Enhancement of Saccharomyces cerevisiae's Production of cis,cis-Muconic Acid by Biosensor-Aided Genome Engineering

Abstract

One possible platform chemical for the synthesis of nylon, polyurethanes, and terephthalic acid is muconic acid. Because of its two
double bonds, it is also a desirable functional copolymer in plastics. There is currently no economically feasible method for producing
muconic acid. We screened UV-mutagenesis libraries of CCM-producing yeast using a CCM-biosensor connected to GFP expression with
a broad dynamic response in order to identify new genetic targets for enhanced synthesis of cis,cis-muconic acid (CCM) in the yeast
Saccharomyces cerevisiae. We discovered a clone Mut131 using fluorescence-activated cell sorting, which had a 164% higher titer of
biosynthetic intermediate-protocatechuic acid (PCA) and a 49.7% higher CCM titer. Seven causative missense mutations of the native
genes (PWP2, EST2, ATG1, DIT1, CDC15, CTS2, and MNE1) and a duplication of two CCM biosynthetic genes, encoding dehydroshikimate
dehydratase and catechol 1,2-dioxygenase, which were previously unknown as flux controlling, were discovered by genome
resequencing of the Mut131 and reverse engineering. By restoring URA3 prototrophy and overexpressing the genes producing PCA
decarboxylase and AROM protein without shikimate dehydrogenase domain (Aro1pΔE), the Mut131 strain was further rationally
developed. With a yield of 66.2 mg/g glucose and a productivity of 139 mg/L/h, the resultant engineered strain generated 20.8 g/L CCM
in controlled fed-batch fermentation. This was the highest production of an aromatic compound in yeast and the highest performance
metrics for de novo CCM production to date. The study advances the possibility of biobased muconic acid and demonstrates the
advantages of biosensor-based selection