Bacteria and Fungi Synergistically Reprogram Flavonoid Metabolites in the Pericarp of <i>Citrus Reticulata</i> 'Chachi' During Storage

The mature pericarp of Citrus reticulata ' Chachi ' (PCRC) is a traditional Chinese medicine known for its enhanced efficacy through long-term storage and processing. However, the specific mechanisms underlying these enhancements remain unclear. This study employed widely targeted metabolomics, microbial amplicon sequencing, and fermentation assays to investigate the microbiome 's influence on PCRC 's flavonoid profile over 0-19 years of storage. Correlation analysis revealed that the accumulation of polymethoxyflavones (PMFs) was closely linked to specific bacterial and fungal communities. Solid-state fermentation showed that Bacillus subtilis N18-1 enhanced the content of certain PMFs, while Aspergillus tubingensis P21-1 reduced them. Liquid-state assays confirmed that A. tubingensis P21-1 converted nobiletin to 3 ' -demethylnobiletin, and B. subtilis N18-1 further converted this to tangeretin. Combined with genome sequencing and molecular docking, four candidate genes were identified. The catalytic activity verification assays demonstrated that At21-68 and At21-21 catalyze the conversion of nobiletin to 3 '-demethylnobiletin, while Bs18-51 and Bs18-84 catalyze the conversion of 3 ' -demethylnobiletin to tangeretin. These findings highlight the synergistic molecular mechanism by which microorganisms modulate PMFs during PCRC aging, providing insights for optimizing medicinal plant aging through microbial biotransformation.