Rational Engineering of Patchoulene Synthase from Pogostemon cablin for Enhanced Patchoulene Production

Patchoulene, the characteristic sesquiterpene of patchouli essential oil, is highly valued in the perfume industry for its distinctive woody note and fixative properties. Beyond its olfactory applications, patchoulene has demonstrated promising biological activities, including anti-inflammatory, antimicrobial, and neuroprotective effects. Current production relies mainly on extraction from <i>Pogostemon cablin</i> plants, which requires long growth cycles (≥8 months), exhibits low yields, and imposes significant environmental constraints. To overcome these limitations, this study aimed to enhance the Whole-cell yield of patchoulene synthase (PcPTS) through structure-informed protein engineering. A semi-rational design approach was employed, combining homology modeling, molecular docking, evolutionary analysis, and molecular dynamics simulations to identify functional residues within the enzyme active site. Ala-scanning mutagenesis highlighted Thr532 as essential for catalytic activity, and coevolutionary analysis indicated synergistic effects between Phe456 and Thr532. Site-directed mutagenesis was conducted to generate single (F456M, T532Y) and double (F456M/T532Y, designated M2) mutants. The double mutant M2 showed a 3.62-fold increase in patchoulene production compared to the wild-type enzyme. In silico analyses suggested that the enhanced performance of M2 originates from improved substrate positioning, reduced structural flexibility, and strengthened molecular interactions, collectively contributing to a lower energy barrier for catalysis. This study provides an effective strategy for the rapid optimization of terpenoid synthases and facilitates the development of microbial cell factories for sustainable and high-yield production of plant-derived terpenoids.