New insights into the butyric acid metabolism of Clostridium acetobutylicum

Abstract

Biosynthesis of acetone and n-butanol is naturally restricted to the group of solventogenic clostridia with Clostridium acetobutylicum being the model organism for acetone-butanol-ethanol (ABE) fermentation. According to limited genetic tools, only a few rational metabolic engineering approaches were conducted in the past to improve the production of butanol, an advanced biofuel. In this study, a phosphotransbutyrylase-(Ptb) negative mutant, C. acetobutylicum ptb::int(87), was generated using the ClosTron methodology for targeted gene knock-out and resulted in a distinct butyrate-negative phenotype. The major end products of fermentation experiments without pH control were acetate (3.2 g/l), lactate (4.0 g/l), and butanol (3.4 g/l). The product pattern of the ptb mutant was altered to high ethanol (12.1 g/l) and butanol (8.0 g/l) titers in pH ≥ 5.0-regulated fermentations. Glucose fed-batch cultivation elevated the ethanol concentration to 32.4 g/l, yielding a more than fourfold increased alcohol to acetone ratio as compared to the wildtype. Although butyrate was never detected in cultures of C. acetobutylicum ptb::int(87), the mutant was still capable to take up butyrate when externally added during the late exponential growth phase. These findings suggest that alternative pathways of butyrate re-assimilation exist in C. acetobutylicum, supposably mediated by acetoacetyl-CoA:acyl-CoA transferase and acetoacetate decarboxylase, as well as reverse reactions of butyrate kinase and Ptb with respect to previous studies.

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum—evidence from a mathematical model

“…As a consequence, the decline of acetate in the mutant after the initiation of the pH-shift is explained by the shift in the prevalent subpopulation. In contrast, the observed decline of butyrate requires an additional reducing process in the model that is interpreted as an CtfA/B-independent mechanism (Desai et al 1999;Lehmann et al 2012a;Lehmann et al 2012b). In the present manuscript, we have accommodated previous suggestions (Hüsemann and Papoutsakis 1989) and assumed a reverse activity of butyrate kinase and phosphotransbutyrylase.…”

“…Biochemical investigations ; Wiesenborn et al 1989a) have suggested that the hetero-dimeric CoA-transferase, CtfA/B, mediates the re-assimilation of the acids acetate and butyrate and the formation of acetoacetate from acetoacetyl-CoA. However, several investigations have led to the notion that a CtfA/B-independent butyrate re-assimilation mechanism is also present in C. acetobutylicum (Desai et al 1999;Lehmann et al 2012a;Lehmann et al 2012b). It has been suggested by Hüsemann and Papoutsakis (1989), that a reverse pH-dependent activity of the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, which facilitate the butanol formation in the forward direction, might provide this alternative mechanism.…”

“…This view is supported by several experimental studies which had observed an increased transcription of the encoding genes, ctfA and ctfB, which are part of the sol operon (Grimmler et al 2011;Jones et al 2008), and an increased intracellular concentration of the protein during the pH-induced shift and during solventogenesis (Janssen et al 2010;Mao et al 2010). However, recent experimental evidence indicates that an alternative re-assimilation mechanism for butyrate could exist which is independent of the CoA-transferase (Desai et al 1999;Lehmann et al 2012a) and may rely on a pH-dependent reverse activity of the butyrate forming enzymes (Desai et al 1999;Lehmann et al 2012b). However, this activity would not be consistent with the results reported in , where the authors concluded from 13 C NMR studies, enzyme assays, and thermodynamic considerations that a CoA-transferase-mediated mechanism is more likely responsible for acid re-assimilation than a reverse action of acetate and butyrate formation pathways, acyl-CoA synthase reactions, or generation of acetyl and butyryl phosphate followed by their direct reduction to the corresponding aldehyde.…”

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) from butyrate using engineered Ralstonia eutropha

“…4). Considering butyrate is easily prepared as the end product of anaerobic fermentation of organic materials by obligate anaerobic bacteria such as Clostridium and Fusobacterium (Lehmann et al 2012;Potrykus et al 2007) and that R. eutropha also prefers butyrate to other volatile acids such as acetate and propionate (Yang et al 2010), P(HB-co-HHx) production from butyrate alone could potentially be more straightforward and cost-effective than the use of oils or refined sugars.…”

“…In this study, we demonstrate that engineered R. eutropha can produce P(HB-co-HHx) from the volatile organic acid, butyrate, which is prepared as the end product of anaerobic fermentation of organic materials by obligate anaerobic bacteria, such as Clostridium and Fusobacterium (Lehmann et al 2012;Potrykus et al 2007). We had previously demonstrated that R. eutropha prefers butyrate to other volatile acids, such as acetate and propionate (Yang et al 2010).…”