S57: Engineering E. coli binary cultures for integrated conversion of hemicellulose to biofuels

Metabolic engineering has created several E. coli biocatalysts for production of biofuels and other useful molecules.  However, the inability of these biocatalysts to directly use polymeric substrates necessitates costly pretreatment and enzymatic hydrolysis prior to fermentation. Consolidated bioprocessing has the potential to simplify the process by combining enzyme production, hydrolysis, and fermentation into a single step but requires a fermenting organism to multitask by producing both necessary enzymes and target molecules.  We demonstrate here a binary strategy for consolidated bioprocessing of xylan, a complex substrate requiring six hemicellulases for complete hydrolysis.

An integrated modular approach was used to design the two strains to function cooperatively. The first strain was engineered to co-express two hemicellulases. Recombinant enzymes were secreted to growth medium by a method of lpp deletion with over 90% efficiency. Secreted enzymes hydrolyzed xylan into xylooligosaccharides, which was taken in by the second strain, designed to use the xylooligosaccharides for ethanol production.  Co-cultivation of the two strains converted xylan hemicellulose to ethanol with yield about 55% of the theoretical value. Inclusion of other three hemicellulases improved the ethanol yield to 70%.  Analysis of the culture broth shown that xylooligosaccharides with four or more xylose units were not utilized, suggesting that improving the use of higher xylo-ogligomers should be the focus in the future efforts.  This is the first demonstration of an engineered binary culture for consolidated bioprocessing of xylan. The modular design should allow the strategy to be adopted for a broad range of biofuel and biorefinery products.