Enabling directed evolution 3.0 by a promoter toolbox, casting epPCR and engineered polymerase

Over the last decades, protein engineering by directed evolution has been applied as a powerful algorithm to improve proteins for applications as biocatalysts, biosensors or therapeutic proteins. Exploring functions and characteristics of these proteins are still often limited by low to no heterologous protein production in the selected hosts. Additionally, improving these proteins by directed evolution is limited by low diversity of mutant libraries and the available high-throughput screening systems. These are introduced in detail in Chapter I. The low diversity of amino acid substitution reduces the chances to find beneficial substitutions for the improved enzyme properties. Furthermore, the lack of an efficient screening method could lead to the loss of potentially improved variants, waste of the previous time and efforts when generating diversified mutant library. All these are tackled in this thesis, aiming for improved methods in directed evolution and protein production.Considering the low or no production of the heterologous protein, in Chapter II, we developed a novel PLICable promoter toolbox which enables in a single cloning and after a screening experiment to identify out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) the most suitable one for high level protein production. The target gene is cloned under the control of different promoters in a single and efficient cloning step using the ligase-free cloning method PLICing (phosphorothioate-based ligase-independent gene cloning). The promoter toolbox was firstly validated using three well producible proteins (a cellulase from a metagenome library, a phytase from Yersinia mollaretii and an alcohol dehydrogenase from Pseudomonas putida) and then applied to two poorly producible enzymes (3D1 DNA polymerase and glutamate dehydrogenase) in Escherichia coli. By applying our PLICable promoter toolbox, we were able to increase production by 2 fold for 3D1 DNA polymerase (lac promoter) and 29 fold for glutamate dehydrogenase (trc promoter) compared to those under the T7 promoter in Escherichia coli.In order to solve the weakness of low diversity generation in directed evolution, in Chapter III, a new random mutagenesis strategy named cepPCR (casting error prone polymerase chain reaction) was developed to generate mutant libraries with high ratio of transversion, high mutation frequency, and increased consecutive mutations to explore more amino acid positions compared to typical epPCR (error prone PCR). In cepPCR, the model gene lipase A from Bacillus subtilis (bsla) (543 bp) was divided into 3 fragments and randomized by epPCR with high mutation load. CepPCR leads to 2 times increased beneficial amino acid positions in only sequencing 500 mutations. Additionally, for the first time, the minimum number of clones that needs to be screened to cover most common generated beneficial amino acid positions was defined and can be carried over for other similar target genes. Although cepPCR allows to generate high diversity libraries, however, it still has a strong transition bias. In Chapter III we could conclude that combining the transversion biased sequence saturation random mutagenesis method (SeSaM) with cepPCR, 39% beneficial positions for improved ionic liquids [BMIM][TfO] could be obtained. Therefore, in Chapter IV, SeSaM was further improved to supplement the mutation patterns in the mutant library generated by typical epPCR. In this Chapter, the aim is to improve the protocol of SeSaM by directed evolution of DNA polymerase and subsequently applied it in the step 3 of SeSaM to increase the fraction of consecutive transversions. According to the codon usage, consecutive substitutions could cover 90% of possible substitutions at amino acid level, the increases of consecutive transversions is crucial for exploring more amino acid substitutions. For engineering a DNA polymerase capable of efficient elongating consecutive transversions, high-throughput screening assay is primarily the prerequisite. A screening assay based on Scorpion real-time PCR and in vitro expression in emulsions cooperating with flow cytometry screening was investigated. However, because of the high fluorescent background even after the optimization of different factors, the screening assay was not successfully established and need to be further optimized. Nevertheless, further literature and experimental studies showed that 5D4 DNA polymerase has higher activity for elongating consecutive mismatches than 3D1 DNA polymerase. It could be used to replace original used 3D1 DNA polymerase in the step 3 of SeSaM to obtain more diverse mutant library.Finally, in Chapter V, we tried to evolve DNA polymerase by incorporating more nucleotide analogs and applied it in typical epPCR for adjusting the mutation patterns (more transversions and consecutive mutations). Prerequisite for the evolution of the DNA polymerase is a reliable and efficient high-throughput screening assay. Here the screening assay is based on the fluorescence from DNA dye intercalated DNA products which were amplified by the DNA polymerase variants incorporating nucleotide analogs. For construction of the most sensitive screening assay, SYBR® Green I was selected by the comparison of 10 DNA dyes. The critical values of the nucleotide analog concentrations were also determined. The validation of the screening assay to screen 3D1 DNA polymerase variants generated by epPCR was performed using model analog dITP, and four mutants with higher incorporation efficiency for nucleotide analogs were selected, concluding that this assay could be used to screen the DNA polymerase libraries for incorporating more nucleotide analogs. The improved DNA polymerase could be further improved by iterative directed evolution and finally used in epPCR to increase the diversity of mutant libraries