Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics

Abstract

BackgroundClostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C. thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools.ResultsClostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h−1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6.98, and intercellular concentration reached 1.8 μmol/g pellet at pH 5.80 (stopped flow).ConclusionsClostridium thermocellum LL1210 can grow under slightly acidic conditions, similar to limits reported for other strains. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1095-y) contains supplementary material, which is available to authorized users.

Metabolic engineering strategies for consolidated production of lactic acid from lignocellulosic biomass

“…One of the main limits toward LA production through native cellulolytic microorganisms is that known anaerobic cellulolytic bacteria, such as C. thermocellum , typically do not grow at pH values <6.0 . Low extracellular pH is toxic because it causes dissipation of the proton gradient across the cytoplasmic membrane.…”

“…In this condition, weak acids such as LA become protonated and can cross the cytoplasmic membrane. Since cytoplasm is more alkaline, weak acids dissociate protons which acidify cytoplasm and collapse the ∆pH . As far as I know, no information on LA tolerance by native cellulolytic microorganisms has been reported.…”

“…A recent transcriptomic/metabolomic study has identified possible protein targets for improving acidic pH tolerance of C. thermocellum that include: (1) improving the expression of F 1 F 0 ‐ATPase, owing to its function in pumping protons out of the cell at the expense of ATP; (2) upregulating proton‐pumping PP i ‐ase; (3) improving the expression of protein chaperones and heat‐shock proteins such as GrpE, Hsp 20, and Hsp33. A further promising target for engineering acid tolerance in this strain seems to be nitrogen metabolism.…”

“…A further promising target for engineering acid tolerance in this strain seems to be nitrogen metabolism. Acidic pH induces intracellular glutamate accumulation, which could be exploited by introducing a heterologous glutamate decarboxylase . Actually, bacterial glutamate decarboxylases are generally involved in neutralizing pH acidity through proton‐consuming decarboxylation of glutamate to γ‐aminobutyrate .…”

“…Actually, bacterial glutamate decarboxylases are generally involved in neutralizing pH acidity through proton‐consuming decarboxylation of glutamate to γ‐aminobutyrate . Furthermore, inactivation of glutamine synthase might also reduce the need for buffering fermentation media of C. thermocellum cultures . Recently, a combination of random chemical mutagenesis and evolutionary engineering has been used to increase acid tolerance in the anaerobic cellulolytic bacterium Fibrobacter succinogenes .…”