Designer biosensors for directed evolution of macrolide biosynthetic enzymes

项目总结 聚酮生物合成装置的复杂性阻碍了获得大环内酯类化合物及其 通过组合生物合成的类似物。作为我们重新编程生物合成的长期目标的一部分 用于合成治疗先导的天然产品，这里的总体目标是使用基因编码 生物传感器，以增强对新型大环内酯类化合物的获取。我们的假设是：(1)既定的诱导者乱交 可以被操纵以提供具有新的诱导剂特异性和 选择性，(2)大环内酯类剪裁酶的专一性可以通过生物传感器引导的定向操作来操纵。 (3)MPHR可用于识别具有改进活动的混合装配线。这些 假设得到以下支持：(1)初步数据显示MPHR变异体具有新的特异性、选择性， 和适当的检测能力，(2)可获得的各种大环内酯类剪裁酶 定向进化的起点和改变底物专一性的定向进化的成功 其他酶，以及(3)证明使用反式作用酶来 配套聚酮装配线。拟议研究的理由是，我们的方法是 利用设计者的生物传感器提供了报告各种大环内酯类生物合成活性的能力 酶，可用于解决与大环内酯类生物合成有关的广泛问题，导致 敬有价值的新大环内酯类。为了解决这些假设，并完成这一总体目标 具体目标如下：(1)获得新型大环内酯O-烷基衍生物；(2) 挽救活跃程度不高的混合动力PKS装配线。我们的方法是高度创新的，因为它 开发了一套目前不可用的筛选工具，可以应用于工程设计 在潜在的任何微生物宿主中合成广泛的大环内酯类化合物。拟议的研究是 意义重大，因为预计它将对天然产物的生物合成和合成产生广泛的积极影响 生物学通过开发生产大环内酯类的新策略，通过扩大我们对生物传感器的理解 特异性，通过开发大环内酯多样化的新方法，并通过扩大 酶工程和合成生物学。

PROJECT SUMMARY The complexity of polyketide biosynthetic machinery has hampered attempts to access macrolides and their analogs via combinatorial biosynthesis. As part of our long-term goal of reprogramming the biosynthesis of natural products for the synthesis of therapeutic leads, the overall objective here is to use genetically encoded biosensors to enhance access to novel macrolides. Our hypotheses are (1) the established inducer promiscuity of the MphR repressor protein can be manipulated to provide biosensors with new inducer specificities and selectivities, (2) the specificity of macrolide tailoring enzymes can be manipulated by biosensor-guided directed evolution, and (3) MphR can be used to identify hybrid assembly lines with improved activities. These hypotheses are supported by (1) preliminary data that shows MphR variants with new specificities, selectivities, and suitable detection capabilities can be generated, (2) the variety of macrolide tailoring enzymes available as starting points for directed evolution and the success of directed evolution for altering substrate specificity of other enzymes, and (3) preliminary data that demonstrates the feasibility of using trans-acting enzymes to complement polyketide assembly lines. The rationale for the proposed research is that our approach of leveraging designer biosensors offers the ability to report the activity of a variety of macrolide biosynthetic enzymes, which can be applied to solving a broad range of problems related to macrolide biosynthesis, leading to valuable new macrolides. To address these hypotheses, and to complete the overall objective of this proposal, the following specific aims will be completed: (1) access novel macrolide O-alkyl derivatives, and (2) rescue the activity of poorly active hybrid PKS assembly lines. Our approach is highly innovative because it develops a set of screening tools that are currently not available and that can be applied to engineering the biosynthesis of a broad range of macrolides in potentially any microbial host. The proposed research is significant because it is expected to have broad positive impact in natural product biosynthesis and synthetic biology by developing new strategies for producing macrolides, by expanding our understanding of biosensor specificity, by developing new approaches for macrolide diversification, and by expanding the capabilities of enzyme engineering and synthetic biology.