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Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Muconic acid is a potential platform chemical for the production of nylon, polyurethanes, and terephthalic acid. It is also an attractive functional copolymer in plastics due to its two double bonds. At this time, no economically viable process for the production of muconic acid exists. To harness novel genetic targets for improved production of cis,cis -muconic acid (CCM) in the yeast Saccharomyces cerevisiae , we employed a CCM-biosensor coupled to GFP expression with a broad dynamic response to screen UV-mutagenesis libraries of CCM-producing yeast. Via fluorescence activated cell sorting we identified a clone Mut131 with a 49.7% higher CCM titer and 164% higher titer of biosynthetic intermediate-protocatechuic acid (PCA). Genome resequencing of the Mut131 and reverse engineering identified seven causal 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 not recognized as flux controlling before. The Mut131 strain was further rationally engineered by overexpression of the genes encoding for PCA decarboxylase and AROM protein without shikimate dehydrogenase domain (Aro1p ΔE ), and by restoring URA3 prototrophy. The resulting engineered strain produced 20.8 g/L CCM in controlled fed-batch fermentation, with a yield of 66.2 mg/g glucose and a productivity of 139 mg/L/h, representing the highest reported performance metrics in a yeast for de novo CCM production to date and the highest production of an aromatic compound in yeast. The study illustrates the benefit of biosensor-based selection and brings closer the prospect of biobased muconic acid.