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Light-based control of metabolic flux through assembly of synthetic organelles

Abstract

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.

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Fig. 1: Light-switchable synthetic organelles for redirecting metabolic flux.
Fig. 2: Light-regulated organelle formation depends on component concentration.
Fig. 3: Redirecting flux in the deoxyviolacein pathway using light-inducible optoClusters.
Fig. 4: Redirecting flux in the prodeoxyviolacein pathway using light-dissociable PixELLs.
Fig. 5: Light-switchable metabolic flux control at an enzymatic branch point.

Data availability

All plasmids, strains and raw data will be made available upon reasonable request to the corresponding authors.

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Acknowledgements

We thank all members of the Toettcher and Avalos laboratories for helpful comments. We also thank J. Dueber for kindly providing violacein enzyme plasmids. This work was supported by the Maeder Graduate Fellowship in Energy and the Environment (to E.M.Z.), NIH grant DP2EB024247 (to J.E.T.) and The Pew Charitable Trusts, the U.S. DOE Office of Biological and Environmental Research, Genomic Science Program Award DESC0019363, and NSF CAREER Award CBET-1751840 (to J.L.A.) and a Schmidt Transformative Technology grant (to J.E.T. and J.L.A).

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Authors and Affiliations

Authors

Contributions

E.M.Z., M.Z.W., J.E.T. and J.L.A. conceived the project and designed the experiments. E.M.Z. and N.S. conducted all metabolic flux experiments. E.M.Z., N.S., M.Z.W., E.D. and N.L.P. cloned constructs and performed microscopy. Z.G. contributed methodology and reagents. E.M.Z., J.E.T. and J.L.A. wrote the paper with editing from all authors. J.E.T. and J.L.A. provided funding and supervised the research.

Corresponding authors

Correspondence to José L. Avalos or Jared E. Toettcher.

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Competing interests

Some of the authors are co-inventors on patent applications harnessing optogenetics for metabolic engineering (J.L.A., J.E.T. and E.M.Z.: patent application no. WO2017177147A1) and establishing optogenetic control of protein clustering (J.E.T.: patent application no. US20170355977A1).

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Supplementary Figures 1–11, Supplementary Note 1

Reporting Summary

Supplementary Video 1

OptoDroplet formation and dissociation in S. cerevisiae.

Supplementary Video 2

OptoCluster formation and dissociation in S. cerevisiae.

Supplementary Video 3

PixELL formation and dissociation in S. cerevisiae.

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Zhao, E.M., Suek, N., Wilson, M.Z. et al. Light-based control of metabolic flux through assembly of synthetic organelles. Nat Chem Biol 15, 589–597 (2019). https://doi.org/10.1038/s41589-019-0284-8

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