title: UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems.
abstract: In this investigation, we succeeded to generate Chlamydomonas mutants that bear dramatically enhanced ability for transgene expression. To yield these mutants, we utilized DNA methyltransferase deficient strain. These mutants must be useful as a plant cell factory. Chlamydomonas reinhardtii (hereafter Chlamydomonas) is a green freshwater microalga. It is a promising cell factory for the production of recombinant proteins because it rapidly grows in simple salt-based media. However, expression of transgenes integrated into the nuclear genome of Chlamydomonas is very poor, probably because of severe transcriptional silencing irrespective of the genomic position. In this study, we generated Chlamydomonas mutants by ultraviolet (UV)-mediated mutagenesis of maintenance-type DNA methyltransferase gene (MET1)-null mutants to overcome this disadvantage. We obtained several mutants with an enhanced ability to overexpress various transgenes irrespective of their integrated genomic positions. In addition, transformation efficiencies were significantly elevated. Our findings indicate that in addition to mechanisms involving MET1, transgene expression is regulated by a DNA methylation-independent transgene silencing system in Chlamydomonas. This is in agreement with the fact that DNA methylation occurs rarely in this organism. The generated mutants may be useful for the low-cost production of therapeutic proteins and eukaryotic enzymes based on their rapid growth in simple salt-based media.
authors.affiliation: School of Environmental Science and Engineering, Kochi University of Technology, Tosayamada, Kochi, 782-8502, Japan.
authors.forename: Sari Dewi
authors.initials: SD
authors.lastname: Kurniasih
authors.affiliation: School of Environmental Science and Engineering, Kochi University of Technology, Tosayamada, Kochi, 782-8502, Japan.
authors.forename: Tomohito
authors.initials: T
authors.lastname: Yamasaki
authors.affiliation: School of Environmental Science and Engineering, Kochi University of Technology, Tosayamada, Kochi, 782-8502, Japan.
authors.forename: Fantao
authors.initials: F
authors.lastname: Kong
authors.affiliation: Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural & Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Sigeru
authors.initials: S
authors.lastname: Okada
authors.affiliation: School of Environmental Science and Engineering, Kochi University of Technology, Tosayamada, Kochi, 782-8502, Japan.
authors.forename: Dwiyantari
authors.initials: D
authors.lastname: Widyaningrum
authors.affiliation: School of Environmental Science and Engineering, Kochi University of Technology, Tosayamada, Kochi, 782-8502, Japan. ohama.takeshi@kochi-tech.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ohama
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000528:radiation effects; D019175:DNA Methylation / Q000235:genetics* / Q000528:radiation effects; D018506:Gene Expression Regulation, Plant / Q000528:radiation effects*; D020868:Gene Silencing / Q000502:physiology; D009154:Mutation / Q000235:genetics* / Q000528:radiation effects; D019076:Transgenes / Q000235:genetics* / Q000528:radiation effects; D014466:Ultraviolet Rays
keywords: Chlamydomonas; Heterologous gene expression; Maintenance-type DNA methyltransferase; Transcriptional gene silencing; UV-mediated mutation