RETROBIOSYNTHETIC ANALYSIS OF CARBON FIXATION IN THE PHOTOTROPHIC EUBACTERIUM CHLOROFLEXUS-AURANTIACUS

The phototrophic bacterium Chloroflexus aurantiacus does not use any o f 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 wit h 3-hydroxy[1-C-13]propionate, [1-C-13]acetate, or [2-C-13]acetate, in addition to unlabeled CO2. The relative C-13 content of individual ca rbon atoms in biosynthetic amino acids and nucleosides was determined by H-1- and C-13-NMR spectroscopy. C-13 coupling patterns were analyze d by two-dimensional C-13-TOCSY experiments which were optimized for t he analysis of multiply C-13-labeled biosynthetic samples. From the C- 13 enrichments of amino acids and nucleosides, the labeling patterns o f central metabolic intermediates were evaluated by a retrobiosyntheti c approach. Both 3-hydroxypropionate and acetate were incorporated int o all central metabolic pools. The C-13 labeling and coupling patterns suggest a novel carbon fixation pathway via 3-hydroxypropionate. Spec ifically, we propose that acetyl-CoA is carboxylated to malonyl-CoA wh ich 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 gl yoxylate is channeled into anabolic metabolism. Assimilation of acetat e can proceed via the CO2 fixation pathway, but also via the glyoxylat e pathway.