DEVELOPING A NANOPORE SEQUENCING WORKFLOW FOR PROTEIN ENGINEERING APPLICATIONS

Abstract

Next generation sequencing (NGS) has become an invaluable tool in protein engineering to rapidly identify the genotype of a desired mutation as it allows multiplexing of different samples, whereby, each DNA strand is being read simultaneously. We evaluated the performance of two commercially available NGS technologies (Illumina NGS and nanopore sequencing) with several mutant libraries. The sequencing results indicate that the barcode hopping phenomenon in nanopore sequencing was less severe as compared to Illumina NGS. We then developed a new library preparation workflow for nanopore sequencing, which further reduced the incidence of barcode hopping. This new workflow was then applied on protein engineering of Candida parapsilosis secondary alcohol dehydrogenase (CpSADH) mutants. By applying the fold change analysis of mutants after two rounds of selections, we identified many improved mutants, among which the best one (L55F/C57G/T158) was 520% more active than the wildtype when (R)-2-butanol was used as the substrate.