Pichia pastoris is greatly used as a protein expression system based on tightly regulated aldehyde oxidase 1 (AOX1) promoter (P-AOX1) that is induced by methanol; however, the use of methanol of highly expensive and dangerous. By deleting transcription repressors and overexpressing transcription activator of the P-AOX1, a methanol-independent strain MF1 (Delta mig1 Delta mig2 Delta nrg1-Mit1) has been previously constructed. This study investigated the transcriptomic and metabolomic profiles of the MF1 strain in response to different carbon sources, including methanol, glucose, and glycerol, in comparison with the wild-type strain. AOX expression was observed in all groups except the wild-type strains cultured in glucose and glycerol. Genes involved in methanol utilization and peroxisome biosynthesis were mainly upregulated in the mutant strain grown on glucose or glycerol. Metabolomics data showed significant increase of the products related to metabolic pathways in amino acid and energy metabolism in the mutant strain grown in glycerol. Genes such as glutamic acid decarboxylase (GAD) and some metabolites were both involved in the pathways such as GABAergic synapse. GAD1, GAD2, and succinate may be associated with the recombinant protein expression system of the mutant MF1 strain of P. pastoris grown on glucose or glycerol.