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

Abstract

BackgroundDirected 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.FindingsHere we report on an investigation into the value of neutral selection in a classical model system for directed evolution, the conversion of the E. coli 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.ConclusionsOur 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.

Cryptic genetic variation accelerates evolution by opening access to diverse adaptive peaks

“…When directed evolution relies on large populations sizes (45), it can lead to repeatable evolutionary outcomes that cannot be further improved (46,47). Small populations with strong neutral drift can be more effective (48,49), but small populations will also accumulate limited diversity. In contrast, large populations subject to stabilizing selection will not only accumulate substantial cryptic variation, but may also uncover different high fitness phenotypes during subsequent directional selection on a new phenotype.…”

Phylogenetic distribution of genes encoding β‐glucuronidase activity in human colonic bacteria and the impact of diet on faecal glycosidase activities

“…The β‐glucuronidases studied here are related to β‐galactosidases and it is questionable whether the β‐glucuronidase activity is indeed the main activity of all the genes described here. Molecular evolution of the gus gene from Escherichia coli to increase its β‐galactosidase activity has revealed key amino acid residues (Matsumura and Ellington, 2001; Smith et al ., 2011). Residues D508 and T509 were strongly conserved in most of the gus sequences examined here, but many of the genes more closely related to BG displayed G and A, respectively, in the corresponding position, which were the changes found in the E. coli enzyme to increase β‐galactosidase activity.…”

LabTrove: A Lightweight, Web Based, Laboratory “Blog” as a Route towards a Marked Up Record of Work in a Bioscience Research Laboratory

“…An example of material extracted from a laboratory notebook. The notebook entries relevant to a specific published paper [40] were extracted and converted to a static html representation, which can be viewed in any web browser. The archive is made available via the figshare service [41].…”