Pentose sugars inhibit metabolism and increase expression of an AgrD-type cyclic pentapeptide in Clostridium thermocellum

Tobin J Verbeke,Adam M Guss,Robert L Hettich,Thomas Rydzak,Nancy L Engle,Steven D Brown,Timothy J Tschaplinski,Dawn M Klingeman,Richard J Giannone,James G Elkins

doi:10.1038/srep43355

Tobin J Verbeke, Adam M Guss + Show 8 more

Open Access

https://doi.org/10.1038/srep43355

Copy DOI

Abstract

Clostridium thermocellum could potentially be used as a microbial biocatalyst to produce renewable fuels directly from lignocellulosic biomass due to its ability to rapidly solubilize plant cell walls. While the organism readily ferments sugars derived from cellulose, pentose sugars from xylan are not metabolized. Here, we show that non-fermentable pentoses inhibit growth and end-product formation during fermentation of cellulose-derived sugars. Metabolomic experiments confirmed that xylose is transported intracellularly and reduced to the dead-end metabolite xylitol. Comparative RNA-seq analysis of xylose-inhibited cultures revealed several up-regulated genes potentially involved in pentose transport and metabolism, which were targeted for disruption. Deletion of the ATP-dependent transporter, CbpD partially alleviated xylose inhibition. A putative xylitol dehydrogenase, encoded by Clo1313_0076, was also deleted resulting in decreased total xylitol production and yield by 41% and 46%, respectively. Finally, xylose-induced inhibition corresponds with the up-regulation and biogenesis of a cyclical AgrD-type, pentapeptide. Medium supplementation with the mature cyclical pentapeptide also inhibits bacterial growth. Together, these findings provide new foundational insights needed for engineering improved pentose utilizing strains of C. thermocellum and reveal the first functional Agr-type cyclic peptide to be produced by a thermophilic member of the Firmicutes.

Highlights

Potential bioenergy crops, such as Miscanthus, switchgrass and Populus, contain xylan-based hemicelluloses[5,6,7]
Given the abundance of pentose sugars in lignocellulosic biomass, and the lack of xylose consumption during C. thermocellum pure culture fermentation on minimally pretreated biomass, the purpose of this study was to assess the impact of xylose and xylo-oligomers on the metabolism of the bacterium
The model substrates xylose and Beechwood xylan were added at increasing concentrations to C. thermocellum M157019 fermentation medium and their effect on end-product formation, as an indicator of metabolic limitation, was assessed (Fig. 1)

Summary

Introduction

Potential bioenergy crops, such as Miscanthus, switchgrass and Populus, contain xylan-based hemicelluloses[5,6,7]. Despite being limited to cellodextrins for fermentation, multiple reports show that C. thermocellum is highly capable of deconstructing and solubilizing hemicellulose polymers as a means of improving accessibility to cellulose[10,11,12]. The products of C. thermocellum mediated hemicellulose deconstruction would typically be consumed by other saccharolytic, pentose-fermenting thermophiles such as Thermoanaerobacter and Thermoanaerobacterium spp[16]. While C. thermocellum is able to hydrolyze hemicellulose polymers, but unable to catabolize pentoses, little effort has been given to understanding how the microorganism interacts with these residual saccharides. Given the abundance of pentose sugars in lignocellulosic biomass, and the lack of xylose consumption during C. thermocellum pure culture fermentation on minimally pretreated biomass, the purpose of this study was to assess the impact of xylose and xylo-oligomers on the metabolism of the bacterium

Objectives

Methods

Results

Conclusion