title: Directed evolution of plant basic helix-loop-helix transcription factors for the improvement of transactivational properties.
abstract: Myc-RP from Perilla frutescens and Delila from Antirrhinum majus, two plant basic helix-loop-helix transcription factors (bHLH TFs) involved in the flavonoid biosynthetic pathway, have been used for the improvement of transactivational properties by directed evolution. Through two rounds of DNA shuffling, Myc-RP variants with up to 70-fold increase in transcriptional activities have been identified using a yeast transactivation system. In a tobacco protoplast transient expression assay, one of the most improved variants, M2-1, also shows significant increase of transactivation. The majority of resulting mutations (approximately 53%) are localized in the acidic (activation) domains of the improved Myc-RP variants. In variant M2-1, three of the four mutations (L301P/N354D/S401F) are in the acidic domain. The fourth mutation (K157M) is localized to a helix within the N-terminal interaction domain. Combinatorial site-directed mutagenesis reveals that, while the acidic domain mutations contribute modestly to the increase in activity, the K157M substitution is responsible for 80% of the improvement observed in variant M2-1. The transactivation activity of the K157M/N354D double mutant is equal to that of M2-1. These results suggest that the interaction domain plays a critical role in transactivation of these bHLH TFs. Delila variants have also been screened for increased activities toward the Arabidopsis chalcone synthase (CHS) promoter, a pathway promoter that responds weakly to the bHLH TFs. Variants with increased activity on the CHS promoter, while maintaining wildtype-level activities on the naturally responsive dihydroflavonol reductase promoter, have been obtained. This study demonstrates that functional properties of TFs can be modified by directed evolution.
authors.affiliation: Department of Plant and Soil Sciences, University of Kentucky, Cooper and University Drives, Lexington, KY 40546, USA.
authors.forename: Sitakanta
authors.initials: S
authors.lastname: Pattanaik
authors.forename: Claire H
authors.initials: CH
authors.lastname: Xie
authors.forename: Que
authors.initials: Q
authors.lastname: Kong
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Shen
authors.forename: Ling
authors.initials: L
authors.lastname: Yuan
mesh_terms: D000217:Acyltransferases / Q000235:genetics; D000595:Amino Acid Sequence; D032161:Antirrhinum / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D018744:DNA, Plant / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D017343:Genes, Plant; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D031344:Perilla frutescens / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010944:Plants / Q000235:genetics* / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins; D014157:Transcription Factors; C400767:myc-rp protein, Perilla frutescens; D000217:Acyltransferases; C020141:flavanone synthetase