Consolidated bioprocessing of hemicellulose to fuels and chemicals through an engineered Bacillus subtilis-Escherichia coli consortium

Lignocellulosic biomass is an inexpensive and abundant renewable carbon feedstock available for the sustainable production of fuels and chemicals. However, the process for obtaining pentose and hexose sugars from the hemicellulosic components of plant biomass requires the use of expensive purified enzymes. In this study, Bacillus subtilis strains were first constructed to enable the extracellular depolymerization of hemicellulose at a higher rate. Three different signal peptides (YwmCsp, SacCsp, and AmyEsp) were explored for the secretion of two endo-1,4-β-xylanases (from Trichoderma reesei and Bacillus pumilis), leading to the identification of an optimal design by which B. subtilis could secrete xylanase and effectively depolymerize xylan (the major hemicellulose component). In situ depolymerization of xylan by the engineered B. subtilis (SSL26) produced a maximum xylose titer of 7.1 g/L, corresponding to 66.7% of the total xylose initially present in 13.3 g/L of xylan. To demonstrate the application of this strain in consolidated bioprocessing, a B. subtilis-Escherichia coli consortium was developed by culturing SSL26 together with an E. coli strain X2S, a xylose assimilating succinate producer. Lastly, to demonstrate the generalizability of this approach for fuels and chemicals production, coculture studies were conducted for the production of ethanol and D-lactate from xylan. Together, this novel coculture consolidated bioprocessing (CCBP) enabled the production of succinate, ethanol, and D-lactate directly from xylan at a maximum titre of 3.9 g/L, 2 g/L, and 2 g/L respectively.