Rad6　is　a　canonical　ubiquitin-conjugating　enzyme　known　for　its　role　in　regulating　chromosome-related　cellular　processes　in　yeast　and　has　been　proven　to　have　multiple　functions　in　Beauveria　bassiana,　including　insect-pathogenic　lifestyle,　UV　damage　repair,　and　conidiation.　However,　previous　studies　have　only　reported　the　key　role　of　Rad6　in　regulating　conidial　production　in　a　nutrient-rich　medium,　without　any　deep　mechanism　analyses.　In　this　study,　we　found　that　the　disruption　of　Rad6　leads　to　a　profound　reduction　in　conidial　production,　irrespective　of　whether　the　fungus　is　cultivated　in　nutrient-rich　or　nutrient-poor　environments.　The　absence　of　rad6　exerts　a　suppressive　effect　on　the　transcription　of　essential　genes　in　the　central　developmental　pathway,　namely,　brlA,　abaA,　and　wetA,　resulting　in　a　direct　downregulation　of　conidiation　capacity.　Additionally,　mutant　strains　exhibited　a　more　pronounced　decline　in　both　conidial　generation　and　hyphal　development　when　cultured　in　nutrient-rich　conditions.　This　observation　correlates　with　the　downregulation　of　the　central　developmental　pathway　(CDP)　downstream　gene　vosA　and　the　upregulation　of　flaA　in　nutrient-rich　cultures.　Moreover,　single-transcriptomics　analyses　indicated　that　irregularities　in　biotin　metabolism,　DNA　repair,　and　tryptophan　metabolism　are　the　underlying　factors　contributing　to　the　reduced　conidial　production.　Comprehensive　dual　transcriptomics　analyses　pinpointed　abnormal　biotin　metabolism　as　the　primary　cause　of　conidial　production　decline.　Subsequently,　we　successfully　restored　conidial　production　in　the　Rad6　mutant　strain　through　the　supplementation　of　biotin,　further　confirming　the　transcriptomic　evidence.　Altogether,　our　findings　underscore　the　pivotal　role　of　Rad6　in　influencing　biotin　metabolism,　subsequently　impacting　the　expression　of　CDP　genes　and　ultimately　shaping　the　asexual　life　cycle　of　B.　bassiana.　©　2024　by　the　authors.