Engineering Escherichia Coli For Succinate Production From Hemicellulose Via Consolidated Bioprocessing

The recalcitrant nature of hemicellulosic materials and the high cost indepolymerization are the primary obstacles preventing the use of xylan as feedstockfor fuel and chemical production. Consolidated bioprocessing (CBP), incorporatingenzyme-generating, biomass-degrading and bioproduct-producing capabilities into asingle microorganism, could potentially avoid the cost of the dedicated enzymegeneration in the process of xylan utilization. In this study, drawing on the basicprinciples of metabolic engineering and synthetic biology, we engineered Escherichiacoli strains capable of exporting three hemicellulases to the broth for the succinateproduction directly from beechwood xylan.Xylanases were extracellular environment-directed by fusing with OsmY.Subsequently, twelve variant OsmY fused endoxylanase-xylosidase combinationswere characterized and tested. The combination of XynC-A from Fibrobactersuccinogenes S85and XyloA from Fusarium graminearum which appeared to haveoptimal enzymatic properties was identified as the best choice for xylan hydrolysis(0.18±0.01g/l protein in the broth with endoxylanase activity of12.14±0.34U/mgprotein and xylosidase activity of92±3mU/mg protein at8h after induction).To improve the hydrolysis efficiency and for better meet the needs of the CBPproduction, further improvements of hemicellulases secretion were investigated bylpp deletion, dsbA overexpression, blc surface displaying and expression leveloptimization. With co-expression of α-arabinofuranosidase, the engineered E. colicould hydrolyze beechwood xylan to pentose monosaccharides.The hemicellulolytic capacity was further integrated with a succinate-producing strainto demonstrate the production of succinate directly from xylan without externallysupplied hydrolases and any other organic nutrient. The resulting E. coli Z6373wasable to produce0.37g/g succinate from xylan anaerobically equivalent to76%of thatfrom xylan acid hydrolysates.This report represents a promising step towards the goal of hemicellulosic chemicalproduction. This engineered E. coli expressing and secreting three hemicellulasesdemonstrated a considerable succinate production on the released monosaccharidesfrom xylan. The ability to use lower-cost crude feedstock will make biological succinate production more economically attractive.