Engineering Komagataella phaffii to biosynthesize cordycepin from methanol which drives global metabolic alterations at the transcription level

Cordycepin has the potential to be an alternative to the disputed herbicide glyphosate. However, current laborious and time-consuming production strategies at low yields based on Cordyceps militaris lead to extremely high cost and restrict its application in the field of agriculture. In this study, Komagataella phaffii (syn. Pichia pastoris) was engineered to biosynthesize cordycepin from methanol, which could be converted from CO2. Combined with fermentation optimization, cordycepin content in broth reached as high as 2.68 ± 0.04 g/L within 168 h, around 15.95 mg/(L·h) in productivity. Additionally, a deaminated product of cordycepin was identified at neutral or weakly alkaline starting pH during fermentation. Transcriptome analysis found the yeast producing cordycepin was experiencing severe inhibition in methanol assimilation and peroxisome biogenesis, responsible for delayed growth and decreased carbon flux to pentose phosphate pathway (PPP) which led to lack of precursor supply. Amino acid interconversion and disruption in RNA metabolism were also due to accumulation of cordycepin. The study provided a unique platform for the manufacture of cordycepin based on the emerging non-conventional yeast and gave practical strategies for further optimization of the microbial cell factory. [Display omitted]