Engineering and evolution of Saccharomyces cerevisiae for muconic acid production

The advent of metabolic engineering and synthetic biology has resulted in a proliferation of microbial cell factories capable of producing valuable chemical products in diverse microbial hosts. This promises to provide a means to produce many of the chemical products which are currently derived from petroleum in an alternative, environmentally friendly, renewable process. Muconic acid is a chemical of particular interest for bioproduction as it can serve as a precursor for many compounds including the polymers nylon and polyethylene terephthalate. My initial research resulted in importing the biosynthetic capacity for muconic acid into the yeast host Saccharomyces cerevisiae. Through this work, we demonstrated the novel production of muconic acid for the first time in yeast and performed subsequent strain engineering to increase titers to 140mg/L, then the highest titer of any product from the shikimate pathway in yeast [1].

To further improve muconic acid titers, we chose to use adaptive laboratory evolution to complement initial, rational metabolic engineering efforts. To facilitate the screening of mutant strains with increased muconic acid production, a transcription-factor based biosensor was created. This biosensor was created to detect aromatic amino acids as a surrogate for flux through the shikimate pathway, the precursor pathway also used for muconic acid biosynthesis. This biosensor was based on the Aro80p transcription factor and demonstrated both tunable induction upon aromatic amino acids as well as a constitutive mode that created ultra-strong promoters capable of two-fold stronger expression that TDH3 (GPD), one of the strongest promoters available in yeast [2].

Finally, the utility of this biosensor coupled with adaptive laboratory evolution was demonstrated in a further approach to increase muconic acid production. Namely, this sensor was used in a biosensor-enabled adaptive laboratory evolution scheme to increase titers in our original strain to over 550 mg/L muconic acid in shake flask and 1.94g/L in a fed-batch bioreactor. This work represents a 14-fold improvement in titer over our previously engineered strain and nearly a 400-fold increase over simple heterologous expression of the pathway. These results demonstrate the power of coupling rationale engineering with adaptive engineering to increase product titers.