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bioRxiv · 10.1101/2022.11.24.517869

Synthetic in vivo compartmentalisation improves metabolic flux and modulates the product profile of promiscuous enzymes

Cheah, L. C.·Liu, L.·Plan, M.·Peng, B.·Lu, Z.·Schenk, G.·Vickers, C. E.·Sainsbury, F.
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Abstract

Enzyme spatial organisation and compartmentalisation are naturally evolved mechanisms for facilitating multi-step biocatalysis. We explored the synthetic in vivo co-encapsulation of two different cargo proteins in yeast using a self-assembling virus-like particle. Co-encapsulation was verified using single particle techniques for both end-to-end fusion of the cargo proteins with the encapsulation anchor at one end, and coexpression of each cargo protein with their individual anchors. The co-encapsulation of a bifunctional geranyl diphosphate/farnesyl diphosphate synthase and a bifunctional linalool/nerolidol synthase delivered nerolidol titres up to 30 times that of an unorganised free enzyme control, a remarkable improvement from a single engineering step. Interestingly, striking differences in the ratio of products (linalool and nerolidol) were observed with each spatial organisation approach. This work presents the largest reported titre fold increases from in vivo enzyme compartmentalisation and suggests that enzyme spatial organisation could be used to modulate the product profile of promiscuous enzymes.

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Cheah, L. C., Liu, L., Plan, M., Peng, B., Lu, Z., Schenk, G., Vickers, C. E., Sainsbury, F.. 2022-11-25. Synthetic in vivo compartmentalisation improves metabolic flux and modulates the product profile of promiscuous enzymes. https://doi.org/10.1101/2022.11.24.517869

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