sb5002399_si_001.pdf (1.09 MB)

Speeding up Directed Evolution: Combining the Advantages of Solid-Phase Combinatorial Gene Synthesis with Statistically Guided Reduction of Screening Effort

Download (1.09 MB)
journal contribution
posted on 20.03.2015, 00:00 by Sabrina Hoebenreich, Felipe E. Zilly, Carlos G. Acevedo-Rocha, Matías Zilly, Manfred T. Reetz
Efficient and economic methods in directed evolution at the protein, metabolic, and genome level are needed for biocatalyst development and the success of synthetic biology. In contrast to random strategies, semirational approaches such as saturation mutagenesis explore the sequence space in a focused manner. Although several combinatorial libraries based on saturation mutagenesis have been reported using solid-phase gene synthesis, direct comparison with traditional PCR-based methods is currently lacking. In this work, we compare combinatorial protein libraries created in-house via PCR versus those generated by commercial solid-phase gene synthesis. Using descriptive statistics and probabilistic distributions on amino acid occurrence frequencies, the quality of the libraries was assessed and compared, revealing that the outsourced libraries are characterized by less bias and outliers than the PCR-based ones. Afterward, we screened all libraries following a traditional algorithm for almost complete library coverage and compared this approach with an emergent statistical concept suggesting screening a lower portion of the protein sequence space. Upon analyzing the biocatalytic landscapes and best hits of all combinatorial libraries, we show that the screening effort could have been reduced in all cases by more than 50%, while still finding at least one of the best mutants.

History