Abstract
(+)-Borneol is a desirable monoterpenoid with effective anti-inflammatory and analgesic effects that is known as soft gold. (+)-bornyl diphosphate synthase is the key enzyme in the (+)-borneol biosynthesis pathway. Despite several reported (+)-bornyl diphosphate synthase genes, relatively low (+)-borneol production hinders the attempts to synthesize it using microbial fermentation. Here, we identified the highly specific (+)-bornyl diphosphate synthase CbTPS1 from Cinnamomum burmanni. An in vitro assay showed that (+)-borneol was the main product of CbTPS1 (88.70% of the total products), and the Km value was 5.11 ± 1.70 μM with a kcat value of 0.01 s–1. Further, we reconstituted the (+)-borneol biosynthetic pathway in Saccharomyces cerevisiae. After tailored truncation and adding Kozak sequences, the (+)-borneol yield was improved by 96.33-fold to 2.89 mg⋅L–1 compared with the initial strain in shake flasks. This work is the first reported attempt to produce (+)-borneol by microbial fermentation. It lays a foundation for further pathway reconstruction and metabolic engineering production of this valuable natural monoterpenoid.
Highlights
The monoterpene borneol is a highly desirable natural product widely used in medicine, spice, and chemical fields since ancient times (Wojtunik-Kulesza et al, 2019)
Homologous alignment analysis showed that CbTPS1 shared highest sequence identities with SBS (41.75%) from Salvia officinalis (Wise et al, 1998; Figure 3), followed by AvBPPS (40.10%) from Amomum villosum (Wang et al, 2018), LaBPPS (38.68%) from Lavandula angustifolia (Despinasse et al, 2017), and LdBPPS (36.20%) from Lippia dulcis (Hurd et al, 2017)
We identified a (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii that catalyzed geranyl diphosphate (GPP) to form (+)-borneol under the hydrolysis of calf intestinal alkaline phosphatase (CIAP)
Summary
The monoterpene borneol is a highly desirable natural product widely used in medicine, spice, and chemical fields since ancient times (Wojtunik-Kulesza et al, 2019). It has a broad spectrum of bidirectional regulation on the central nervous system (Zhang et al, 2017; Zheng et al, 2018); anti-inflammatory (Zou et al, 2017; Ji et al, 2020) and antimicrobial activities (Xin et al, 2020); and increases biofilm barrier permeability (Song et al, 2018; Chen et al, 2019). It is necessary to explore other methods to produce natural (+)-borneol
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