Industrial Robustness: Understanding the Mechanism of Tolerance for the Populus Hydrolysate-Tolerant Mutant Strain of Clostridium thermocellum

Abstract

BackgroundAn industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes.Methodology/Principal FindingsIn this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v).Conclusion/SignificanceThe findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially robust strains of consolidated bioprocessing microorganisms.

Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum

“…Escherichia coli strains were grown on LB medium supplemented with 12 μg ml -1 chloramphenicol as needed. For C. thermocellum, strains were grown either in modified DSM122 rich medium (Tripathi et al, 2010) or defined Medium for Thermophilic Clostridia (MTC) (Linville et al, 2013), with cellobiose, crystalline cellulose (Avicel), or pretreated biomass as the carbon source. To make MTC, Solution A was made in 162 mL serum bottles with cellobiose, Avicel PH105 or dilute-acid pretreated biomass as the carbon source.…”

Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass

“…Without the active removal/bioconversion of these inhibitory compounds and/or increased tolerance, the potential application of this cellulolytic organism for effective biofuel production will be limited, especially considering the planned industrial use of complex lignocellulosic substrates like switchgrass. Fortunately, studies have shown that it is possible to generate C. thermocellum strains with improved tolerance to complex lignocellulosic substrates, such as Populus hydrolysates [34]. …”

Toxicological challenges to microbial bioethanol production and strategies for improved tolerance

“…The directed evolution approach expedites this process by treating seed cultures with mutagens such as N-methyl N-nitro N-nitrosoguanidine or ultraviolet light, via whole genome shuffling, or through the expression of deletion libraries (for a review of this approach, see Nicolaou et al 2010). For both methods, genomic re-sequencing of the isolated tolerant mutants is employed following selection to provide insight towards the mechanisms that have evolved to improve tolerance (Brown et al 2011;Linville et al 2013). …”

“…Other production hosts, such as Clostridium species, also present significant alterations to gene expression. While the majority of work with these species has highlighted functional expression changes such as nutrient sensing and cellulosome synthesis (Akinosho et al 2014), additional studies demonstrate that these expression dynamics can influence energy and redox metabolism as well (Linville et al 2013;Wilson et al 2013). …”