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.

Keywords: ATPase; Hfq chaperone; butyrate; heat shock proteins; stringent response.

FIGURE 1

Growth kinetics (A) and specific growth rate (μ, B) of C. cellulovorans grown in media supplemented with different butanol concentration. Bars represent standard errors. Data are the averages of three biological replicates. Asterisks indicate values that significantly (* p < 0.05; ** p < 0.005) differ from that measured in the control condition (no added butanol).

FIGURE 2

Glucose consumption and catabolite production of C. cellulovorans grown in control condition (no added butanol, A) or in butanol-supplemented medium (B). (C) Fermentation end-product yield (g per g of consumed glucose). Data are the mean of triplicate measurements. Bars represent standard errors. Asterisks indicate values that significantly (^∗p-value < 0.05; ^∗∗p-value < 0.01) differ between control (green) and butanol-supplemented (red) culture conditions.

FIGURE 3

Schematic overview of the proteomic workflow used to study C. cellulovorans responses to butanol stress. (1) C. cellulovorans was grown in media supplemented with different concentrations of butanol (0–8 g/L). Whole-cell soluble protein extraction was performed on C. cellulovorans cells grown in control (0 g/L butanol) or butanol-challenged (6 g/L butanol) cultures. (2) Differential proteomics were analyzed through Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS). Phylogenetic protein classification by COGs (Clusters of Orthologous Groups of proteins) was performed and qRT-PCR was used to confirm differential gene expression of a gene pool. (3) Butanol-challenged C. cellulovorans metabolism was described by discussing the differentially expressed proteins (up- and down-regulated in butanol-stressed bacteria). This information led to identification of target genes for rational metabolic engineering strategies to obtain a butanol-hypertolerant C. cellulovorans strain for large scale bio-production.

FIGURE 4

COG categories overrepresentation in differentially expressed proteins. The treemap displays the fold enrichment of each COG category in (A) down-regulated and (B) up-regulated proteins. The size and color of the rectangles are proportional to the registered fold enrichment. The proportion of regulated proteins that are annotated to each COG category is shown in brackets.

FIGURE 5

Protein content of cells grown in control (green) or butanol-supplemented medium (red) in different growth phases. Data are the mean of triplicate measurements. Asterisks indicate values that significantly (**p-value < 0.01) differ between the two growth conditions.

FIGURE 6

The expression of several heat shock proteins (HSPs) is affected in butanol-challenged C. cellulovorans. The gene loci encoding the main C. cellulovorans HSPs are indicated in red or green depending on if their protein products were up-regulated or down-regulated in butanol challenged cultures. Products of the genes indicated in blue were not identified in the present study.

FIGURE 7

Schematic representation of purine (A) and pyrimidine (B) metabolic pathways of C. cellulovorans. C. cellulovorans genes encoding each pathway enzyme is indicated. Red and green colors were used for gene loci whose protein product was over- or under-expressed in butanol-challenged cells, respectively. Geni loci whose protein product was identified in this study but was in similar amounts in butanol-supplemented and control cultures were indicated in black, while blue indicates geni loci whose protein product was not identified in the present investigation. Add, adenosine deaminase; Adk, adenylate kinase; AICAR, 1-(5′-Phosphoribosyl)-5-amino-4-imidazolecarboxamide; AIR, Aminoimidazole ribotide; CAIR, 1-(5-Phospho-D-ribosyl)-5-amino-4-imidazolecarboxylate; CarA, carbamoyl-phosphate synthase, small subunit; CarB, carbamoyl-phosphate synthase, large subunit; Cdd, cytidine deaminase; Cmk, cytidylate kinase; FAICAR, 1-(5′-Phosphoribosyl)-5-formamido-4-imidazolecarboxamide; FGAM, 2-(Formamido)-N1-(5′-phosphoribosyl)acetamidine; FGAR, 5′-Phosphoribosyl-N-formylglycinamide; GAR, 5′-Phosphoribosylglycinamide; GuaA, GMP synthase; GuaB, inosine-5′-monophosphate dehydrogenase; IMP, inosine monophosphate; Ndk, nucleoside-diphosphate kinase; NdrD, anaerobic ribonucleoside-triphosphate reductase; NrdJ, ribonucleoside-diphosphate reductase; P_i, inorganic phosphate; Pdp, pyrimidine-nucleoside phosphorylase; PK, pyruvate kinase; PRPP, 5-phosphoribosyl 1-pyrophosphate; PRPS, ribose-phosphate pyrophosphokinase; PurB, adenylosuccinate lyase; PurC, phosphoribosylaminoimidazole-succinocarboxamide synthase; PurD, phosphoribosylamine/glycine ligase; PurE, phosphoribosylaminoimidazole carboxylase; PurF, amidophosphoribosyltransferase; PurH, phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase; PurK, 5-(carboxyamino)imidazole ribonucleotide synthase; PurL, phosphoribosylformylglycinamidine synthase; PurM, phosphoribosylformylglycinamidine cyclo-ligase; PurN, phosphoribosylamine–glycine ligase; PyrB, aspartate carbamoyltransferase; PyrC, dihydroorotase; PyrD, dihydroorotate dehydrogenase; PyrE, orotate phosphoribosyltransferase; PyrF, orotidine 5′-phosphate decarboxylase; PyrG, CTP synthase; PyrH, uridylate kinase; PyrR, uracil phosphoribosyltransferase; SAICAR, 1-(5′-Phosphoribosyl)-5-amino-4- (N-succinocarboxamide)-imidazole; SurE, 5′-nucleotidase; Trdx, thioredoxin; Udp, uridine phosphorylase; XMP, xanthosine 5′-phosphate.

FIGURE 8

The acetyl-CoA–butyrate pathway is down-regulated in butanol-challenged C. cellulovorans. Gene loci encoding the pathway enzymes are represented in colors indicating their level of relative expression in butanol-supplemented cultures (green, down-regulated; black, identified but not differentially expressed, blue, not identified). Abbreviations: Bcd/EftAB, butyryl-CoA dehydrogenase/electron transfer protein; Buk, butyrate kinase; Crt, crotonase; Fd, ferredoxin; H₂ase, hydrogenase; Hbd, 3-hydroxybutyryl-CoA dehydrogenase; Pbt, phosphate butyryltransferase; Thl, thiolase.

FIGURE 9

Growth curve and intracellular ATP content of C. cellulovorans grown in control condition (green) or in butanol-supplemented medium (red). Bars represent standard deviations (n = 3). Asterisks indicate values that significantly (^∗p-value < 0.01) differ between the two growth conditions.