Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose

Efficient cofermentation ofd -glucose,d -xylose, andl -arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacteriumClostridium acetobutylicum , known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predictedglcG gene, encoding enzyme II of thed -glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strainClostridium acetobutylicum ATCC 824, resulting in greatly improvedd -xylose andl -arabinose consumption in the presence ofd -glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermentedd -glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result ofglcG disruption. Furthermore, the inherent rate-limiting steps of thed -xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of thed -xylose proton-symporter (cac1345),d -xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integratingglcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures ofd -glucose,d -xylose, andl -arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.