Numerous kaurene synthase-like cyclases have been identified in Isodon rubescens, providing new perspectives into understanding the evolution of diterpenoid che

Borneol is a precious monoterpenoid with two chiral structures, (-)-borneol and (+)-borneol. Bornyl diphosphate synthase is the key enzyme in the borneol biosynthesis pathway. Many (+)-bornyl diphosphate synthases have been reported, but no (-)-bornyl diphosphate synthases have been identified. Blumea balsamifera leaves are rich in borneol, almost all of which is (-)-borneol. In this study, we identified a high-efficiency (-)-bornyl diphosphate synthase (BbTPS3) from B. balsamifera that converts geranyl diphosphate (GPP) to (-)-bornyl diphosphate, which is then converted to (-)-borneol after dephosphorylation in vitro. BbTPS3 exhibited a K_m value of 4.93 ± 1.38 μM for GPP, and the corresponding k_cat value was 1.49 s⁻¹. Multiple strategies were applied to obtain a high-yielding (-)-borneol producing yeast strain. A codon-optimized BbTPS3 protein was introduced into the GPP high-yield strain MD, and the resulting MD-B1 strain produced 1.24 mg·L^-1 (-)-borneol. After truncating the N-terminus of BbTPS3 and adding a Kozak sequence, the (-)-borneol yield was further improved by 4-fold to 4.87 mg·L^-1. Moreover, the (-)-borneol yield was improved by expressing the fusion protein module of ERG20^F96W-N127W-YRSQI-t14-BbTPS3K2, resulting in a final yield of 12.68 mg·L^-1 in shake flasks and 148.59 mg·L^-1 in a 5-L bioreactor. This work is the first reported attempt to produce (-)-borneol by microbial fermentation.