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

Abstract

Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium 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 predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-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 of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-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.

Redox-Responsive Repressor Rex Modulates Alcohol Production and Oxidative Stress Tolerance in Clostridium acetobutylicum

“…Briefly, a 350-bp fragment for retargeting an intron to insert within the rex gene (CAC2713) was generated by one-step assembly PCR using the primers shown in Table S1 in the supplemental material according to the protocol of the TargeTron gene knockout system (Sigma). The PCR product was digested and ligated to targetron vector pWJ1 (31), yielding the plasmid pWJ1-rex. The plasmid was methylated in vivo in Escherichia coli ER2275(pAN1) (32) and electroporated into C. acetobutylicum ATCC 824.…”

Ribulokinase and Transcriptional Regulation of Arabinose Metabolism in Clostridium acetobutylicum

“…Briefly, the 350-bp fragments for retargeting introns to insert within the araK (CAC1344) or araR (CAC1340) genes, respectively, were generated by one-step assembly PCR using the primers shown in Table S1 in the supplemental material according to the protocol of the TargeTron gene knockout system (Sigma). The PCR products were then digested and ligated to a targetron plasmid, pWJ1 (39), yielding the plasmids pWJ1-araK and pWJ1-araR. Each plasmid was methylated in vivo in E. coli ER2275(pAN1) (18) and electroporated into C. acetobutylicum ATCC 824.…”

Rewiring carbon catabolite repression for microbial cell factory

“…Disruption of the PTS in C. acetobutylicum leads to an improved co-utilization of arabinose and xylose without greatly impairing the glucose metabolism. Over-expression of xylose metabolic pathway together with the PTS knockout enhanced the co-metabolism of the three sugars (55). CCR system observed in C. acetobutylicum was relatively simple than that observed in E. coli.…”

“…Few other strains were shown to co-metabolize a mixture of carbon sources. (8,(53)(54)(55)(56). Several genetic and evolutionary engineering approaches helped achieve efficient pentose utilization in Z. mobilis and S. cerevisiae (57).…”