Clostridium cellulovorans Proteomic Responses to Butanol Stress

Abstract

Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, one of the most promising liquid biofuels. Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n-butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative gel-free proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS) analysis allowed identification and quantification of differentially expressed soluble proteins. The study data are available via ProteomeXchange with the identifier PXD024183. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (HSPs), involved in protein quality control, were up-regulated such as the chaperones GroES (Cpn10), Hsp90, and DnaJ. Globally, our data indicate that protein biosynthesis is reduced, likely not to overload HSPs. Several additional metabolic adaptations were triggered by butanol exposure such as the up-regulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid (e.g., arginine, lysine, methionine, and branched chain amino acids) biosynthesis and proteins involved in cell envelope re-arrangement (e.g., the products of Clocel_4136, Clocel_4137, Clocel_4144, Clocel_4162 and Clocel_4352, involved in the biosynthesis of saturated fatty acids) and a redistribution of carbon flux through fermentative pathways (acetate and formate yields were increased and decreased, respectively). Based on these experimental findings, several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans are suggested. This includes overexpression of HSPs (e.g., GroES, Hsp90, DnaJ, ClpC), RNA chaperone Hfq, V- and F-type ATPases and a number of genes whose function in C. cellulovorans is currently unknown.

Clostridium acetobutylicum atpG-Knockdown Mutants Increase Extracellular pH in Batch Cultures

“…The increase in extracellular pH shown in this study is presumed to be due to inhibition of proton pumping across the membrane by knockdown of the atpG gene. This seems to be closely related to the recent report that ATPase is inhibited by butanol, which resulted in a low intracellular pH and reduction of PMF (Costa et al, 2021).…”

“…Depending on the situation, F-ATPase can reversibly synthesize or degrade ATP ( Löbau et al, 1998 ; Bowler et al, 2006 ; Hayashi et al, 2012 ). ATP is hydrolyzed to create a proton gradient through the plasma membrane, while PMF is used for ATP synthesis ( Costa et al, 2021 ). The increase in extracellular pH shown in this study is presumed to be due to inhibition of proton pumping across the membrane by knockdown of the atpG gene.…”

Current progress on engineering microbial strains and consortia for production of cellulosic butanol through consolidated bioprocessing

“…The butyryl‐CoA pathway of clostridia is generally downregulated under a low intracellular NADH/NAD + ratio through the transcriptional repressor Rex (Hu et al, 2016 ; Nguyen et al, 2018 ). As this seems to occur also in C. cellulovorans (Costa et al, 2021 ), it would be worth testing if disruption of the rex gene may increase butanol production in this strain as previously reported in C. acetobutylicum (Nguyen et al, 2018 ).…”

“…Native butanol‐producers such as C. acetobutylicum generally show rather low tolerance (i.e., 1–2% v/v butanol) (Huang et al, 2010 ; Nicolaou et al, 2010 ). Cellulolytic clostridia such as C. thermocellum (Tian, Cervenka, et al, 2019 ) or C. cellulovorans (Costa et al, 2021 ; Yang et al, 2015 ) show even lower resistance since they cannot grow at butanol concentrations higher than 5–8 g L −1 (i.e., 0.6–1% v/v), respectively. Among other potential hosts for recombinant butanol production, E. coli growth is inhibited at 1% v/v (Si et al, 2016 ) while microbes tolerating the highest butanol concentrations include bacteria belonging to the Pseudomonas genus (2%–3% v/v) (Cuenca et al, 2016 ; Halan et al, 2017 ) and lactic acid bacteria (3.5%–4% v/v) (Li et al, 2021 ).…”

Current Progress in Production of Building-Block Organic Acids by Consolidated Bioprocessing of Lignocellulose

“…Cytosolic proton-consuming reactions have been identified in several microorganisms such as LAB, E. coli, and Helicobacter pylori and include urease reaction (urea + 2 H + + H 2 O → 2 NH 4 + + CO 2 ) [202], arginine deiminase pathway (arginine + H + +ADP + P i → ornithine + 2 NH 4 + + CO 2 + ATP), amino acid decarboxylation (amino acid + H + → amine + CO 2 ), and malolactic fermentation (MA + H + → LA + CO 2 ) [203]. Mechanisms for modulating cytoplasmic membrane fluidity/permeability include modification of lipid composition such as length and degree of unsaturation of fatty acids, ratio of cis/trans unsaturated fatty acids, presence of branched chain, and/or cyclopropane fatty acids [199,204]. In addition, protein chaperones (e.g., HdeAB, DnaKJ, GrpE, GroELS) and DNA repair systems (e.g., RecA, RecO, UvrABCD) are used by acid-challenged microorganisms to alleviate toxic effects such as protein denaturation and DNA damages (e.g., abasic sites), respectively [190,199,205].…”