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).

中文翻译：

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使用适应性实验室进化和组学技术提高大肠杆菌对沙宾的耐受性。

背景通过引入异源途径和使用可再生糖作为碳源，已在工程微生物中完成了双环单萜栎烯的生物合成。然而，这种方法的效率和滴度受限于宿主对栎烯的低耐受性（在真核生物和原核生物中）。结果本研究选择大肠杆菌BL21(DE3)作为适应性实验室进化菌株。该菌株在逐渐增加栀子浓度的培养基中连续传代进化，获得了对栀子具有显着耐受性的进化菌株XYF(DE3)。然后，将XYF（DE3）用作桧木烯生产的宿主，与亲本BL21（DE3）相比，桧木烯的产量提高了8.43倍，达到191.76 mg / L。全基因组重测序表明 XYF(DE3) 菌株是一种超突变体。对XYF(DE3)和BL21(DE3)的转录组进行了比较分析，揭示了提高桤木耐受性的机制，鉴定出734个上调基因和857个下调基因。我们通过逆向工程进一步测试了已识别基因在桧烯耐受性中的作用。结果表明，过表达DLP12家族的ybcK基因、内膜蛋白基因ygiZ和甲基丙二酰辅酶A变位酶基因scpA可使BL21(DE3)对桧木烯的耐受性分别提高127.7%、71.1%和75.4%。此外，应用扫描电子显微镜来监测细胞形态。在桧烯胁迫下，亲本 BL21(DE3) 的细胞长度增加，而 XYF(DE3) 显示正常的细胞形态。此外，ybcK、ygiZ 或 scpA 的过表达可以部分挽救桧烯胁迫下的细胞形态，而 ygiZ 或 scpA 的过表达可以增加 BL21(DE3) 中桧木烯的产生。结论 本研究不仅获得了用于生产栉木烯的微生物生产桧木烯耐受菌株，而且揭示了对桧木烯耐受性很重要的潜在调控机制。此外，首次发现 ybcK、ygiZ 和 scpA 对大肠杆菌 BL21(DE3) 的萜烯耐受性很重要。结论 本研究不仅获得了用于生产栉木烯的微生物生产桧木烯耐受菌株，而且揭示了对桧木烯耐受性很重要的潜在调控机制。此外，首次发现 ybcK、ygiZ 和 scpA 对大肠杆菌 BL21(DE3) 的萜烯耐受性很重要。结论 本研究不仅获得了用于生产栉木烯的微生物生产桧木烯耐受菌株，而且揭示了对桧木烯耐受性很重要的潜在调控机制。此外，首次发现 ybcK、ygiZ 和 scpA 对大肠杆菌 BL21(DE3) 的萜烯耐受性很重要。