Development of E. Coli Cell Factories for Consolidated Bioprocessing of Hemicellulose to Biosuccinic Acid Through Metabolic Engineering and Cell-Surface Display

Bio-based succinate is predicted to be one of the future platform chemicals that can be derived from renewable resources, but it is still faced with the challenge of becoming cost competitive against petrochemical-based alternatives. Consolidated bioprocessing (CBP), incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single microorganism, could potentially avoid the cost of the dedicated enzyme generation in the process of hemicellulosic materials utilization. In this study, drawing on the basic principles of metabolic engineering, synthetic biology and cell-surface display, we engineered Escherichia coli strains capable of exporting three hemicellulases to the broth for the succinate production directly from beechwood xylan.

Xylanases were extracellular environment-directed by fusing with OsmY. Subsequently, twelve variant OsmY fused endoxylanase-xylosidase combinations were characterized and tested. The combination of XynC-A from Fibrobacter succinogenes S85 and XyloA from Fusarium graminearum which appeared to have optimal enzymatic properties was identified as the best choice for xylan hydrolysis.

   To improve the hydrolysis efficiency and for better meet the needs of the CBP production, further improvements of hemicellulases secretion were investigated by lpp deletion, dsbA overexpression, blc surface displaying. Three xylanases (β-xylosidase, endoxylanase, and α-arabinofuranosidase ) were expressed on the surface of the E. coli. The xylanases were displayed on the surface of the cells by fusing with anchor proteins, Blc.

The hemicellulolytic capacity was further integrated with a succinate-producing strain to demonstrate the production of succinate directly from xylan without externally supplied hydrolases and any other organic nutrient. The resulting E. coli was able to produce 0.37 g/g succinate from xylan anaerobically equivalent to 76% of that from xylan acid hydrolysates. This engineered E. coli expressing and secreting three hemicellulases demonstrated a considerable succinate production on the released monosaccharides from xylan. Because a cost-effective process cannot be realized until all three of titer, rate (or productivity), and yield (TRY) have been optimized, so it will need much more work to improve it to the point that it can support a commercial process.