Mutagenesis-visualization: analysis of site saturation mutagenesis datasets in Python

Site saturation mutagenesis (SSM) experiments have been transformative in our study of protein function. Despite the rich data generated from such experiments, current tools for processing, analyzing, and visualizing the data offer only a limited set of static plots that are difficult to customize. Furthermore, usage of the tools requires extensive experience and programming. This slows the research process for those in the biological field who are unfamiliar with programming. Here, we introduce mutagenesis-visualization, a Python API for the creation of publication quality figures for SSM datasets which requires no prior Python or statistics experience. The plots can be rendered as native matplotlib objects (easy to stylize) or as Plotly objects (interactive graphs). Additionally, the software offers the possibility to visualize the datasets on Pymol. Availability and implementation: The software can be installed from PyPI or GitHub using the pip package manager and is compatible with Python ≥ 3.8. The documentation can be found at readthedocs and the source code can be found on GitHub.

Identification, Characterization and Site-Saturation Mutagenesis of a Thermostable ω-Transaminase From Chloroflexi Bacterium

In present study, we have mined a ω-transaminase (ω-TA) from Chloroflexi bacterium from genome database by using an ω-TA sequence ATA117 Arrmut11 from Arthrobacter sp . KNK168 and an amine transaminase from Aspergillus terreus as templates in a BLASTP search and motif sequence alignment. The protein sequence of the ω-TA from C. bacterium shows 38% sequence identity to ATA117 Arrmut11. The gene sequence of the ω-TA was inserted into pRSF-Duet1 and functionally expressed in E. coli BL21(DE3). Results showed that the recombinant ω-TA has a specific activity of 1.19 U/mg at pH 8.5, 40 °C. The substrate acceptability test showed that ω-TA has significant reactivity to aromatic amino donors and amino receptors. More importantly, the ω-TA also exhibited a good affinity towards some cyclic substrates such as 1-Boc-3-piperidone. The homology model of the ω-TA was built by Discovery Studio and docking was performed to describe the relative activity towards some substrates. The ω-TA was evolved by site-saturation mutagenesis and found that the Q192G mutant increased the activity to the (R)-α-methylbenzylamine (MBA) by around seven-fold. The Q192G mutant was then used to convert two cyclic ketones, N -Boc-3-pyrrolidinone and N -Boc-3-aminopiperidine, and the conversions were both improved compared to the parental ω-TA.

Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

Site-saturation libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method—termed SpliMLiB for Split-and-Mix Library on Beads—was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.