De Novo Biosynthesis of cis -1,4-Polyisoprene from Methanol in Pichia pastoris via Systematic Metabolic Engineering

Abstract cis-1,4-Polyisoprene, the primary component of natural rubber, is a critical industrial polymer traditionally sourced from Hevea brasiliensis or petroleum. Microbial production offers a sustainable alternative, but achieving efficient synthesis and high molecular weight remains challenging. Here, we report the de novo biosynthesis of cis-1,4-polyisoprene in Pichia pastoris (P. pastoris) using methanol as a carbon source. Our systematic metabolic engineering strategy featured the structure-guided selection of HbHMGR2 to amplify precursor supply, the strategic tuning of initiators to identify FPP as the primary unit, and the assembly of a heterologous rubber biosynthetic complex by co-expressing REF1 and SRPP1. This integrated approach elevated the titer to 87.29 mg/L in fed-batch fermentation and substantially increased the polymer molecular weight to ∼10,000 Da, representing one of the highest molecular weights achieved heterologously in a non-model microorganism. This work establishes P. pastoris as a potent C1-based biorefinery platform for sustainable polymer production, paving the way for bio-based rubber synthesis.