Constructing CRISPR-Cas9 system for metabolic reprogramming and cordycepin biomanufacturing in Pichia pastoris.

Cordycepin, a nucleoside analog mainly produced byCordyceps militaris, is widely used in food, medicine, and feed industries.Conventional microbial engineering faces challenges from antibiotic resistance genes, which increase environmental risks.Here, we engineeredPichia pastorisusing an optimized CRISPR-Cas9 system with gRNA-tRNA array and Brex27-enhanced homologous recombination, achieving antibiotic marker-free cordycepin biosynthesis. Through modular metabolic engineering strategies that optimized promoter combinations, gene copy numbers, methanol assimilation, precursor supply, and ATP/NADPH balance, strain PC19 achieved 2509.7 mg/L cordycepin in shake-flask fermentation. In fed-batch fermentation, PC19 achieved the highest production of 18.3 g/L (3.05 g/L/d and 122.2 mg/g DCW) to date in a 10 L bioreactor, and the CO2-eq emissions were 3.3-57.6 times lower than C. militaris and other microbial cell factories. This CRISPR-Cas9 system lays the foundation for low-carbon and efficient biosynthesis of cordycepin and other nucleoside analogs inP. pastoris.