UC Irvine

Acetyl-CoA is an abundant metabolite in microorganisms and is a common precursor for many native and non-native products produced in microbial hosts. Acetyl-CoA is formed in four distinct locations in the yeast Saccharomyces cerevisiae creating three distinct pools of acetyl-CoA: the cytosolic pool, the peroxisomal pool, and the mitochondrial pool. Increasing access to the acetyl-CoA contained in those pools will increase the production of products synthesized from acetyl-CoA. One such product is the polyketide triacetic acid lactone (TAL) which is formed from acetyl-CoA and malonyl-CoA (also made from acetyl-CoA) via the enzyme 2-pyrone synthase (2PS) from Gerbera hybrida. This work focuses on improving production of TAL by enhancing access of 2PS to each distinct pool of acetyl-CoA in S. cerevisiae. A combinatorial CRISPR knockout approach was used to knockout random combinations of pathway and regulatory genes related to acetyl and malonyl-CoA utilization in the cell for the purpose of identifying gene knockout combinations that increase the cytosolic pool of these metabolites and the production of TAL in the cytosol. Conditions for both construction of guide RNA and cell cultivation were optimized to reduce bias. Expression of 2PS in the knockout library strains gave a distribution of TAL production with several strains producing significantly more TAL than the unmodified strain. To increase access to the peroxisomal pool of acetyl-CoA, formed from β-oxidation, three genes in the TAL production pathway, the acetylcarnitine transferase, Cat2; the modified acetyl-CoA carboxylase, Acc1S1157A; and 2PS, were anchored on the surface of the peroxisome using the native Pex15 anchoring motif. This method utilizes the advantages of both enzyme colocalization and the capture and rapid use of the β-oxidation produced acetyl-CoA, exported from the peroxisome in the form of acetylcarnitine. Anchoring all three enzymes on the peroxisomal membrane surface significantly improved TAL production. The mitochondria both creates acetyl-CoA and uses it in the TCA cycle, leading to a large mitochondrial acetyl-CoA pool. Targeting 2PS to the mitochondria allows access to that large pool in the confined volume of an organelle. Multiple variants of the mitochondrial leader sequence (MLS), CoxIV, were assessed for 2PS targeting in S. cerevisiae. The mitochondrial targeting method was expanded into the industrially relevant non-conventional yeast Kluyveromyces marxianus by targeting both 2PS and the S. cerevisiae Acc1S1157A.