Applying neutral drift to the directed molecular evolution of a β-glucuronidase into a β-galactosidase: Two different evolutionary pathways lead to the same variant

<p>Abstract</p> <p>Background</p> <p>Directed protein evolution has been used to modify protein activity and research has been carried out to enhance the production of high quality mutant libraries. Many theoretical approaches suggest that allowing a population to undergo neutral selection may be valuable in directed evolution experiments.</p> <p>Findings</p> <p>Here we report on an investigation into the value of neutral selection in a classical model system for directed evolution, the conversion of the <it>E. coli </it>β-glucuronidase to a β-galactosidase activity. We find that neutral selection, i.e. selection for retaining glucuronidase activity, can efficiently identify the majority of sites of mutation that have been identified as beneficial for galactosidase activity in previous experiments. Each variant demonstrating increased galactosidase activity identified by our neutral drift experiments contained a mutation at one of four sites, T509, S557, N566 or W529. All of these sites have previously been identified using direct selection for beta galactosidase activity.</p> <p>Conclusions</p> <p>Our results are consistent with others that show that a neutral selection approach can be effective in selecting improved variants. However, we interpret our results to show that neutral selection is, in this case, not a more efficient approach than conventional directed evolution approaches. However, the neutral approach is likely to be beneficial when the resulting library can be screened for a range of related activities. More detailed statistical studies to resolve the apparent differences between this system and others are likely to be a fruitful avenue for future research.</p

Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alphasynuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways.MGH/MIT Morris Udall Center of Excellence in PD Researc