title: Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains.
abstract: Chlamydomonas is a very attractive candidate plant cell factory. However, its main drawback is the difficulty to find the transformants that robustly express heterologous genes randomly inserted in the nuclear genome. We previously showed that domestic squalene synthase (SQS) gene of Chlamydomonas was much more efficiently overexpressed in a mutant strain [UV-mediated mutant (UVM) 4] than in wild type. In this study, we evaluated the possibility of a new mutant strain, met1, which contains a tag in the maintenance type methyltransferase gene that is expected to play a key role in the maintenance of transcriptional gene silencing. The versatile usefulness of the UVM4 strain to express heterologous genes was also analyzed. We failed to overexpress CrSSL3 cDNA, which is the codon-adjusted squalene synthase-like gene originated from Botryococcus braunii, using the common expression cassette in the wild-type CC-1690 and UVM4 strains. However, we succeeded in isolating western blot-positive transformants through the combinational use of the UVM4 strain and ble2A expression system of which expression cassette bears a fused ORF of the target gene and the antibiotic resistance gene ble via the foot-and-mouth disease virus (FMDV) self-cleaving 2A sequence. It is noteworthy that even with this system, huge deviations in the accumulated protein levels were still observed among the UVM4 transformants.
authors: School of Environmental Science and Engineering, Kochi University of Technology (KUT), Tosayamada, Kochi 782-8502, Japan.
authors: Fantao
authors: F
authors: Kong
authors: School of Environmental Science and Engineering, Kochi University of Technology (KUT), Tosayamada, Kochi 782-8502, Japan.
authors: Tomohito
authors: T
authors: Yamasaki
authors: School of Environmental Science and Engineering, Kochi University of Technology (KUT), Tosayamada, Kochi 782-8502, Japan.
authors: Sari Dewi
authors: SD
authors: Kurniasih
authors: School of Environmental Science and Engineering, Kochi University of Technology (KUT), Tosayamada, Kochi 782-8502, Japan.
authors: Liyuan
authors: L
authors: Hou
authors: Carnegie Institution for Science, 260 Panama St. Stanford, CA 94305, USA.
authors: Xiaobo
authors: X
authors: Li
authors: Carnegie Institution for Science, 260 Panama St. Stanford, CA 94305, USA.
authors: Nina
authors: N
authors: Ivanova
authors: 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; Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda, Tokyo 102-0075, Japan.
authors: Shigeru
authors: S
authors: Okada
authors: School of Environmental Science and Engineering, Kochi University of Technology (KUT), Tosayamada, Kochi 782-8502, Japan. Electronic address: ohama.takeshi@kochi-tech.ac.jp.
authors: Takeshi
authors: T
authors: Ohama
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000378:metabolism; D000460:Chlorophyta / Q000235:genetics; D003062:Codon / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics; D015870:Gene Expression; D020868:Gene Silencing; D005819:Genetic Markers / Q000235:genetics*; D008780:Methyltransferases / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*
chemical_list: D003062:Codon; D018076:DNA, Complementary; D005819:Genetic Markers; D008780:Methyltransferases; D013186:Farnesyl-Diphosphate Farnesyltransferase
keywords: Botryococcus braunii; Methyltransferase gene; Position effect; Squalene synthase-like gene; Transgene silencing