Directed evolution of polymerases and its application in sequence saturation mutagenesis
Chung, Mu-En; Schwaneberg, Ulrich (Thesis advisor); Elling, Lothar (Thesis advisor)
Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2021
Directed evolution plays a crucial role in advancing modern biotechnological, medical, and industrial technologies. The importance of directed evolution was recently acknowledged by Novel prize in chemistry in 2018. Directed evolution consists of iterative steps of diversity generation and isolation of the variants with improved protein property. Each round of evolution can be seen as one step toward the "hilltop of desired property" in the protein sequence space. As the sequence space of a regular sized protein already far exceeds the throughput of existing screening methods, a gene diversification method that generates a diverse and unbiased library is crucial to an efficient walk to the "hilltop" with minimal number of steps. Sequence saturation mutagenesis (SeSaM) was reported to generate a distinct mutational spectrum unobtainable by epPCR, which is widely used for mutagenesis. However, SeSaM as well as epPCR rarely generates consecutive transversions (TvTv). TvTv mutations are particularly important for generating enzyme libraries with diverse amino acid substitutions and broad chemical diversity. 91.4% of amino acid substitutions generated by TvTv mutations are distinct, and of which 90% are chemically different. The challenge of generating TvTv mutations lies in the structure of the DNA, in which transversion mutations form bulky or wobbly mispairs that destabilize double helix formation and are structurally unfavored for DNA polymerases to read over comparing to transition mutations. In the first part of the thesis, we developed SeSaM-DV, an advanced SeSaM method which employs a new mismatch-tolerant polymerase and generated the highest amount of TvTv mutations ever reported (217% of the best reported number). In the development of SeSaM-DV, a DNA polymerase assay targeting mismatch tolerance ability was developed. Subsequently, thirteen DNA polymerases were evaluated for their ability to read over consecutive phosphorothioated mismatches, which is considered a "bottle neck" for increasing consecutive substitutions in SeSaM libraries. Employing the polymerase mismatch tolerance assay S. islandicus DNA polymerase IV (Sis. Dpo4) exhibited the best ability of extending consecutive mismatches and was therefore subjected to an additional PCR-based assay which directly validated the ability of Sis. Dpo4 to extend four types of consecutive mismatches thus generate TvTv, TvTs, TsTv, and TsTs substitutions. Finally, Sis. Dpo4 was incorporated into SeSaM step 3 with the addition of Vent exo- to achieve efficient with extension after reading over mismatches. The resulted SeSaM-DV library was benchmarked by sequencing and 10% of the identified mutations were TvTv mutations, which is 2.17-fold better than the best reported number. With the new type of TvTv-enriched mutational bias, SeSaM-DV can efficiently evolve enzymes by exploring natures sequence space in a new "non-natural" way with an expanded chemical diversity, which might be a better approach to engineer enzymes for industrial productions which often involve non-natural conditions such as organic solvents and altered pH. Microbial cell surface display (CSD) is a powerful platform to present and immobilize the protein of interest on microbial surfaces. CSD provides a genotype-phenotype linkage without the need of artificial compartmentalization of the protein and its coding gene thus enables FACS-based ultra-high throughput screening of protein libraries. Various proteins have been displayed on microbial surface, however, to the best of our knowledge, no nucleic acid (NA) polymerase has been successfully displayed on microbial surface. In the second part of the thesis, we developed a first of its kind functional cell surface display of nucleic acid polymerase and its directed evolution to efficiently incorporate 2’-O-methyl nucleotide triphosphates (2’-OMe-NTPs). In the development of polymerase cell surface display, two autotransporter proteins (Escherichia coli Adhesin Involved in Diffuse Adherence and Pseudomonas aeruginosa Esterase A, EstA) were employed to transport and anchor the 68 kDa Klenow fragment of E. coli DNA polymerase I (KF) on the surface of E. coli. The localization and function of the displayed KF were verified by analysis of cell outer membrane fractions, immunostaining and fluorometric detection of synthesized DNA products. The EstA cell surface display system was applied to evolve KF for incorporation of 2’-OMe-NTPs and a KF variant with 50.7-fold increased ability to successively incorporate 2’-OMe-NTPs was discovered. Expanding the scope of cell-surface displayable proteins to the realm of polymerases provides a novel screening tool for tailoring polymerases to diverse application demands in PCR and sequencing based biotechnological and medical applications. Especially, cell surface display enables novel polymerase screening strategies in which heat-lysis step are bypassed and thus allows the screening of mesophilic polymerases with broad application potentials ranging from diagnostics and DNA sequencing to replication of synthetic genetic polymers.
- Department of Biology 
- Chair of Biotechnology