利用PKS的固有混杂和/或反式ATS的固有/工程混杂来构建用于区域选择性地将非自然延伸单元安装到聚酮中的原型细菌菌株。这些假说得到以下支持：(1)强有力的初步数据表明丙二酰辅酶A合成酶的底物专一性可以扩展，(2)初步数据和文献先例暗示PKS和反式ATS可能混杂，(3)在异源宿主中产生天然多酮的能力和所需小分子前体的已知细胞渗透性。这项研究的基本原理是，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延伸单元的能力，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。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-CoA合成酶；(2)表征和改变PKS及其相关生物合成机制的专一性；(3)构建用于扩展单元生成和安装到聚酮中的原型细菌菌株。这项研究的预期结果包括：(1)用于PKS底物合成的突变酶；(2)用于PKS及其相关机械的新的特异性；(3)用于异源生产聚酮类似物的细菌菌株；(4)提高对PKS底物专一性和催化作用的理解。这些结果有望产生广泛的积极影响，并通过以下方式在天然产物合成、合成生物学和药物发现方面取得垂直进展：(1)提供天然产物多样化的新策略，(2)扩大我们对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)获得常规有机合成或基因操作不易获得的具有生物活性的天然产物。

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.