High-Throughput Site-Directed Mutagenesis

Abstract

Protein engineering has an array of uses; whether you are studying a disease mutation, removing undesirable sequences, adding stabilizing mutations for structural purposes, or simply dissecting protein function. Protein engineering is almost exclusively performed using site-directed mutagenesis (SDM) as this provides targeted modification of specific amino acids, as well as the option of re-writing the native sequence to include or exclude certain regions. Despite its widespread use, SDM has often proved to be a bottleneck, requiring precision manipulation on a sample-by-sample basis to make it work. When dealing with large volumes of samples it is not possible to use such a low-throughput approach. Here we describe a high-throughput (HTP) method for SDM, optimized and used by the Structural Genomics Consortium (SGC) to complement structural studies.

CLADE: Cluster learning-assisted directed evolution

“…In contrast to the current MLDE protocols where site-directed mutagenesis is conducted to generate the variants used to train ML models, the CLADE protocol requires making specific variants throughout the whole process from the initial sampling and the training sets to the predicted set, which would increase experimental cost in making constructs. However, with the rapid decrease in the cost of gene synthesis and development of highthroughput site-directed mutagenesis [45], making hundreds of variants harboring multiple mutations would still be efficient and affordable. The increased cost would be sufficiently compensated by the significantly improved performance of the supervised learning with the increased expected max fitness and global max hitting rate.…”

Enzyme and Pathway Engineering for Improved Betanin Production in Saccharomyces cerevisiae

“…Meanwhile, the capacity rational enzyme design has been rapidly advancing with the use of computational protein structure simulations (like AlphaFold) and molecular docking . The emerging strategy of combining alanine scanning and site-directed saturation mutagenesis streamlines an efficient methodology for directed evolution, only involving a small but smart mutant library, to improve the catalytic efficiency and enantioselectivity of enzymes. − …”

The use of phylogenetics and ancestral sequence reconstruction to identify improved halophilic enzymes for the biodegradation of poly(R-3-hydroxybutyrate)

“…The traditional route to fine-tuning the kinetic and stability parameters of an enzyme is through the use of site directed mutagenesis, often employing a high throughput methodology [26] or by directed evolution methods [27]. An emerging method that can be used separately or in conjunction with these techniques is through the reconstruction of ancestral sequences [28] [29] [30] [31].…”