Isoprenol is the basis for industrial flavor and vitamin synthesis and also a promising biofuel. Biotechnological production of isoprenol with E. coli is currently limited by the high toxicity of the final product. Adaptive laboratory evolution (ALE) is a promising method to address complex biological problems such as toxicity. Here we applied this method successfully to evolve E. coli towards higher tolerance against isoprenol, increasing growth at the half-maximal inhibitory concentration by 47%. Whole-genome re-sequencing of strains isolated from three replicate evolutions at seven time-points identified four major target genes for isoprenol tolerance: fabF, marC, yghB, and rob. We could show that knock-out of marC and expression of mutated Rob H(48) → frameshift increased tolerance against isoprenol and butanol. RNA-sequencing showed that the deletion identified upstream of yghB correlated with a strong overexpression of the gene. The knock-out of yghB demonstrated that it was essential for isoprenol tolerance. The mutated Rob protein and yghB deletion also lead to increased vanillin tolerance. Through ALE, novel targets for strain optimization in isoprenol production and also the production of other fuels, such as butanol, could be obtained. Their effectiveness could be shown through re-engineering. This paves the way for further optimization of E. coli for biofuel production.

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大肠杆菌MG1655对异戊二烯的耐受性的进化工程

异戊二烯是工业香料和维生素合成的基础，也是一种有前途的生物燃料。目前，大肠杆菌对异戊烯醇的生物技术生产受到最终产品高毒性的限制。自适应实验室进化（ALE）是解决复杂生物问题（如毒性）的一种有前途的方法。在这里，我们成功地应用了这种方法，使大肠杆菌对异戊二烯具有更高的耐受性，在半数最大抑制浓度下，其生长量增加了47％。在七个时间点从三个重复的进化过程中分离出的菌株进行全基因组重测序，确定了对异戊二烯耐受性的四个主要靶基因：fabF，marC，yghB和rob。我们可以证明，marC的敲除和突变的Rob H（48）→移码的表达增加了对异戊二烯和丁醇的耐受性。RNA测序表明，在yghB上游鉴定出的缺失与该基因的强烈过表达有关。敲除yghB证明这对异戊二烯耐受性至关重要。突变的Rob蛋白和yghB缺失也导致香草醛耐受性增加。通过ALE，可以获得异戊二烯生产以及其他燃料（例如丁醇）生产中的应变优化的新目标。通过重新设计可以证明其有效性。这为进一步优化用于生物燃料生产的大肠杆菌铺平了道路。可以获得异戊二烯生产以及其他燃料（例如丁醇）生产中的应变优化的新目标。通过重新设计可以证明其有效性。这为进一步优化用于生物燃料生产的大肠杆菌铺平了道路。可以获得异戊二烯生产以及其他燃料（例如丁醇）生产中的应变优化的新目标。通过重新设计可以证明其有效性。这为进一步优化用于生物燃料生产的大肠杆菌铺平了道路。