Enhancing fructosylated chondroitin production in Escherichia coli K4 by balancing the UDP-precursors

Abstract Microbial production of chondroitin and chondroitin-like polysaccharides from renewable feedstock is a promising and sustainable alternative to extraction from animal tissues. In this study, we attempted to improve production of fructosylated chondroitin in Escherichia coli K4 by balancing intracellular levels of the precursors UDP-GalNAc and UDP-GlcA. To this end, we deleted pfkA to favor the production of Fru-6-P. Then, we identified rate-limiting enzymes in the synthesis of UDP-precursors. Third, UDP-GalNAc synthesis, UDP-GlcA synthesis, and chondroitin polymerization were combinatorially optimized by altering the expression of relevant enzymes. The ratio of intracellular UDP-GalNAc to UDP-GlcA increased from 0.17 in the wild-type strain to 1.05 in a 30-L fed-batch culture of the engineered strain. Titer and productivity of fructosylated chondroitin also increased to 8.43 g/L and 227.84 mg/L/h; the latter represented the highest productivity level achieved to date.