High-yield production of D-1,2,4-butanetriol from lignocellulose-derived xylose by using a synthetic enzyme cascade in a cell-free system

Approaches using metabolic engineering to produce D-1, 2, 4-butanetriol (BT) from renewable biomass in microbial systems have achieved initial success. However, due to the lack of incomplete understanding of the complex branch pathway, the efficient fermentation system for BT production was difficult to develop. Here we reconstituted a cell-free system in vitro using purified enzymes to produce BT from D-xylose. The factors that influencing the efficiency of cell-free system, including enzyme concentration, reaction buffer, pH, temperature, metal ion additives and cofactors were first identified to define optimal reaction conditions and essential components for the cascade reaction. Meanwhile, a natural cofactor recycling system was found in cell-free system. Finally, we were able to convert 18 g/L xylose to 6.1 g/L BT within 40 h with a yield of 48.0%. The feasibility of cell-free system to produce BT in corncob hydrolysates was also determined.