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

Abstract

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 downregulated 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).

Programming adaptive laboratory evolution of 4-hydroxyisoleucine production driven by a lysine biosensor in Corynebacterium glutamicum

“…For example, a C. glutamicum strain with improved tolerance to high concentrations of methanol was successfully obtained by ALE, thereby enhancing methanol biotransformation, meanwhile, the cell growth was also improved (Tuyishime et al 2018 ; Wang et al 2020 ). ALE was successfully applied for improving the tolerance of E. coli to sabinene, and the sabinene production was increased to 8.43-fold (Wu et al 2020 ). ALE was also applied for generating a C. glutamicum strain with higher growth rates on glucose minimal medium (Pfeifer et al 2017 ).…”

Programming Adaptive Laboratory Evolution of 4-Hydroxyisoleucine Production Driven by a Lysine Biosensor in Corynebacterium Glutamicum

“…For example, a C. glutamicum strain with improved tolerance to high concentrations of methanol was successfully obtained by ALE, thereby enhancing methanol biotransformation, meanwhile, the cell growth was also improved (Tuyishime et al 2018;Wang et al 2020). ALE was successfully applied for improving the tolerance of E. coli to sabinene, and the sabinene production was increased to 8.43-fold (Wu et al 2020). ALE was also applied for generating a C. glutamicum strain with higher growth rates on glucose minimal medium (Pfeifer et al 2017).…”

Omics-Driven Biotechnology for Industrial Applications

“…While ALE can precede the Test and Learn steps in the DBTL cycle, the information gained from these steps can also be utilized by ALE to produce strains with better properties. In this way, ALE may benefit from using “omics” technologies during the Test phase of the DBTL cycle (Horinouchi et al, 2018 ; Long and Antoniewicz, 2018 ; Walker et al, 2019 ; Wu et al, 2020 ).…”