Successful Enzyme Colocalization Strategies in Yeast for Increased Synthesis of Non-native Products

Abstract

Yeast cell factories, particularly Saccharomyces cerevisiae, have proven valuable for the synthesis of non-native compounds, ranging from commodity chemicals to complex natural products. One significant challenge has been ensuring sufficient carbon flux to the desired product. Traditionally, this has been addressed by strategies involving “pushing” and “pulling” the carbon flux toward the products by overexpression while “blocking” competing pathways via downregulation or gene deletion. Colocalization of enzymes is an alternate and complementary metabolic engineering strategy to control flux and increase pathway efficiency toward the synthesis of non-native products. Spatially controlling the pathway enzymes of interest, and thus positioning them in close proximity, increases the likelihood of reaction along that pathway. This mini-review focuses on the recent developments and applications of colocalization strategies, including enzyme scaffolding, construction of synthetic organelles, and organelle targeting, in both S. cerevisiae and non-conventional yeast hosts. Challenges with these techniques and future directions will also be discussed.

Translational fusion of terpene synthases enhances metabolic flux by increasing protein stability

“…The observed enhancements from spatially organised enzymes have been hypothesised to originate from either (i) the probabilistic diffusion of the intermediate metabolite towards the second enzyme (rather than into competing pathways), or (ii) the direct transfer of the intermediate metabolite between enzymes without diffusion into the bulk solution 1,[9][10][11][12][13] . Specifically for translationally fused enzymes, the former explanation is favoured in the literature 14,15 ; this is supported by a study on an FPPS and patchoulol synthase fusion that found constructs designed with short linker peptides slightly outperforming constructs with long linker peptides 4 . Another study investigating the fusion of geranyl diphosphate synthase (GPPS) with pinene synthase also found a small decrease in pinene titre as the linker length increases 16 .…”

Advances in Metabolic Engineering Paving the Way for the Efficient Biosynthesis of Terpenes in Yeasts

“…Enzyme colocalization strategies increase the local concentration of substrates and minimize the efflux of intermediate metabolites by improving the binding efficiency of substrates to enzymes, which is an effective strategy to promote the conversion of different pyrophosphate precursors into diverse terpenes in S. cerevisiae and Y. lipolytica . , For example, in order to further improve the utilization of FPP after increasing the copy number of ERG20, Hu et al fused ERG20 with sesquiterpene synthase to shorten the distance between the enzyme and substrate in space . The results showed that after controlling for a single variable, the forward fusion enzyme containing the flexible linker GSG significantly increased the production of germacrene A in S. cerevisiae to 190.7 mg/L, which was six times higher than without the connecting peptide .…”

Enzyme Catalysis in Non‐Native Environment with Unnatural Selectivity Using Polymeric Nanoreactors

“…Reverse micelles, stabilized by amphiphilic molecules, can form the basis for distributing enzymes in apolar organic solvents [33–38] . In this approach, the enzymes can be directly encapsulated inside without the need of any functional group modification.…”