Utilization of Monosaccharides by Hungateiclostridium thermocellum ATCC 27405 through Adaptive Evolution

Abstract

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden–Mayerhof–Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage.

Deciphering Cellodextrin and Glucose Uptake in Clostridium thermocellum

“…Contrary to previous reports ( 17 ), transporter A in C. thermocellum was confirmed to be the sole glucose transporter in our study, instead of transporter C. This finding is indeed consistent with those of two recent studies involving the adaptive evolution of C. thermocellum on glucose and fructose, which showed that transporter A genes contained the most frequent mutations in the adaptive strains ( 21 , 22 ). Unlike other monosaccharide-binding proteins with strong affinities to the substrate ( K D in the μM range) ( 51 ), the binding of CbpA to glucose is remarkably weak as shown by ITC experiments.…”

“…These findings are quite unexpected, because many microorganisms are known to have a multiplicity of redundant sugar transporters for their major carbon source ( 51 , 52 ). C. thermocellum takes up cellodextrins derived from cellulose as its major carbon source, while monosaccharides, such as glucose and fructose, can also be utilized by the bacterium after a lengthy adaptation period ( 21 , 22 ). Although the physiological and evolutionary benefits of these restrictive transporters are still unknown, these findings suggest that engineering sugar transporters in C. thermocellum could be easier than in species with multiple redundant transporters.…”