LAUSR

Previous efforts to engineer a heterologous MA pathway in Saccharomyces cerevisiae have been hindered by a bottleneck at the PCA decarboxylation step and the creation of aromatic amino acid auxotrophy through deleterious manipulation of the pentafunctional Aro1 protein. In light of these studies, this work was undertaken with the central objective of preserving amino acid prototrophy, which we achieved by employing an Aro1 degradation strategy. Moreover, resolution of the key PCA decarboxylase bottleneck, as detailed herein, advances our understanding of yeast MA biosynthesis and will guide future strain engineering efforts. These strategies resulted in the highest titer reported to date for muconic acid produced in yeast. Overall, our study showcases the effectiveness of careful tuning of yeast Aro1 activity and the importance of host-pathway dynamics. ABSTRACT Muconic acid (MA) is a chemical building block and precursor to adipic and terephthalic acids used in the production of nylon and polyethylene terephthalate polymer families. Global demand for these important materials, coupled to their dependence on petrochemical resources, provides substantial motivation for the microbial synthesis of MA and its derivatives. In this context, the Saccharomyces cerevisiae yeast shikimate pathway can be sourced as a precursor for the formation of MA. Here we report a novel strategy to balance MA pathway performance with aromatic amino acid prototrophy by destabilizing Aro1 through C-terminal degron tagging. Coupling of a composite MA production pathway to degron-tagged Aro1 in an aro3Δ aro4Δ mutant background led to the accumulation of 5.6 g/liter protocatechuic acid (PCA). However, metabolites downstream of PCA were not detected, despite the inclusion of genes mediating their biosynthesis. Because CEN.PK family strains of S. cerevisiae lack the activity of Pad1, a key enzyme supporting PCA decarboxylase activity, chromosomal expression of intact PAD1 alleviated this bottleneck, resulting in nearly stoichiometric conversion (95%) of PCA to downstream products. In a fed-batch bioreactor, the resulting strain produced 1.2 g/liter MA under prototrophic conditions and 5.1 g/liter MA when supplemented with amino acids, corresponding to a yield of 58 mg/g sugar. IMPORTANCE Previous efforts to engineer a heterologous MA pathway in Saccharomyces cerevisiae have been hindered by a bottleneck at the PCA decarboxylation step and the creation of aromatic amino acid auxotrophy through deleterious manipulation of the pentafunctional Aro1 protein. In light of these studies, this work was undertaken with the central objective of preserving amino acid prototrophy, which we achieved by employing an Aro1 degradation strategy. Moreover, resolution of the key PCA decarboxylase bottleneck, as detailed herein, advances our understanding of yeast MA biosynthesis and will guide future strain engineering efforts. These strategies resulted in the highest titer reported to date for muconic acid produced in yeast. Overall, our study showcases the effectiveness of careful tuning of yeast Aro1 activity and the importance of host-pathway dynamics.