Improving membrane protein expression and function using genomic edits

Abstract

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.Membrane proteins are critical to cellular functions such as response to environmental changes, membrane stability, nutrient transport, redox balance, energy generation, and cellular defense. However, cells must carefully balance the necessary functions of many membrane proteins against the capacity to produce and translocate membrane proteins maintaining membrane physiology. This is evidenced by many bacterial studies [1][2][3] showing that overexpression of membrane proteins tends to have far stronger deleterious effects on cell health than similar overexpression of cytosolic proteins. Hypotheses that have been proposed to explain this phenomenon include the overutilization of membrane translocation systems or chaperones, and alterations in central metabolism revolving around respiration 1,4,5 , but no unifying theory has emerged.The deleterious effect of membrane protein overexpression and the relatively poor understanding of the underlying cell biology translates into significant challenges in biological engineering. Membrane proteins (and complexes) are frequent targets for cellular engineering applications, such as membrane-spanning electron conduits 6,7 , responses to environmental cues 8,9 , and transport of nutrients or products during bioproduction 10-14 . Additionally, biochemical and structural studies of membrane proteins require expression of large amounts of the protein in bacterial cells, and these studies are hampered by the deleterious effects of expression on cell growth. Current approaches to improving membrane protein expression largely use tighter transcriptional control to minimize the effect on cell viability [15][16][17][18] . These approaches require significant optimization of growth conditions, i...

Restoration of biofuel production levels and increased tolerance under ionic liquid stress is enabled by a mutation in the essential Escherichia coli gene cydC

“…The assumption that the function of cydC - D86G involves cytochrome bd-I activity also leads to two testable predictions. The first prediction is that E. coli cydC - D86G strains may have increased hydrogen peroxide resistance, the same phenotype as cells with increased cytochrome bd-I activity [ 38 ]. We subjected wild-type E. coli and E. coli cydC - D86G strains to increasing concentrations of exogenous hydrogen peroxide (“ Methods ”).…”

“…The second prediction is that strains with increased cytochrome bd-I activity may also have increased tolerance to the IL [EMIM]OAc. We tested this hypothesis using E. coli strains which we have previously shown to have increased cytochrome bd-I activity as measured by quantitatively improved hydrogen peroxide resistance [ 38 ]. This strain ( E. coli ∆ oppF P lacUV5 - cydAB ) was grown in the presence of [EMIM]OAc (Fig.…”

A Pseudomonas putida efflux pump acts on short-chain alcohols

“…1 b) indicates that directly transferring TtgABC to other strains may be challenging. Recently identified mutations in E. coli leading to a reduced burden of expressing membrane proteins may help to reduce the toxicity of the efflux pump [ 36 ], as it may chaperone overexpression [ 37 ]. Strategies for regulating membrane protein production via genetic circuits are also promising [ 6 ].…”

Genetic analyses link the conserved inner membrane protein CvpA to the σEextracytoplasmic stress response

“…Two of the screens relied on transposoninsertion mutagenesis, and one used spontaneous suppressor analysis. Similar genetic approaches have been fruitful for other membrane proteins (33)(34)(35)(36), which are often difficult to probe using biochemical approaches to identify protein-protein interactions.…”