Abstract
BackgroundBiosynthesis 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).ResultsIn 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).ConclusionsThis 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).
Highlights
Biosynthesis of sabinene, a bicyclic monoterpene, has been accomplished in engineered microorganisms by introducing heterologous pathways and using renewable sugar as a carbon source
Enhancing sabinene tolerance by Adaptive laboratory evolution (ALE) To determine the minimum inhibitory concentration of sabinene to E. coli BL21(DE3), growth curve analysis was performed with different concentrations of sabinene
The improved sabinene tolerance and production of XYF(DE3) demonstrated that ALE is an effective strategy for metabolic engineering
Summary
Biosynthesis of sabinene, a bicyclic monoterpene, has been accomplished in engineered microorganisms by introducing heterologous pathways and using renewable sugar as a carbon source. Terpenoids is the largest class and the most widely distributed secondary metabolites that have been discovered to date [1]. These compounds are widely used in medicine, food additives, perfume, energy industry, and many other fields [2, 3]. A bicyclic monoterpene, is widely applied in the above fields [4, 5]. The chemical catalysis-based synthetic route for sabinene had already been established by organic chemists in last century [9]. Sabinene is mainly isolated from plants which greatly limits the large-scale production and application of these compounds due to its low content in plant materials [10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
