Techniques for High-Throughput Directed Enzyme Evolution

Abstract

Enzymes are versatile biological catalysts that play crucial roles in numerous industrial processes, including the production of biofuels, pharmaceuticals, and fine chemicals. However, natural enzymes often face limitations in stability and efficiency under industrial conditions, necessitating their evolution for enhanced performance. High-throughput directed evolution schemes typically involve four key steps: mutagenesis library construction, library screening or selection, variant sequencing, and variant characterization. In this thesis, we present a series of techniques relevant to enhancing the efficiency and effectiveness of high-throughput directed evolution. In Chapter 2, we introduce a platform that utilizes nCas9 and mutagenic polymerase to achieve autonomous gene diversification. This platform, combined with a customized lab-on-chip device, enables continuous screening and thus facilitates controlled continuous evolution. Chapter 3 discusses the development of Random Saturation Mutagenesis (RSM) libraries. This method allows for the generation of chemically diverse libraries with minimal bias, broadening the exploration of the enzyme fitness landscape. We demonstrate the design, construction, and superiority of these libraries over traditional error-prone PCR techniques in terms of diversity and efficiency. In Chapter 4, we address the challenge of maintaining functional conditions in complex droplet environments by developing a hydrogel-bead based method for high-throughput screening of IVTT-expressed enzymes in each droplet. And finally in Chapter 5, we present an economical method for sequencing mutagenesis libraries at full gene length using Illumina sequencers, significantly reducing costs for validating mutations in large libraries.