Abstract Enzyme spatial organization is an evolved mechanism for facilitating multi‐step biocatalysis and can play an important role in the regulation of promiscuous enzymes. The latter function suggests that artificial spatial organization can be an untapped avenue for controlling the specificity of bioengineered metabolic pathways. A promiscuous terpene synthase (nerolidol synthase) is co‐localized and spatially organized with the preceding enzyme (farnesyl diphosphate synthase) in a heterologous production pathway, via translational protein fusion and/or co‐encapsulation in a self‐assembling protein cage. Spatial organization enhances nerolidol production by ≈11‐ to ≈62‐fold relative to unorganized enzymes. More interestingly, striking differences in the ratio of end products (nerolidol and linalool) are observed with each spatial organization approach. This demonstrates that artificial spatial organization approaches can be harnessed to modulate the product profiles of promiscuous enzymes in engineered pathways in vivo. This extends the application of spatial organization beyond situations where multiple enzymes compete for a single substrate to cases where there is competition among multiple substrates for a single enzyme.
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