Enzymes isolating from nature have been adapted to diverse applications, like biomedicals, catalytic industry, agriculture, molecular biology, etc. However, applications of these enzymes are limited by other characteristics obtained from nature, which can be solved by enzyme engineering techniques. For example, the extraordinary strand displacement capability and high processivity of wildtype (WT) phi29 DNA polymerase make it a promising candidate in isothermal amplification applications, for instance, in second strand amplification of Next-Generation Sequencing (NGS). However, the application of this WT enzyme in NGS is limited by its low stability which leads to a shelf life of less than 10 months (1-2 times less than thermophilic polymerases), and low replication activity (0.8-0.9 kb/min of WT phi29 vs 2-3 kb/min of thermophilic polymerases) which leads to longer second strand amplification time in NGS. To improve its performance in the application, WT phi29 DNA polymerase was engineered in its stability and replication activity. Firstly, a cell supernatant-based stability screening method was developed by calculating the residual activity percentage after temperature stress treatment. In addition, thermostable variants prediction methods were developed based on rational design strategies, including surface interaction analysis, docking analysis and molecular dynamics simulation analysis. In this study, 208 amino acid sites of WT phi29 DNA polymerase were analysed in silico, 150 single mutations were selected for screening, and finally 35 variants were pointed out with better thermostability. The top 5 hits were Y369R, E515R, E515N, Y224K, and Y347G, with 2.9±0.2 ℃, 2.5±0.1 ℃, 2.3±0.1 ℃, 2.2±0.4 ℃, 2.2±0.3 ℃ elevation in melting temperature compared to WT. This increasement indicated significantly improvement to thermostability of the variants. For example, Y224K increased the residual activity percentage from 22.0% to 44.0% at stability stress evaluation experiment carried out under 37 ℃. To further improve the stability and activity, the 15 hot-spots from obtained thermostable variants were then combined with R96, M97, L123 from the screened activity improved variants. The combinational mutation libraries were then screened with droplet based isothermal compartmentalization self-replication (iCSR) method, which can accumulate positive variants over the rest with a theoretically throughput of 2.2x107 cells per test. In total, 5 variants were identified by Sanger sequencing from the enriched library after 2 rounds of iCSR screening. Among the 5 variants, phi29_cC (Y224K-I474K-E515P) was obtained as the most stable functional variant, with 5.1±0.3 ℃ improvement in Tm; and phi29_cE (M97T-L123S-E515A) was obtained as the most active variant, with 52% increasement in replication activity as well as 2.8±0.2 ℃ elevation in Tm. Phi29_cE was successfully applied in second strand amplification of NGS sequencing, with extended shelf life (27 months, >170% improvement compared to WT), and reduced reaction time (15 min, half of WT). In addition, sequencing data of phi29_cE presented 7-fold decrease in mismatch rate in 15 min amplification compared to WT. Phi29_cC and phi29_cE were also successfully applied in other isothermal amplifications like whole genome amplification for single cell sequencing and plasmid amplification for Sanger sequencing. The successful engineering of phi29 DNA polymerase with improved thermostability and replication activity proves the great potential of the enzyme engineering pipeline I constructed, with a more efficient and focused combinational mutation library formed of sites from positive variants, which offers a new solution of polymerase engineering to fulfil versatile applications.
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