Abstract
Carbonic anhydrase catalyses the interconversion of carbon dioxide and water to bicarbonate and protons. It was unknown if the industrial-relevant acetogen Clostridium autoethanogenum possesses these enzymes. We identified two putative carbonic anhydrase genes in its genome, one of the β class and one of the γ class. Carbonic anhydrase activity was found for the purified β class enzyme, but not the γ class candidate. Functional complementation of an Escherichia coli carbonic anhydrase knock-out mutant showed that the β class carbonic anhydrase could complement this activity, but not the γ class candidate gene. Phylogenetic analysis showed that the β class carbonic anhydrase of Clostridium autoethanogenum represents a novel sub-class of β class carbonic anhydrases that form the F-clade. The members of this clade have the shortest primary structure of any known carbonic anhydrase.
Highlights
Clostridium autoethanogenum fixes carbon dioxide through the Wood–Ljungdahl pathway (WLP) and produces acetate, ethanol and 2,3-butanediol natively (Abrini et al 1994; Köpke et al 2011)
We assembled consensus sequences of the α, β- and γCA classes and used amino acid sequences and the assembled consensus sequences to search the genome of C. autoethanogenum
To assess carbonic anhydrase (CA) activity for the proteins encoded by these genes, these were heterologously expressed in a Can disruption mutant of E. coli
Summary
Clostridium autoethanogenum fixes carbon dioxide through the Wood–Ljungdahl pathway (WLP) and produces acetate, ethanol and 2,3-butanediol natively (Abrini et al 1994; Köpke et al 2011). Besides capture in the WLP, CO2 is fixed at other metabolic steps, and it was shown, for instance, that elevated CO2 partial pressures benefit the production of 2,3-butanediol (Simpson et al 2014). Many reactions in microbial metabolism exist where CO2 or bicarbonate are substrates or products (Smith and Ferry 2000). It was proposed that without a mechanism for the rapid interconversion of carbon dioxide and bicarbonate, the turnover rates of common carboxylation reactions that consume bicarbonate would not be feasible in Escherichia coli (Merlin et al 2003). To optimise product formation and carbon fixation, knowledge about CA activity is important (Hawkins et al 2013; Lian et al 2016)
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