Regulation of Carbon and Electron Flow in Clostridium butyricum VPI 3266 Grown on Glucose-Glycerol Mixtures

Abstract

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H ؉ ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.By-products of the food industry have great potential for the production of intermediates for the chemical industry; however, until now only a few applications have been found. Glycerol, a by-product with three atoms of carbon, can easily enter the metabolic pathways of several microorganisms to produce a wide range of compounds.Bioconversion of glycerol to 1,3-propanediol is already known for several bacterial strains, e.g., Lactobacillus brevis and Lactobacillus buchnerii (44,46), Bacillus welchii (23), Citrobacter freundii and Klebsiella pneumoniae (32,40,47), Clostridium acetobutylicum (14), Clostridium pasteurianum (35), and Clostridium butyricum (3, 21, 43). Anaerobic metabolic pathways of glycerol catabolism have, until now, only been completely characterized in the genera Klebsiella (11,12,13,36) and Citrobacter (7,8,9,45). According to the experiments done in these microorganisms, glycerol is metabolized in two simultaneous pathways. In the first, an NAD ϩ -dependent glycerol dehydrogenase catalyzes the oxidation of glycerol to dihydrox...

Novel metabolism in Chlamydomonas through the lens of genomics

“…The presence of both the acetaldehyde and acetyl-CoA pathways for ethanol production and the PFOR and PFL pathways for acetyl-CoA generation is remarkable for a eukaryote. Oxidation of NADH could also be coupled to H 2 production, as observed for Clostridia [43].…”

“…The presence of both the acetaldehyde and acetyl-CoA pathways for ethanol production and the PFOR and PFL pathways for acetyl-CoA generation is remarkable for a eukaryote. Oxidation of NADH could also be coupled to H 2 production, as observed for Clostridia [43].Interestingly, the Chlamydomonas genome has two copies of PTA and ACK. PTA1 and ACK2 are localized to mitochondria, whereas PTA2 and ACK1 are probably in the chloroplast [40].…”

Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals

“…A number of microorganisms have been shown to ferment glycerol in a 1,3-PDO-dependent manner, with the biological production of 1,3-PDO from glycerol demonstrated in Lactobacillus brevis, Lactobacillus buchnerii [21,22], Bacillus welchii [23], Citrobacter freundii, Klebsiella pneumoniae [24,25], Clostridium pasteurianum [26], and Clostridium butyricum [27][28][29]. 1,3-PDO is more reduced than glycerol resulting in glycerol fermentation producing 1,3-PDO and a more oxidized co-product.…”

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

“…We recently reported that the glycerol dehydratase of Clostridium butyricum VPI1718, extracted from 1,3-PD-producing cells, was not stimulated by coenzyme B12 and was extremely oxygen sensitive, which suggested it might be a coenzyme B12-independent enzyme (11). A B12-independent diol dehydratase, inactive on glycerol, was previously purified from Clostridium glycolycum and was proposed to be a diiron-tyrosyl radical protein (12).…”