Aspergillus niger uses the peroxisomal CoA-dependent ß-oxidative genes to degrade the hydroxycinnamic acids caffeic acid, ferulic acid, and p-coumaric acid.

Aromatic compounds are important molecules which are widely applied in many industries and are mainly produced from nonrenewable sources. Renewable sources such as plant biomass are interesting alternatives for the production of aromatic compounds. Ferulic acid and p-coumaric acid, a precursor for vanillin and p-vinyl phenol, respectively, can be released from plant biomass by the fungus Aspergillus niger. The degradation of hydroxycinnamic acids such as caffeic acid, ferulic acid, and p-coumaric acid has been observed in many fungi. In A. niger, multiple metabolic pathways were suggested for the degradation of hydroxycinnamic acids. However, no genes were identified for these hydroxycinnamic acid metabolic pathways. In this study, several pathway genes were identified using whole-genome transcriptomic data of A. niger grown on different hydroxycinnamic acids. The genes are involved in the CoA-dependent ß-oxidative pathway in fungi. This pathway is well known for the degradation of fatty acids, but not for hydroxycinnamic acids. However, in plants, it has been shown that hydroxycinnamic acids are degraded through this pathway. We identified genes encoding hydroxycinnamate-CoA synthase (hcsA), multifunctional ß-oxidation hydratase/dehydrogenase (foxA), 3-ketoacyl CoA thiolase (katA), and four thioesterases (theA-D) of A. niger, which were highly induced by all three tested hydroxycinnamic acids. Deletion mutants revealed that these genes were indeed involved in the degradation of several hydroxycinnamic acids. In addition, foxA and theB are also involved in the degradation of fatty acids. HcsA, FoxA, and KatA contained a peroxisomal targeting signal and are therefore predicted to be localized in peroxisomes. KEY POINTS: • Metabolism of hydroxycinnamic acid was investigated in Aspergillus niger • Using transcriptome data, multiple CoA-dependent ß-oxidative genes were identified. • Both foxA and theB are involved in hydroxycinnamate but also fatty acid metabolism.

Evolutionary adaptation of Aspergillus niger for increased ferulic acid tolerance.

AIMS: To create an Aspergillus niger mutant with increased tolerance against ferulic acid using evolutionary adaptation. METHODS AND RESULTS: Evolutionary adaptation of A. niger N402 was performed by consecutive growth on increasing concentrations of ferulic acid in the presence of 25 mmol l-1 d-fructose, starting from 0·5 mmol l-1 and ending with 5 mmol l-1 ferulic acid. The A. niger mutant obtained after six months, named Fa6, showed increased ferulic acid tolerance compared to the parent. In addition, Fa6 has increased ferulic acid consumption and a higher conversion rate, suggesting that the mutation affects aromatic metabolism of this species. Transcriptome analysis of the evolutionary mutant on ferulic acid revealed a distinct gene expression profile compared to the wild type. Further analysis of this mutant and the parent strain provided the first experimental confirmation that A. niger converts coniferyl alcohol to ferulic acid. CONCLUSIONS: The evolutionary adaptive A. niger mutant Fa6 has beneficial mutations that increase the tolerance, conversion rate and uptake of ferulic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that evolutionary adaptation is a powerful tool to modify micro-organisms towards increased tolerance to harsh conditions, which is beneficial for various industrial applications.