Pyruvate Carboxylase-Mediated Regulation of Aspergillus flavus Development and Aflatoxin Biosynthesis

Pyruvate carboxylase (PYC), a multifunctional biotin-dependent enzyme, is essential in diverse metabolic cascades including gluconeogenesis and the tricarboxylic acid (TCA) cycle. This study aimed to dissect the physiological roles of PYC and its succinylated site in Aspergillus flavus (A. flavus). In A. flavus, biological functions of PYC were characterized through gene deletion and site-directed mutagenesis. Phenotypic analysis and qRT-PCR demonstrated that PYC positively regulated sporulation and sclerotial formation, while negatively regulating aflatoxin B1 biosynthesis. The transcriptomics revealed that development-related DEGs of abaA, wetA, vosA, atfB, creA, crzA, and areA were downregulated, whereas veA and are2 were increased in Δpyc1. Meanwhile, aflatoxin-related DEGs of nor1, aflW, agiB, aflO, and aflM were upregulated, while cfoC and ucdC were lower in Δpyc1. In addition, a succinylated site on PYC (K1068) was characterized by noncovalent interactions. Combined with the transcriptomics analysis of this site, this study revealed that DEGs in pyc1K1068R (mimic desuccinylation) were enriched in fundamental metabolic pathways of glycolysis/gluconeogenesis, carbon metabolism, fructose and mannose metabolism, pentose phosphate pathway, etc., which mainly affected A. flavus development. This research provided insights into the regulatory mechanisms underlying A. flavus development and mycotoxin contamination.