Carbaryl is a widely used carbamate pesticide in agriculture. The strain Rhizobium sp. X9 possesses the typical carbaryl degradation pathway in which carbaryl is mineralized via 1-naphthol, salicylate, and gentisate. In this study, we cloned a carbaryl hydrolase gene cehA and a novel two-component 1-naphthol hydroxylase gene cehC1C2. CehA mediates carbaryl hydrolysis to 1-naphthol and CehC1, an FMNH 2 or FADH 2 -dependent monooxygenase belonging to the HpaB superfamily, and hydroxylates 1-naphthol in the presence of reduced nicotinamide-adenine dinucleotide (FMN)/flavin adenine dinucleotide (FAD), and the reductase CehC2. CehC1 has the highest amino acid similarity (58%) with the oxygenase component of a two-component 4nitrophenol 2-monooxygenase, while CehC2 has the highest amino acid similarity (46%) with its reductase component. CehC1C2 could utilize both FAD and FMN as the cofactor during the hydroxylation, although higher catalytic activity was observed with FAD as the cofactor. The optimal molar ratio of CehC1 to CehC2 was 2:1. The K m and K cat /K m values of CehC1 for 1-naphthol were 74.71 ± 16.07 μM and (8.29 ± 2.44) × 10 −4 s −1 •μM −1 , respectively. Moreover, the enzyme activities and substrate spectrum between CehC1C2 and previously reported 1-naphthol hydroxylase McbC were compared. The results suggested that McbC had a higher 1-naphthol hydroxylation activity, while CehC1C2 had a broader substrate spectrum.

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Shiitake mushroom, Lentinula edodes, is the second largest edible fungus in the world, with a characteristic aroma. 1,2,3,5,6-pentathioheterocycloheptane, commonly known as lenthionine, is the main source of this aroma. Lenthionine has high commercial value, and if we explore the possible induction mechanism of citric acid in lenthionine synthesis, we can provide a reference for the effective application of citric acid as an inducer. In this paper, the single-factor treatment of Lentinula edodes with variable citric acid concentration and treatment duration showed that the best citric acid concentration for L. edodes was 300 μM, and the best treatment duration was 15 days. Additionally, the optimal design conditions were obtained using the response surface method (RSM); the treatment concentration was 406 μM/L, the treatment duration was 15.6 days, and the lenthionine content was 130 μg/g. γ-Glutamyl transpeptidase (LEGGT) and cystine sulfoxide lyase (LECSL) are the key enzymes involved in the biosynthesis of lanthionine. The expression levels of LEGGT and LECSL genes increased significantly under citric acid treatment. Additionally, the lenthionine content of the silenced strains of LEGGT and LECSL was significantly decreased.