Advancing Terpenoid Biosynthesis in Saccharomyces cerevisiae: Synthetic Biology Approaches for Efficient Longifolene Production.

Abstract

The sustainable microbial production of high-value terpenoids like longifolene represents a critical frontier in green manufacturing, yet it is often hindered by systemic metabolic inefficiencies in microbial hosts. To address this, we present a comprehensive systems metabolic engineering framework in Saccharomyces cerevisiae that integrates three complementary modules to overcome the key bottlenecks of enzyme catalysis, cofactor supply, and precursor availability. First, we enhanced the catalytic module by employing the RII dimerization/docking domain-RI anchoring disruptor (RIDD-RIAD) peptide-mediated enzyme assembly and structure-guided Pinus sylvestris longifolene synthase (psTPS) variant screening, `which improved whole-cell longifolene production under the tested conditions. Second, to resolve nicotinamide adenine dinucleotide phosphate (NADPH) cofactor imbalance, we engineered a dual-regeneration system by introducing an exogenous formate dehydrogenase (FDH) and simultaneously amplifying endogenous pentose phosphate pathway (PPP) flux. Third, we reprogrammed central carbon metabolism by integrating the non-oxidative glycolysis (NOG) pathway and deleting the transcriptional repressors DOS2 and YNR063W, and was associated with a larger steady-state cytosolic acetyl-CoA pool, the essential terpenoid precursor. This multi-pronged strategy culminated in a highly optimized strain that achieved a titer of 92.84 mg/L in shake-flask cultures. Critically, the process was successfully scaled to a 5 L bioreactor under fed-batch conditions, yielding 4181.51 mg/L total longifolene at 204 h (3484.79 mg/L extracellular and 696.72 mg/L cell-associated), the highest total titer among the microbial reports identified in Supplementary Table S4. Differences in product recovery and cultivation protocols limit direct process comparisons. Our work demonstrates that coordinated optimization of enzyme assembly, cofactor regeneration, and carbon-flow reprogramming can substantially improve longifolene biosynthesis in S. cerevisiae. This study provides a longifolene-focused case study for modular pathway optimization, while broader application of this strategy to other terpenoids or natural products will require further pathway-specific validation.