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.

Colocalization of enzymes is a proven approach to increase pathway flux and the synthesis of nonnative products. Here, we develop a method for enzyme colocalization using the yeast peroxisomal membrane as an anchor point. Pathway enzymes were fused to the native Pex15 anchoring motif to enable display on the surface of the peroxisome facing the cytosol. The peroxisome is the sole location of β-oxidation in Saccharomyces cerevisiae, and acetyl-CoA is a by-product that is exported in the form of acetyl-carnitine. To access this untapped acetyl-CoA pool, we surface-anchored the native peroxisomal/mitochondrial enzyme Cat2 to convert acetyl-carnitine to acetyl-CoA directly upon export across the peroxisomal membrane; this increased acetyl-CoA levels 3.7-fold. Subsequent surface attachment of three pathway enzymes – Cat2, a high stability Acc1 (for conversion of acetyl-CoA to malonyl-CoA), and the type III PKS 2-pyrone synthase – demonstrated the success of peroxisomal surface display for both enzyme colocalization and access to acetyl-CoA from exported acetyl-carnitine. Synthesis of the polyketide triacetic acid lactone increased by 21% over cytosolic expression at low gene copy number, and an additional 11-fold (to 766 mg/L) after further optimization. Finally, we explored increasing peroxisomal membrane area through overexpression of the peroxisomal biogenesis protein Pex11. Our findings establish peroxisomal surface display as an efficient strategy for enzyme colocalization and for accessing the peroxisomal acetyl-CoA pool to increase synthesis of acetyl-CoA-based products.