The fixation of inorganic carbon into organic material (autotrophy) is a prerequisite for life and sets the starting point of biological evolution. In the extant biosphere the reductive pentose phosphate (Calvin-Benson) cycle is the predominant mechanism by which many prokaryotes and all plants fix CO₂ into biomass. However, the fact that five alternative autotrophic pathways exist in prokaryotes is often neglected. This bias may lead to serious misjudgments in models of the global carbon cycle, in hypotheses on the evolution of metabolism, and in interpretations of geological records. Here, I review these alternative pathways that differ fundamentally from the Calvin-Benson cycle. Revealingly, these five alternative pathways pivot on acetyl-coenzyme A, the turntable of metabolism, demanding a gluconeogenic pathway starting from acetyl-coenzyme A and CO₂. It appears that the formation of an activated acetic acid from inorganic carbon represents the initial step toward metabolism. Consequently, biosyntheses likely started from activated acetic acid and gluconeogenesis preceded glycolysis.

Aromatic compounds constitute the second most abundant class of organic substrates and environmental pollutants, a substantial part of which (e.g., phenylalanine or styrene) is metabolized by bacteria via phenylacetate. Surprisingly, the bacterial catabolism of phenylalanine and phenylacetate remained an unsolved problem. Although a phenylacetate metabolic gene cluster had been identified, the underlying biochemistry remained largely unknown. Here we elucidate the catabolic pathway functioning in 16% of all bacteria whose genome has been sequenced, including Escherichia coli and Pseudomonas putida . This strategy is exceptional in several aspects. Intermediates are processed as CoA thioesters, and the aromatic ring of phenylacetyl-CoA becomes activated to a ring 1,2-epoxide by a distinct multicomponent oxygenase. The reactive nonaromatic epoxide is isomerized to a seven-member O-heterocyclic enol ether, an oxepin. This isomerization is followed by hydrolytic ring cleavage and β-oxidation steps, leading to acetyl-CoA and succinyl-CoA. This widespread paradigm differs significantly from the established chemistry of aerobic aromatic catabolism, thus widening our view of how organisms exploit such inert substrates. It provides insight into the natural remediation of man-made environmental contaminants such as styrene. Furthermore, this pathway occurs in various pathogens, where its reactive early intermediates may contribute to virulence.

The phototrophic bacterium Chloroflexus aurantiacus uses a yet unsolved 3-hydroxypropionate cycle for autotrophic CO ₂ fixation. It starts from acetyl-CoA, with acetyl-CoA and propionyl-CoA carboxylases acting as carboxylating enzymes. In a first cycle, ( S )-malyl-CoA is formed from acetyl-CoA and 2 molecules of bicarbonate. ( S )-Malyl-CoA cleavage releases the CO ₂ fixation product glyoxylate and regenerates the starting molecule acetyl-CoA. Here we complete the missing steps devoted to glyoxylate assimilation. In a second cycle, glyoxylate is combined with propionyl-CoA, an intermediate of the first cycle, to form β-methylmalyl-CoA. This condensation is followed by dehydration to mesaconyl-C1-CoA. An unprecedented CoA transferase catalyzes the intramolecular transfer of the CoA moiety to the C4 carboxyl group of mesaconate. Mesaconyl-C4-CoA then is hydrated by an enoyl-CoA hydratase to ( S )-citramalyl-CoA. ( S )-Citramalyl-CoA is cleaved into acetyl-CoA and pyruvate by a tri-functional lyase, which previously cleaved ( S )-malyl-CoA and formed β-methylmalyl-CoA. Thus, the enigmatic disproportionation of glyoxylate and propionyl-CoA into acetyl-CoA and pyruvate is solved in an elegant and economic way requiring only 3 additional enzymes. The whole bicyclic pathway results in pyruvate formation from 3 molecules of bicarbonate and involves 19 steps but only 13 enzymes. Elements of the 3-hydroxypropionate cycle may be used for the assimilation of small organic molecules. The 3-hydroxypropionate cycle is compared with the Calvin–Benson–Bassham cycle and other autotrophic pathways.