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

The Mutagenesis Assistant Program

Mutagenesis Assistant Program (MAP) is a web-based statistical tool to develop directed evolution strategies by investigating the consequences at the amino acid level of the mutational biases of random mutagenesis methods on any given gene. The latest development of the program, the MAP2.03D server, correlates the generated amino acid substitution patterns of a specific random mutagenesis method to the sequence and structural information of the target protein. The combined information can be used to select an experimental strategy that improves the chances of obtaining functionally efficient and/or stable enzyme variants. Hence, the MAP2.03D server facilitates the “in silico” prescreening of the target gene by predicting the amino acid diversity generated in a random mutagenesis library. Here, we describe the features of MAP2.03D server by analyzing, as an example, the cytochrome P450BM3 monooxygenase (CYP102A1). The MAP2.03D server is available publicly at http://​map.​jacobs-university.​de/​map3d.​html

Engineering and application of a biosensor with focused ligand specificity

Della Corte D, van Beek HL, Syberg F, et al. Engineering and application of a biosensor with focused ligand specificity. Nature communications. 2020;11(1): 4851.Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors