Caffeic Acid Production By Simultaneous Saccharification and Fermentation of Kraft Pulp Using Recombinant Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) is a major hydroxycinnamic acids present in plant cells, and expected to serve as an important precursor for a variety of biologically active compounds due to the anticancer, anti-inflammatory, and antiviral activities. In the present study, Escherichia coli was metabolically engineered to produce caffeic acid from cellulosic feedstocks. To establish plant-specific phenylpropanoid pathway in a metabolically engineered E. coli, hpaBC genes encoding two component flavin dependent monooxygenase derived from Pseudomonas aeruginosa PAO1 was expressed in combination with fevV gene encoding tyrosine ammonia lyase derived from Streptomyces sp. WK-5344 under T7 promoter in a tyrosine overproducing E. coli AN219, which compensates a metabolic pathway from tyrosine to caffeic acid through a metabolic intermediate p-coumaric acid. A resulting recombinant strain YK01 produced caffeic acid from glucose as the sole carbon source. Kraft pulp was employed as a model cellulosic feedstock for caffeic acid production. After 84 h of cultivation, separate hydrolysis and fermentation (SHF) with kraft pulp hydrolysate yielded less caffeic acid than SHF with filter paper hydrolysate (3 and 6 mg/L, respectively). To avoid fermentation inhibition observed in SHF with kraft pulp hydrolysate, simultaneous saccharification and fermentation (SSF) of kraft pulp supplemented with cellulase cocktails was demonstrated. Notably, caffeic acid titer was markedly increased with reducing loads of cellulase, reaching at 240 mg/L in culture with the minimum cellulase load at 2.5 FPU/g kraft pulp after 310 h of cultivation. In the optimal cultivation conditions, glucose accumulation was no longer observed after 16 h of cultivation, indicating that the E. coli cells experienced glucose limitation under the conditions. In addition, the accumulations of a precursor p-coumaric acid and a variety of potential fermentation inhibitors were markedly lower and the formation of by-products of acetate, lactate, and succinate was significantly reduced as compared to both SHF with kraft pulp hydrolysate and SSF with higher loads of cellulase. The caffeic acid yield was negatively correlated with the initial glucose concentration, with a five-fold higher caffeic acid yield observed in culture medium containing 5 g/L glucose compared to 100 g/L. These results suggest that gradual enzymatic hydrolysis during SSF enhances caffeic acid production under glucose limitation and reduces the accumulation of fermentation inhibitors and metabolic overflow to by-products, collectively leading to increased caffeic acid yield.