title: Novel strategy for protein exploration: high-throughput screening assisted with fuzzy neural network.
abstract: To engineer proteins with desirable characteristics from a naturally occurring protein, high-throughput screening (HTS) combined with directed evolutional approach is the essential technology. However, most HTS techniques are simple positive screenings. The information obtained from the positive candidates is used only as results but rarely as clues for understanding the structural rules, which may explain the protein activity. In here, we have attempted to establish a novel strategy for exploring functional proteins associated with computational analysis. As a model case, we explored lipases with inverted enantioselectivity for a substrate p-nitrophenyl 3-phenylbutyrate from the wild-type lipase of Burkhorderia cepacia KWI-56, which is originally selective for (S)-configuration of the substrate. Data from our previous work on (R)-enantioselective lipase screening were applied to fuzzy neural network (FNN), bioinformatic algorithm, to extract guidelines for screening and engineering processes to be followed. FNN has an advantageous feature of extracting hidden rules that lie between sequences of variants and their enzyme activity to gain high prediction accuracy. Without any prior knowledge, FNN predicted a rule indicating that "size at position L167," among four positions (L17, F119, L167, and L266) in the substrate binding core region, is the most influential factor for obtaining lipase with inverted (R)-enantioselectivity. Based on the guidelines obtained, newly engineered novel variants, which were not found in the actual screening, were experimentally proven to gain high (R)-enantioselectivity by engineering the size at position L167. We also designed and assayed two novel variants, namely FIGV (L17F, F119I, L167G, and L266V) and FFGI (L17F, L167G, and L266I), which were compatible with the guideline obtained from FNN analysis, and confirmed that these designed lipases could acquire high inverted enantioselectivity. The results have shown that with the aid of bioinformatic analysis, high-throughput screening can expand its potential for exploring vast combinatorial sequence spaces of proteins.
authors: School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
authors: Ryuji
authors: R
authors: Kato
authors: Hideo
authors: H
authors: Nakano
authors: Hiroyuki
authors: H
authors: Konishi
authors: Katsuya
authors: K
authors: Kato
authors: Yuchi
authors: Y
authors: Koga
authors: Tsuneo
authors: T
authors: Yamane
authors: Takeshi
authors: T
authors: Kobayashi
authors: Hiroyuki
authors: H
authors: Honda
mesh_terms: D017143:Fuzzy Logic; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D016571:Neural Networks, Computer; D015202:Protein Engineering; D013237:Stereoisomerism
chemical_list: D008049:Lipase