Development of a Pichia pastoris cell factory for efficient production of germacrene A: a precursor of β-elemene

Abstract

Abstractβ-Elemene, an active ingredient found in medicinal plants like turmeric and zedoary, is a sesquiterpene compound with antitumor activity against various cancers. However, its current mode of production through plant extraction suffers from low efficiency and limited natural resources. Recently, there has been an increased interest in establishing microbial cell factories to produce germacrene A, which can be converted to β-elemene by a one-step reaction in vitro. In this study, we constructed an engineered Pichia pastoris cell factory for producing germacrene A. We rerouted the fluxes towards germacrene A biosynthesis through the optimization of the linker sequences between germacrene A synthase (GAS) and farnesyl pyrophosphate synthase (ERG20), overexpression of important pathway genes (i.e., IDI1, tHMG1, and ACS), and multi-copy integration of related expression cassettes. In combination with medium optimization and bioprocess engineering, the final titer of germacrene A in a 1 L fermenter reached 1.9 g/L through fed-batch fermentation. This represents the first report on the production of germacrene A in P. pastoris and demonstrates its advantage in producing terpenoids and other value-added natural products. Graphical Abstract

Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris

“…The establishment of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐CRISPR‐associated System (CRISPR/Cas) (Gao et al, 2021; Gu et al, 2019) and the characterization of a panel of neutral integration sites (Cai et al, 2021; Gao et al, 2022) have positioned P. pastoris as an attractive chassis for the biosynthesis of bulk chemicals and natural products. Recent successes include the biosynthesis of α‑santalene (Zuo et al, 2022), α‑farnesene (Liu et al, 2021), lycopene (Bhataya et al, 2009), germacrene A (Cheng et al, 2023), and the anticancer drug precursor catharanthine (Cheng et al, 2023), which involves more than 30 enzymes.…”