Evolutionary engineering of Pichia pastoris for α-farnesene biosynthesis from methanol

Abstract

α-Farnesene is a valuable sesquiterpene with wide applications in cosmetics, biofuels, and pharmaceuticals. Microbial production of α-farnesene from low-cost carbon sources represents a sustainable alternative to chemical synthesis. In this study, Pichia pastoris was engineered to efficiently produce α-farnesene using methanol as the sole carbon source. First, the native mevalonate pathway was systematically optimized by stepwise overexpression of ERG10, ERG13, a truncated HMG1, and key downstream enzymes, resulting in P. pastoris cfn3 with α-farnesene production up to 172.1 mg/L. Then, adaptive laboratory evolution was conducted to improve methanol tolerance, and the evolved strain P. pastoris evo13-4 grew more rapidly than that of P. pastoris cfn3. To further boost α-farnesene production, ARTP mutagenesis was used to generate numerous mutants for screening strains with high performance, among which P. pastoris ccg3-8 exhibited the highest production of α-farnesene up to 449.4 mg/L. Finally, in a 5-L bioreactor, α-farnesene production with the resulting strain P. pastoris ccg3-8 reached 3.28 g/L. This study presents an effective strategy for engineering P. pastoris for methanol-based biosynthesis of α-farnesene, providing a promising platform for the effective and sustainable production of isoprenoid compounds.

Keywords

Pichia pastoris / Α-farnesene / Metabolic engineering / Evolutionary engineering

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