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Design of a redox-proficient Escherichia coli for screening terpenoids and modifying cytochrome P450s

Nature Catalysis volume 6, pages 1016–1029 (2023)Cite this article

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Abstract

High-value terpenoids are found in plants, animals and microbes, with applications spanning health to agriculture. However, moving their biosynthetic pathways to a new host is challenging when cytochrome P450 (CYP) enzymes are needed for function. Here we engineer Escherichia coli to facilitate discovery by introducing 31 recombinant genes that enhance precursor supply, combine electron transfer pathways and implement regulatory control. We successfully produce terpenoids from different classes and species. By screening 64 bacterial CYPs found in genomes near terpenoid cyclase genes, we identify 40 functional CYPs and combine them with 17 cyclases to create 1,088 pathways. Using a kaurene scaffold, we show that bacterial CYPs can substitute 16 of 44 modifications made by plants. This strain enables high-throughput exploration of terpenoids and their chemical diversification, with a high success rate and reliable titres.

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Fig. 1: Design of E. coli MEV.
Fig. 2: Production of terpenoid scaffolds in E. coli MEV15/20.
Fig. 3: Characterization of the redox array.
Fig. 4: Terpenoid titre optimization.
Fig. 5: Production of LRD scaffolds in E. coli MEV20.
Fig. 6: Production of bacterial CYP-modified LRDs in E. coli MEV20.
Fig. 7: Comparison of plant and bacterial CYP modification of ent-kaurene (LRD04).

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Data availability

E. coli MEV15 (https://www.addgene.org/197112/) and E. coli MEV20 (https://www.addgene.org/197113/) are available from Addgene. All the raw MS and NMR data can be accessed from https://doi.org/10.5281/zenodo.8243444. The data that support the findings of this study are available within the main text and its Supplementary Information file. Source data are provided with this paper. Data are also available from the corresponding author upon request.

Code availability

The Jupyter Notebooks used for analysing GNN results and metabolomic analysis are available at https://doi.org/10.5281/zenodo.8353604.

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Acknowledgements

This research was funded by a research award from Novartis Institute for BioMedical Research (Cambridge, USA), Defense Advanced Research Projects Agency Biological Technologies Office (BTO) Program: ReVector Issued by DARPA/CMO under Cooperative Agreement No. HR00112020030, and US Defense Advanced Research Projects Agency’s Living Foundries Program Award (HR0011-15-C-0084). We appreciate W. Massefski, B. Adams and J. Grimes in the Department of Chemistry Instrumentation Facility at the Massachusetts Institute of Technology for their assistance in acquiring NMR spectra.

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  1. Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Geng-Min Lin & Christopher A. Voigt

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Contributions

G.-M.L. and C.A.V. conceived the study, designed the experiments and wrote the manuscript. G.-M.L. performed the experiments and analysed the data.

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Correspondence to Christopher A. Voigt.

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Supplementary Information

Supplementary Figs. 1–33, Tables 1–38 and Methods.

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Supplementary Data 1

Genetic parts and plasmids used in this study.

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All data required to reproduce supplementary figures and tables.

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Statistical source data.

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Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

GC–MS data.

Source Data Fig. 6

List of biosynthetic gene clusters and CYPs, statistical source data.

Source Data Fig. 7

Statistical source data.

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Lin, GM., Voigt, C.A. Design of a redox-proficient Escherichia coli for screening terpenoids and modifying cytochrome P450s. Nat Catal 6, 1016–1029 (2023). https://doi.org/10.1038/s41929-023-01049-5

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