Retrobiosynthetic analysis of carbon fixation in the phototrophic eubacterium Chloroflexus aurantiacus.

The phototrophic bacterium Chloroflexus aurantiacus does not use any of the known autotrophic CO2 fixation pathways. There is evidence for a new cyclic autotrophic pathway in which acetyl-CoA is converted to 3-hydroxypropionate and further to succinate and malate. This hypothesis was tested by feeding growing cultures during several generations with 3-hydroxy[1-13C]propionate, [1-13C]acetate, or [2-13C]acetate, in addition to unlabeled CO2. The relative 13C content of individual carbon atoms in biosynthetic amino acids and nucleosides was determined by 1H- and 13C-NMR spectroscopy. 13C coupling patterns were analyzed by two-dimensional 13C-TOCSY experiments which were optimized for the analysis of multiply 13C-labeled biosynthetic samples. From the 13C enrichments of amino acids and nucleosides, the labeling patterns of central metabolic intermediates were evaluated by a retrobiosynthetic approach. Both 3-hydroxypropionate and acetate were incorporated into all central metabolic pools. The 13C labeling and coupling patterns suggest a novel carbon fixation pathway via 3-hydroxypropionate. Specifically, we propose that acetyl-CoA is carboxylated to malonyl-CoA which is reduced under formation of 3-hydroxypropionyl-CoA. Dehydration and reduction yield propionyl-CoA which is converted to succinate by a second carboxylation reaction. The net product of autotrophic carbon fixation appears to be glyoxylate. However, it is not yet known how glyoxylate is channeled into anabolic metabolism. Assimilation of acetate can proceed via the CO2 fixation pathway, but also via the glyoxylate pathway.