EXTRACELLULAR MELIBIOSE AND FRUCTOSE ARE INTERMEDIATES IN RAFFINOSE CATABOLISM DURING FERMENTATION TO ETHANOL BY ENGINEERED ENTERIC BACTERIA

Contrary to general concepts of bacterial saccharide metabolism, melib iose (25 to 32 g/liter) and fructose (5 to 14 g/liter) accumulated as extracellular intermediates during the catabolism of raffinose ,6-alph a-D-glucopyranosyl-beta-D-fructofuranoside) (90 g/liter) by ethanologe nic recombinants of Escherichia coli B, Klebsiella oxytoca M5A1, and E rwinia chrysanthemi EC16. Both hydrolysis products (melibiose and fruc tose) were subsequently transported and further metabolized by all thr ee organisms, Raffinose catabolism was initiated by beta-fructosidase; melibiose was subsequently hydrolyzed to galactose and glucose by alp ha-galactosidase. Glucose and fructose were completely metabolized by all three organisms, but galactose accumulated in the fermentation bro th with EC16(pLOI555) and P2. MM2 (a raffinose-positive E. coli mutant ) was the most effective biocatalyst for ethanol production (38 g/lite r) from raffinose, All organisms rapidly fermented sucrose (90 g/liter ) to ethanol (48 g/liter) at more than 90% of the theoretical yield, D uring sucrose catabolism, both hydrolysis products (glucose and fructo se) were metabolized concurrently by EC16(pLOI555) and P2 without suga r leakage. However, fructose accumulated extracellularly (27 to 28 g/l iter) at early stages of fermentation with KO11 and MM2. Sequential ut ilization of glucose and fructose correlated with a diauxie in base ut ilization (pH maintenance), The mechanism of sugar escape remains unkn own but may involve downhill leakage via permease which transports pre cursor saccharides or novel sugar export proteins, If sugar escape occ urs in nature with wild organisms, it could facilitate the development of complex bacterial communities which are based on the sequence of s accharide catabolism and the hierarchy of sugar utilization.