Development of a Genetically Encoded Biosensor for Detection of Polyketide Synthase Extender Units in Escherichia coli
Development of a Genetically Encoded Biosensor for Detection of Polyketide Synthase Extender Units in Escherichia coli
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DOI：
10.1021/acssynbio.9b00078
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发表时间：
2019-06-01
影响因子：
4.7
通讯作者：
Williams, Gavin J.
中科院分区：
文献类型：
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作者：
Kalkreuter, Edward;Keeler, Aaron M.;Williams, Gavin J.
聚酮的支架是通过组装基于丙二酰辅酶A及其衍生物的延伸单元来构建的，丙二酰辅酶A及其衍生物被取代在C2-位，具有不同的化学功能。随后，基于转录因子的丙二酰辅酶A生物传感器被证明是检测丙二酰辅酶A的有力工具，有助于丙二酰辅酶A生物合成的动态调节，并指导丙二酰辅酶A依赖过程的高通量工程。然而，用于检测丙二酰辅酶A衍生物的生物传感器还没有报道，这严重限制了高通量合成生物学方法在工程扩链剂单元生物合成中的应用，并限制了动态调节依赖于这种α-羧基辅酶A的聚酮产物的生物合成的能力。在这里，FAPR生物传感器被重新设计和优化，以适应一组大肠杆菌菌株的MCOA浓度范围。通过无细胞转录翻译探讨了FAPR的效应性，揭示了多种非天然和非天然的酰基硫酸酯是FAPR的效应物。事实证明，这种FAPR的混杂足以在大肠杆菌中检测到聚酮延伸单元甲基丙二酰辅酶A，为这一重要代谢物提供了第一个报告的基因编码生物传感器。因此，FAPR以前未知的广泛效应物的混杂为开发新的工具和方法提供了一个平台，可以利用这些工具和方法来克服构建各种α-羧酰-COA的途径和依赖它们的途径的限制，包括生物燃料、抗生素、抗癌药物和其他增值产品。
The scaffolds of polyketides are constructed via assembly of extender units based on malonyl-CoA and its derivatives that are substituted at the C2-position with diverse chemical functionality. Subsequently, a transcription-factor-based biosensor for malonyl-CoA has proven to be a powerful tool for detecting malonyl-CoA, facilitating the dynamic regulation of malonyl-CoA biosynthesis and guiding high-throughput engineering of malonyl-CoA-dependent processes. Yet, a biosensor for the detection of malonyl-CoA derivatives has yet to be reported, severely restricting the application of high-throughput synthetic biology approaches to engineering extender unit biosynthesis and limiting the ability to dynamically regulate the biosynthesis of polyketide products that are dependent on such alpha-carboxyacyl-CoAs. Herein, the FapR biosensor was re-engineered and optimized for a range of mCoA concentrations across a panel of E. coli strains. The effector specificity of FapR was probed by cell-free transcription-translation, revealing that a variety of non-native and non-natural acyl-thioesters are FapR effectors. This FapR promiscuity proved sufficient for the detection of the polyketide extender unit methylmalonyl-CoA in E. coli, providing the first reported genetically encoded biosensor for this important metabolite. As such, the previously unknown broad effector promiscuity of FapR provides a platform to develop new tools and approaches that can be leveraged to overcome limitations of pathways that construct diverse alpha-carboxyacyl-CoAs and those that are dependent on them, including biofuels, antibiotics, anticancer drugs, and other value-added products.