Chitosan Increases Lysine Content through Amino Acid Transporters in Flammulina filiformis

HIGHLIGHTS: Chitosan treatment increased the lysine content of F. filiformis. Results from this study provide insights for industrial-scale trials potentially resulting in higher-quality F. filiformis. Response surface methodology (RSM) was used to optimize the conditions of chitosan-induced amino acid content in F. filiformis for the first time. The amino acid transporters in F. filiformis were systematically screened for the first time. The involvement of AAT3 and AAT4 genes in chitosan-induced lysine biosynthesis in F. filiformis was investigated by gene-silenced strains. ABSTRACT: Lysine content is considered an important indicator of the quality of Flammulina filiformis. In this study, chitosan was used to improve lysine content of F. filiformis. Optimal design conditions were obtained using central combination design (CCD): treatment concentration was 14.61 μg/mL, treatment time was 52.90 h, and the theoretical value of lysine content was 30.95 mg/g. We used Basic Local Alignment Search Tool Protein (BLASTP) to search the F. filiformis genome database using known AATs in the NCBI database. There were 11 members of AAT in F. filiformis. The expression levels of AAT3 and AAT4 genes increased significantly with chitosan treatment. Subsequently, AAT3 and AAT4 silencing strains were constructed using RNAi technology. The lysine content of the wild-type (WT) strain treated with chitosan increased by 26.41%. Compared with the chitosan-induced WT strain, chitosan-induced lysine content decreased by approximately 24.87% in the AAT3 silencing strain, and chitosan-induced lysine content in the AAT4 silencing strain increased by approximately 13.55%. The results indicate that AAT3 and AAT4 are involved in the regulation of the biosynthesis of lysine induced by chitosan in F. filiformis. AAT3 may participate in the absorption of lysine, and AAT4 may be involved in the excretion of lysine with chitosan treatment.