A new amidohydrolase and β-oxidation–like pathway for piperine catabolism in soil actinomycetes

Understanding the microbial degradation of plant-derived specialized metabolites is important for gaining insight into plant microbial diseases. Piperine is a unique alkaloid found in peppers and plays a protective role essential for pepper tree growth. However, how environmental microbiomes interact with pepper trees, especially via the interaction with piperine, has remained completely unknown. To elucidate microbial piperine metabolism, we previously screened Rhodococcus ruber No. 14, an actinomycete that catabolizes piperine from a surface soil sample containing detritus. In this actinomycete, here, we discovered a piperine hydrolase (named PipM) that catalyzes the hydrolysis of the tertiary amide bond in piperine. While no enzyme had been reported to hydrolyze the piperine-type tertiary amide bond, PipM showed amino acid sequence similarity to amidohydrolase family enzymes and, like them, required metal ions for its activity. The mRNA-Seq and genome analysis revealed a gene cluster of 48 genes, among which 42 genes, including pipM, were upregulated by adding piperine into the culture medium. Through gene expression and biochemical analysis, four genes (named pipU, V, W, and X) were identified to encode enzymes that catalyze the β-oxidation-like reaction to convert piperic acid to piperonylic acid. Our study unraveled microbial piperine metabolism and would provide insight into interactions between pepper trees and environmental microbiomes.