(161d) Colocalization of Enzymes on the Yeast Peroxisome Surface to Improve Polyketide Production

Colocalization of enzymes in yeast cells is a proven method of increasing non-native product production. Colocalization can be done by fusing enzymes together, creating an enzyme scaffold, or by securing enzymes together in an organelle. Our method uses the peroxisome as a “scaffold” to hold the enzymes in close proximity. The yeast peroxisome harbors membrane proteins that anchor in the peroxisomal membrane with the active site in the cytosol. The anchoring motif of one such protein, Pex15, has been shown to anchor GFP as well as other non-peroxisomal membrane proteins on the peroxisomal surface. Therefore, we utilized this Pex15 anchoring motif to colocalize pathway enzymes on the surface of the peroxisome. By anchoring two of the enzymes in the proviolacein pathway using the Pex15 anchoring motif, we were able to divert the pathway to increase production of proviolacein relative to prodeoxyviolacein (the major product for all free enzymes). Proximity to the peroxisome is also advantageous for the production of products formed from acetyl-CoA; the peroxisome is the sole location of β-oxidation in yeast and results in acetyl-CoA as an endproduct. Capturing the acetyl-CoA released from the peroxisome is an untapped pool of acetyl-CoA in yeast. Therefore, we also studied the production of the polyketide triacetic acid lactone (TAL) formed from the reaction of one acetyl-CoA and two malonyl-CoA by G. hybrida enzyme 2 pyrone synthase (2PS). As acetyl-CoA from β-oxidation is released from the peroxisome as acetylcarnatine, the S. cerevisiae native enzyme, Cat2, can be employed to covert the acetylcarnatine to acetyl-CoA. Production of TAL needs malonyl-CoA in addition to acetyl-CoA; thus the S. cerevisiae native acetyl-CoA carboxylase, Acc1, is employed to convert acetyl-CoA to malonyl-CoA. Anchoring these three enzymes (Acc1, Cat2, and 2PS) in the peroxisomal membrane using the Pex15 anchoring motif in the production of (TAL) over the unanchored enzyme case. This work illustrates the promise of a new colocalization strategy in yeast.