(430h) Engineering S. Cerevisiae for Production of Cis,Cis-Muconic Acid

Muconic acid is a six-carbon dicarboxylic acid with two insaturations gaining industrial attention due to its potential in the manufacturing of a variety of chemicals. As a precursor molecule, muconic acid can be converted into terephtalic acid and adipic acid; which in turn, represent a highly important market for the plastic industry. Although E. coli is the microorganism with the highest reported muconic acid productivity, the required pH control by addition of strong bases, represents not only an economic disadvantage but also an environmental burden owing to undesired formation of by-products. In contrast, yeasts represent a good alternative to bacteria, as they possess higher acid tolerance, an invaluable feature for chemical and fuel production. In the present work, Saccharomyces cerevisiae was genetically engineered to produce muconic acid via the ortho-cleavage of catechol using glucose as a carbon source. Strong endogenous promoters were selected to control the expression of the three genes comprising the pathway. To improve the production of muconic acid, high throughput strain optimization strategies were applied: the expression level of the genes was tuned by modulating the promoter strength; the upstream pathway providing the precursor for synthesizing muconic acid was manipulated; furthermore, the rate limiting enzyme in the pathway, PCAD, was improved by directed evolution. After these improvements, the muconic acid titers increased 4-fold compared to the highest reported titer in S. cerevisiae strain, representing the highest titer achieved for compounds derived from aromatic amino acid biosynthesis in S. cerevisiae.