Overflow metabolism and growth cessation in Clostridium thermocellum DSM1313 during high cellulose loading fermentations

Abstract

As a model thermophilic bacterium for the production of second-generation biofuels, the metabolism of Clostridium thermocellum has been widely studied. However, most studies have characterized C. thermocellum metabolism for growth at relatively low substrate concentrations. This outlook is not industrially relevant, however, as commercial viability requires substrate loadings of at least 100 g/L cellulosic materials. Recently, a wild-type C. thermocellum DSM1313 was cultured on high cellulose loading batch fermentations and reported to produce a wide range of fermentative products not seen at lower substrate concentrations, opening the door for a more in-depth analysis of how this organism will behave in industrially relevant conditions. In this work, we elucidated the interconnectedness of overflow metabolism and growth cessation in C. thermocellum during high cellulose loading batch fermentations (100 g/L). Metabolic flux and thermodynamic analyses suggested that hydrogen and formate accumulation perturbed the complex redox metabolism and limited conversion of pyruvate to acetyl-CoA conversion, likely leading to overflow metabolism and growth cessation in C. thermocellum. Pyruvate formate lyase (PFL) acts as an important redox valve and its flux is inhibited by formate accumulation. Finally, we demonstrated that manipulation of fermentation conditions to alleviate hydrogen accumulation could dramatically alter the fate of pyruvate, providing valuable insight into process design for enhanced C. thermocellum production of chemicals and biofuels. Biotechnol. Bioeng. 2017;114: 2592-2604. © 2017 Wiley Periodicals, Inc.

Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites

“…Pyruvate accumulation and subsequent overflow metabolism has been observed and studied in detail in C. thermocellum (Deng et al., ; Holwerda et al., ; Olson et al., ; Thompson et al., ). Accumulation of formate and hydrogen in C. thermocellum collectively restrict the reoxidation of ferredoxin and limit its availability for pyruvate:ferredoxin oxidoreductase‐enabled conversion of pyruvate to acetyl‐CoA, causing accumulation of pyruvate and pyruvate‐derived overflow metabolism products (Holwerda et al., ; Thompson & Trinh, ; Thompson et al., ). C. cellulolyticum growth was shown to be inhibited by high concentrations of NADH, which inhibits glyceraldehyde 3‐phosphate dehydrogenase activity and limits glycolytic flux (Payot, Guedon, Cailliez, Gelhaye, & Petitdemange, ).…”

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures

“…This thermophile was chosen, because it has a high cellulolytic activity suitable for CBP, a one-step process configuration for cellulase production, cellulose hydrolysis, and fermentation for direct conversion of lignocellulosic biomass to fuels and chemicals [27]. Furthermore, studies have demonstrated that the wild-type C. thermocellum has native metabolism capable of endogenously producing precursor metabolites for ester biosynthesis, such as acetyl-CoA, isobutyryl-CoA, as well as ethanol [28] and higher alcohols (e.g., isobutanol) under high cellulose loading fermentation [29][30][31] (Fig. 4a, Additional file 1: Figure S5A).…”

Development of a genome-scale metabolic model ofClostridium thermocellumand its applications for integration of multi-omics datasets and strain design

“…The isobutanol pathway can consume NADPH through several enzymes 6 and has increased flux during overflow metabolism at high-substrate loading. 18,37 The model also predicted a decrease in valine secretion (EX val L e), since the isobutanol path-way competes with the valine pathway after KARA1. Remarkably, this prediction is consistent with the lower valine secretion measured in Δ hydG Δ ech .…”

“…17 The iAT601 model was used to identify genetic manipulations for high ethanol, isobutanol, and hydrogen production, 16 and to understand growth cessation prior to substrate depletion observed under high-substrate loading fermentations that simulate industrial conditions. 18 In addition to these core and genome-scale steady-state metabolic models, a kinetic model of central metabolism, k-ctherm118, was recently developed and used to elucidate the mechanisms of nitrogen limitation and ethanol stress. 15 Due to the biotechnological relevance of the Clostridium genus, GSMs have also been developed for other species, 19 including C. acetubutylicum , 20–26 C. beijerinckii , 27 C. butyricum , 28 C. cellulolyticum , 22 and C. ljungdahlii .…”