Abstract
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.
Highlights
Muconic acid (2,4‐hexadienedioic acid) is a six‐carbon dicarboxylic acid with two conjugated double bonds
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
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
Summary
Muconic acid (2,4‐hexadienedioic acid) is a six‐carbon dicarboxylic acid with two conjugated double bonds. Oxidation of phenol for 10 days or o‐coumaric acid for 14 days using peracetic acid generates CCM at a molar yield of 35% and 4%, respectively (Böeseken, 1932; Khalil et al, 2020) Some microbial strains, such as Pseudomonas putida KT2440, Corynebacterium glutamicum, and Amycolatopsis sp. P. putida KT2440 strain, engineered with phenol hydroxylase and catechol‐induced catechol 1,2‐dioxygenase, produced 13 g/L CCM from the hydrothermally depolymerized softwood lignin (Kohlstedt et al, 2018). In these bioconversion processes, glucose or another fermentable carbon source must be added to support cellular growth and energy generation. We combined yeast strain engineering, controlled fed‐batch fermentation and scale‐up, and CCM recovery and purification, to establish an integrated process for CCM production by yeast fermentation
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