Adaptive laboratory evolution is often used to improve the performance of microbial cell factories. Reverse engineering of evolved strains enables learning and subsequent incorporation of novel design strategies via the design-build-test-learn cycle. Here, we reverse engineer a strain of Escherichia coli previously evolved for increased tolerance of octanoic acid (C8), an attractive biorenewable chemical, resulting in increased C8 production, increased butanol tolerance, and altered membrane properties. Here, evolution was determined to have occurred first through the restoration of WaaG activity, involved in the production of lipopolysaccharides, then an amino acid change in RpoC, a subunit of RNA polymerase, and finally mutation of the BasS-BasR two component system. All three mutations were required in order to reproduce the increased growth rate in the presence of 20 mM C8 and increased cell surface hydrophobicity; the WaaG and RpoC mutations both contributed to increased C8 titers, with the RpoC mutation appearing to be the major driver of this effect. Each of these mutations contributed to changes in the cell membrane. Increased membrane integrity and rigidity and decreased abundance of extracellular polymeric substances can be attributed to the restoration of WaaG. The increase in average lipid tail length can be attributed to the RpoC^H419P mutation, which also confers tolerance to other industrially-relevant inhibitors, such as furfural, vanillin and n-butanol. The RpoC^H419P mutation may impact binding or function of the stringent response alarmone ppGpp to RpoC site 1. Each of these mutations provides novel strategies for engineering microbial robustness, particularly at the level of the microbial cell membrane.

适应性实验室进化通常用于提高微生物细胞工厂的性能。进化菌株的逆向工程可以通过设计-构建-测试-学习循环来学习和随后纳入新的设计策略。在这里，我们对一株大肠杆菌进行逆向工程先前进化是为了增加对辛酸 (C8) 的耐受性，这是一种有吸引力的生物可再生化学物质，导致 C8 产量增加、丁醇耐受性增加和膜性质改变。在这里，进化被确定首先通过 WaaG 活性的恢复发生，参与脂多糖的产生，然后是 RpoC（RNA 聚合酶的亚基）的氨基酸变化，最后是 BasS-BasR 两组分系统的突变。需要所有三种突变才能在 20 mM C8 存在下重现增加的生长速率和增加的细胞表面疏水性；WaaG 和 RpoC 突变都有助于增加 C8 滴度，RpoC 突变似乎是这种效果的主要驱动因素。这些突变中的每一个都会导致细胞膜的变化。膜完整性和刚度的增加以及胞外聚合物的丰度减少可归因于 WaaG 的恢复。平均脂质尾长的增加可归因于 RpoC^H419P突变，这也赋予了对其他工业相关抑制剂的耐受性，例如糠醛、香草醛和正丁醇。RpoC ^H419P突变可能会影响严格响应警报 ppGpp 与 RpoC 位点 1 的结合或功能。这些突变中的每一个都为工程微生物稳健性提供了新的策略，尤其是在微生物细胞膜水平。