Sabinene

Biosynthesis and production of sabinene: current state and perspectives

Sabinene is an important naturally occurring bicyclic monoterpene which can be used as flavorings, perfume additives, fine chemicals, and advanced biofuels. Up to now, this valuable terpene is commercially unavailable since there is no applicable manufacturing process. Microbial synthesis can be a promising route for sabinene production. In this review, we summarize knowledge about the metabolic pathway and key enzymes for sabinene biosynthesis. Recent advances that have been made in production of sabinene by microbial fermentation are highlighted. In these studies, researchers have identified the general synthetic pathway of sabinene from simple intermediate metabolites. Sabinene synthases of different origins were also cloned and characterized. Additionally, heterologous systems of the model microbes Escherichia coli and Saccharomyces cerevisiae were constructed to produce sabinene. This review also suggests new directions and attempts to gain some insights for achieving an industrial level production of sabinene. The combination of traditional molecular biology with new genome and proteome analysis tools will provide a better view of sabinene biosynthesis and a greater potential of microbial production.

RIFM fragrance ingredient safety assessment, sabinene, CAS Registry Number 3387-41-5

Improvement of sabinene tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies

Background: Biosynthesis of sabinene, a bicyclic monoterpene, has been accomplished in engineered microorganisms by introducing heterologous pathways and using renewable sugar as a carbon source. However, the efficiency and titers of this method are limited by the low host tolerance to sabinene (in both eukaryotes and prokaryotes).
Results: In this study, Escherichia coli BL21(DE3) was selected as the strain for adaptive laboratory evolution. The strain was evolved by serial passaging in the medium supplemented with gradually increasing concentration of sabinene, and the evolved strain XYF(DE3), which exhibited significant tolerance to sabinene, was obtained. Then, XYF(DE3) was used as the host for sabinene production and an 8.43-fold higher sabinene production was achieved compared with the parental BL21(DE3), reaching 191.76 mg/L. Whole genomes resequencing suggested the XYF(DE3) strain is a hypermutator. A comparative analysis of transcriptomes of XYF(DE3) and BL21(DE3) was carried out to reveal the mechanism underlying the improvement of sabinene tolerance, and 734 up-regulated genes and 857 down-regulated genes were identified. We further tested the roles of the identified genes in sabinene tolerance via reverse engineering. The results demonstrated that overexpressions of ybcK gene of the DLP12 family, the inner membrane protein gene ygiZ, and the methylmalonyl-CoA mutase gene scpA could increase sabinene tolerance of BL21(DE3) by 127.7%, 71.1%, and 75.4%, respectively. Furthermore, scanning electron microscopy was applied to monitor cell morphology. Under sabinene stress, the parental BL21(DE3) showed increased cell length, whereas XYF(DE3) showed normal cell morphology. In addition, overexpression of ybcK, ygiZ or scpA could partially rescue cell morphology under sabinene stress and overexpression of ygiZ or scpA could increase sabinene production in BL21(DE3).
Conclusions: This study not only obtained a sabinene-tolerant strain for microbial production of sabinene but also revealed potential regulatory mechanisms that are important for sabinene tolerance. In addition, for the first time, ybcK, ygiZ, and scpA were identified to be important for terpene tolerance in E. coli BL21(DE3).

Sabinene Prevents Skeletal Muscle Atrophy by Inhibiting the MAPK-MuRF-1 Pathway in Rats

Chrysanthemum boreale Makino essential oil (CBMEO) has diverse biological activities including a skin regenerating effect. However, its role in muscle atrophy remains unknown. This study explored the effects of CBMEO and its active ingredients on skeletal muscle atrophy using in vitro and in vivo models of muscle atrophy. CBMEO reversed the size decrease of L6 myoblasts under starvation. Among the eight monoterpene compounds of CBMEO without cytotoxicity for L6 cells, sabinene induced predominant recovery of reductions of myotube diameters under starvation. Sabinene diminished the elevated E3 ubiquitin ligase muscle ring-finger protein-1 (MuRF-1) expression and p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylations in starved myotubes. Moreover, sabinene decreased the increased level of reactive oxygen species (ROS) in myotubes under starvation. The ROS inhibitor antagonized expression of MuRF-1 and phosphorylation of MAPKs, which were elevated in starved myotubes. In addition, levels of muscle fiber atrophy and MuRF-1 expression in gastrocnemius from fasted rats were reduced after administration of sabinene. These findings demonstrate that sabinene, a bioactive component from CBMEO, may attenuate skeletal muscle atrophy by regulating the activation mechanism of ROS-mediated MAPK/MuRF-1 pathways in starved myotubes, probably leading to the reverse of reduced muscle fiber size in fasted rats.

Atmospheric Oxidation Mechanism of Sabinene Initiated by the Hydroxyl Radicals

The atmospheric oxidation mechanism of sabinene initiated by the OH radical has been studied using quantum chemistry calculations at the CBS-QB3 level and reaction kinetic calculations using transition state theory and unimolecular rate theory coupled with collisional energy transfer. The oxidation is initiated by OH radical additions to the CH₂═C< bond with a branching ratio of ?(92-96)%, while all the hydrogen atom abstractions count for ?(4-8)% of branching ratio, which was estimated by comparing the rate coefficients of the reactions of sabinene and sabinaketon with the OH radical. Addition of OH to the ═C< carbon forms radical adduct Ra, while addition of OH to the terminal CH₂═ carbon forms radical adduct Rb, which would break the three-membered ring promptly and almost completely to radical Re. RRKM-ME calculations obtained fractional yields of 0.40, 0.09, and 0.51 for radicals syn-Ra, anti-Ra, and Re, respectively, at 298 K and 760 Torr. In the atmosphere, the syn/ anti-Ra radical would ultimately transform to sabinaketone in the presence of ppbv levels of NO, while in the transformation of the Re radical, both bimolecular reactions and unimolecular H-migrations could occur competitively for the peroxy radicals formed. The H-migrations in peroxy radicals result in the formation of unsaturated multifunctional compounds containing >C═O, -OH, and/or -OOH groups. Formation of sabinaketone from syn- and anti-Ra and formation of acetone from Re are predicted with yields of ?0.37 and ?0.38 in the presence of high NO, being larger than while in reasonable agreement with the experimental values of 0.19-0.23 and of 0.21-0.27, respectively.