Cellular membranes provide a barrier to toxins, play a central role in metabolism, and are essential for cell viability, making membrane proteins of key interest for microbial strain engineering. However, membrane protein expression often causes decreased cell viability and alterations to central metabolism. We hypothesized that some non-essential proteins may be antagonistic towards the expression of a desired membrane protein and thus expression stress may be alleviated by the deletion of the corresponding genes. To identify mutant Escherichia coli that tolerate greater expression of membrane proteins, we developed a generalizable method, FlowSeq, that utilizes a pooled, bar-coded transposon insertion library in tandem with cell sorting to access genome-wide impact on membrane protein expression. Five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST) fusted to GFP were screened with FlowSeq. We identified 15 gene deletion strains capable of increased membrane protein expresison, and 3 of these knockout strains also increased membrane protein function. To our knowledge, this study represents the first systematic approach to identify genetic deteminants of membrane protein expression. Overall design: Examination of barcoded strain abundance after membrane protein expression stress.