The aromatic amino acid pathway is exemplary for hosting a great diversity of chemicals relevant to the polymer, pharmaceutical, and nutraceutical industries. The precursor module, composed mainly of the shikimic acid pathway, houses the important metabolites shikimic acid (SA), used as a drug precursor, and dehydroshikimic acid (DHS), the branching point for production of the polymer precursor muconic acid (MA). A multilevel approach to engineer the precursor module of this pathway in Saccharomyces cerevisiae was implemented. It consisted of (i) relieving the pathway from strong transcriptional repression, (ii) removal of competing pathways to ensure high carbon capturing, and (iii) rewiring of precursor pathways to increase the carbon funneling to the desired target. By combining computational tools for the prediction of novel transcriptional repression targets, optimization-based OptForce procedure for the design of pathway interventions, and implementation of the CRISPR/Cas9 platform for fast and efficient gene deletion, we constructed S. cerevisiae strains with titers as high as 2.48 g L-1 SA, 13-fold higher than the initial strain. Further expansion of the platform led to the production of MA and intermediates together close to 1.6 g L-1. The novel approach presented here that combines identification of both the transcriptional regulators and novel metabolic targets can be easily extrapolated for engineering the downstream modules in the aromatic pathway leading towards high-titer production of plant-sourced secondary metabolites.

Multilevel engineering of the upstream aromatic module in Saccharomyces cerevisiae for high production of polymer and drug precursors

253rd National Meeting (2017)

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