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Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae

2021; Elsevier BV; Volume: 7; Issue: 1 Linguagem: Inglês

10.1016/j.synbio.2021.12.004

2097-1206

Rui Ma, Ping Su, Qing Ma, Juan Guo, Suiqing Chen, Baolong Jin, Haiyan Zhang, Jinfu Tang, Tao Zhou, Chenghong Xiao, Guanghong Cui, Luqi Huang,

Microbial Natural Products and Biosynthesis

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 Km value of 4.93 ± 1.38 μM for GPP, and the corresponding kcat 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 ERG20F96W-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.