Enzymology of the Wood–Ljungdahl Pathway of Acetogenesis

Abstract

A procedure was developed for the enrichment of auxotrophs in the antibiotic-insensitive archaebacte-rium Methanococcus. After mutagenesis with ethyl methane-sulfonate, growing cells were selectively killed upon exposure to the base analogs 6-azauracil and 8-azahypoxanthine for 48 hr. Using this method, eight independent acetate auxotrophs of Methanococcus maripaludis were isolated. Six of the auxo-trophs had an absolute growth requirement for acetate and contained 1-16% of the wild-ype levels of CO dehydrogenase. Three of these six also contained 14-29% of the wild-type levels of pyruvate oxidoreductase and 12-30% of the wild-type levels of pyruvate synthase. Two spontaneous revertants of these latter auxotrophs regained the ability to grow normally in the absence of acetate and wild-type levels of CO dehydrogenase, acetyl-CoA synthase, pyruvate oxidoreductase, and pyruvate synthase. Likewise, a spontaneous revertant of an auxotroph with reduced levels of CO dehydrogenase and wild-type levels of pyruvate oxidoreductase regained the ability to grow normally in the absence of acetate and wild-type levels of CO dehydrogenase and acetyl-CoA synthase. Two additional aux-otrophs grew poorly in the absence of acetate but contained wild-type levels of CO dehydrogenase and pyruvate oxidore-ductase. These results provide direct genetic evidence for the

Carboxylases in Natural and Synthetic Microbial Pathways

“…The only exception is the reductive acetyl-CoA pathway (see Fig. S1 in the supplemental material), in which two molecules of CO 2 are initially converted into acetyl-CoA by reduction via formate dehydrogenase and CO dehydrogenase/acetyl-CoA synthase (98). However, the reductive acetyl-CoA pathway may contribute up to only 2% of the global carbon fixation (119).…”

“…However, the reductive acetyl-CoA pathway may contribute up to only 2% of the global carbon fixation (119). Moreover, transformation of the initial acetyl-CoA fixation product into more complex molecular compounds through this pathway in fact requires a carboxylation reaction (via pyruvate:ferredoxin oxidoreductase; see below) such that, in organisms using the reductive acetyl-CoA pathway, approximately five-sixths of the carbon is fixed via CO 2 reduction and one-sixth is fixed via carboxylation (54,98,100).…”

Syngas Fermentation: A Microbial Conversion Process of Gaseous Substrates to Various Products

“…In the water-gas shift reaction in Equation (17), CO is oxidized to CO 2 , and H + is reduced to H 2 . The two half-reactions are shown in Equations (23) and (24).…”

“…The acetyl-CoA pathway is also referred to as the Wood-Ljungdahl pathway of autotrophic growth. A detailed description of the history of the discovery of acetogenesis is given in a review [2], and the enzymology is reviewed by Ragsdale [24].…”

“…The acetyl-CoA pathway has been defined over 70 years of research [24,56] and is shown in Figure 2. Energy and carbon from syngas are used to produce acetyl-CoA.…”