Joint BioEnergy Institute, Emeryville, CA, 94608, USA.
Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
Sci Rep. 2017 Oct 12;7(1):13030. doi: 10.1038/s41598-017-12901-7.
Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. We hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression of five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.
膜蛋白的表达通常会导致生长抑制，并扰乱中心代谢，这种负担因被过度表达的蛋白而异。也有已知的菌株背景可以允许更大程度地表达膜蛋白，但在蛋白之间的效果不同。我们假设，对于任何膜蛋白，都有可能确定一个经过修饰的菌株背景，使其在表达时可以承受更小的负担。为了直接检验这一假设，我们使用了带有细胞分选的条形码转座子插入文库，以评估基因缺失对膜蛋白表达的全基因组影响。检测了五种膜蛋白（CyoB、CydB、MdlB、YidC 和 LepI）和一种可溶性蛋白（GST）的表达情况，这些蛋白都与 GFP 融合。我们鉴定出了与野生型相比，膜蛋白表达增加的大肠杆菌突变体。对于其中两种蛋白（CyoB 和 CydB），我们进行了功能测定以确认蛋白表达的增加也导致了功能上的表型改善。这项研究代表了一种广泛识别可用于改善膜蛋白表达的遗传基因座的系统方法，并且我们的方法可以用于改善任何造成细胞负担的蛋白的表达。
