Designer biosensors for directed evolution of macrolide biosynthetic enzymes

用于大环内酯生物合成酶定向进化的设计生物传感器

基本信息

批准号：
10115752
负责人：
THOMAS ASHTON CROPP
金额：
$ 27.63万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-05 至 2023-02-28
项目状态：
已结题

项目摘要

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.

项目摘要聚酮化合物生物合成机械的复杂性阻碍了进入大环内酯类的尝试通过联合生物合成的类似物。作为我们重编程生物合成的长期目标的一部分用于合成治疗铅的天然产品，这里的总体目的是使用遗传编码的生物传感器可增强获得新型大环内酯类的获取。我们的假设是（1）已建立的诱导者滥交可以操纵MPHR抑制剂蛋白，以向生物传感器提供新的诱导剂特异性和选择性，（2）可以通过生物传感器引导来操纵大花环裁缝酶的特异性进化，（3）MPHR可用于识别具有改进活动的混合装配线。这些假设由（1）初步数据支持，该数据显示了具有新的特异性，选择性的MPHR变体可以生成合适的检测功能，（2）可用的大花洛琳裁缝酶的种类定向演变的起点和定向进化的成功，以改变的底物特异性其他酶和（3）证明使用反式酶的可行性补充聚酮化合物组合线。拟议研究的理由是我们的方法利用设计师生物传感器提供了报告各种大环生物合成活动的活性的能力酶，可用于解决与大洛莱德生物合成有关的广泛问题，领先到有价值的新大环内酯类药物。解决这些假设，并完成总体目标提案，将完成以下特定目标：（1）访问新型的大花环O-烷基衍生物，以及（2）营救活性较差的混合PKS组装线的活性。我们的方法是高度创新的，因为它开发了一组筛选工具，这些工具当前尚不可用，可以应用于工程在任何微生物宿主中，多种大花环的生物合成。拟议的研究是重要的是因为预计它将在天然产物生物合成和合成中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，通过扩展我们对生物传感器的理解特异性，通过开发新的大环内酯类多样性方法，并扩大能力酶工程和合成生物学。