Abstract

cis, cis Muconic acid (CCM) is a compound that can be chemically converted to adipic acid, a major component of several copolymer plastics. Biological CCM-producing pathways with high yields have been characterized in Escherichia coli; however the downstream extraction process is prohibitively expensive at an industrial scale. Saccharomyces cerevisiae is a better industrial production host for organic acids; however biological CCM-producing pathways in S. cerevisiae have been plagued with significantly lower yields as compared to production in E. coli. In this thesis CCM production was explored in S. cerevisiae by construction of a heterologous pathway expressing AroZ from K. pneumoniae, AroY from P. anserina and Hqd2 from C. albicans, which resulted in the production of 2.9 mg/L of CCM. Accumulation of a native metabolite, shikimate, was observed, implying inefficient channeling of carbon into the heterologous pathway. To test this hypothesis, the diversion of carbon flux into the heterologous pathway was explored by modulation of the pentafunctional Aro1 protein. Replacing the wild-type aro1 gene with a mutant version eliminated the accumulation of shikimate and increased the carbon flux into the heterologous pathway by 59%. In addition, two-step fermentations with aerobic and anaerobic phases were explored demonstrating the imbalance in oxygen requirements in the heterologous CCM pathway and ways to overcome it.