title: Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris.
abstract: BACKGROUND
  Random mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. Error-prone PCR is often employed for random mutagenesis in bacterial protein expression systems, but has rarely been used in the methylotrophic yeast Pichia pastoris system, despite its significant advantages, mainly because large (μg-level) amounts of plasmids are required for transformation.
  
  
  RESULTS
  We developed a quick and easy technique for random mutagenesis in P. pastoris by sequential Phi29 DNA polymerase-based amplification methods, error-prone rolling circle amplification (RCA) and multiple displacement amplification (MDA). The methodology was validated by applying it for random mutation of the gene encoding cellulase from the basidiomycete Phanerochaete chrysosporium (PcCel6A), a key enzyme in degradation of cellulosic biomass. In the error-prone RCA step, the concentrations of manganese ion (Mn(2+)) and cellulase gene-containing plasmid were varied, and the products obtained under each condition were subjected to the second MDA step in the absence of Mn(2+). The maximum error rate was 2.6 mutations/kb, as evaluated from the results of large-scale sequencing. Several μg of MDA products was transformed by electroporation into Pichia cells, and the activities of extracellularly expressed PcCel6A mutants towards crystalline and amorphous celluloses were compared with those of wild-type enzyme to identify key amino acid residues affecting degradation of crystalline cellulose.
  
  
  CONCLUSIONS
  We present a rapid and convenient random mutagenesis method that does not require laborious steps such as ligation, cloning, and synthesis of specific primers. This method was successfully applied to the protein expression system in P. pastoris.
authors.affiliation: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Tachioka
authors.affiliation: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan ; Biomaterial in Tokyo Co., Ltd., Fukuoka Lab, Ōnojō, Fukuoka 816-0905 Japan.
authors.forename: Naohisa
authors.initials: N
authors.lastname: Sugimoto
authors.affiliation: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan ; Institute for Molecular Science, National Institute of Natural Sciences, Okazaki, 444-8787 Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakamura
authors.affiliation: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Sunagawa
authors.affiliation: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Ishida
authors.affiliation: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Uchiyama
authors.affiliation: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan.
authors.forename: Kiyohiko
authors.identifier: 0000-0001-5152-7177
authors.initials: K
authors.lastname: Igarashi
authors.affiliation: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Samejima
keywords: Cellulase; Error-prone RCA; Phi29 DNA polymerase; Pichia pastoris; Random mutagenesis