Insights into the Degradation of Medium-Chain-Length Dicarboxylic Acids in Cupriavidus necator H16 Reveal β-Oxidation Differences between Dicarboxylic Acids and Fatty Acids.

Many homologous genes encoding β-oxidation enzymes have been found in the genome of Cupriavidus necator H16 (synonym Ralstonia eutropha H16). By proteome analysis, the degradation of adipic acid was investigated and showed differences from the degradation of hexanoic acid. During β-oxidation of adipic acid, activation with coenzyme A (CoA) is catalyzed by the two-subunit acyl-CoA ligase encoded by B0198 and B0199. The operon is completed by B0200 encoding a thiolase catalyzing the cleavage of acetyl-CoA at the end of the β-oxidation cycle. C. necator ΔB0198-B0200 strain showed improved growth on adipic acid. Potential substitutes are B1239 for B0198-B0199 and A0170 as well as A1445 for B0200. A deletion mutant without all three thiolases showed diminished growth. The deletion of detected acyl-CoA dehydrogenase encoded by B2555 has an altered phenotype grown with sebacic acid but not adipic acid. With hexanoic acid, acyl-CoA dehydrogenase encoded by B0087 was detected on two-dimensional (2D) gels. Both enzymes are active with adipoyl-CoA and hexanoyl-CoA as substrates, but specific activity indicates a higher activity of B2555 with adipoyl-CoA. 2D gels, growth experiments, and enzyme assays suggest the specific expression of B2555 for the degradation of dicarboxylic acids. In C. necator H16, the degradation of carboxylic acids potentially changes with an increasing chain length. Two operons involved in growth with long-chain fatty acids seem to be replaced during growth on medium-chain carboxylic acids. Only two deletion mutants showed diminished growth. Replacement of deleted genes with one of the numerous homologous is likely. The biotechnologically interesting bacterium Cupriavidus necator H16 has been thoroughly investigated. Fifteen years ago, it was sequenced entirely and annotated (A. Pohlmann, W. F. Fricke, F. Reinecke, B. Kusian, et al., Nat Biotechnol 24:1257-1262, 2006, https://doi.org/10.1038/nbt1244). Nevertheless, the degradation of monocarboxylic fatty acids and dicarboxylic acids has not been elucidated completely. C. necator is used to produce value-added products from affordable substrates. One of our investigations' primary targets is the biotechnological production of organic acids with different and specific chain lengths. The versatile metabolism of carboxylic acids recommends C. necator H16 as a candidate for producing value-added organic products. Therefore, the metabolism of these compounds is of interest, and, for different applications in industry, understanding such central metabolic pathways is crucial.