title: Screening of accurate clones for gene synthesis in yeast.
abstract: Methods for error-less gene synthesis are desired because synthesized genes often contain mutations. By cloning PCR-assembled oligonucleotide fragments fused to a selection marker in yeast, we developed a novel method to screen accurate clones in gene synthesis. As a model case, the 555-bp luciferase gene from Gaussia princeps (GLuc) was synthesized to contain yeast-optimized codons (called yGLuc hereafter). After standard PCR-mediated oligonucleotide assembly, many clones showed no luciferase activity. Of these clones, most contained randomly located nucleotide deletions that produced frameshifts and resulted in premature termination. To exclude clones with premature termination, the synthesized yGLuc gene was cloned in-frame to fuse with the URA3 coding sequence, which served as a selection marker in the yeast Kluyveromyces marxianus. Ura(+) transformation selection was expected to eliminate clones with frameshift errors. The results showed that in-frame marker selection increased the frequency of active yGLuc gene to 79%. We used this strategy to synthesize the 1812-bp gene from Rhizopus oryzae that encodes glucoamylase. Five out of seven Ura(+) clones exhibited amylase activity. Of the functional clones, one contained the correct sequence, and four contained sequences with nucleotide changes, suggesting that in-frame selection frequently produced functional mutants. The K. marxianus non-homologous end joining mediated cloning method for gene synthesis will be useful for synthetic biological studies.
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai 2-16-1, Ube 755-8611, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yarimizu
authors.affiliation: Innovation Center, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan.
authors.forename: Mikiko
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai 2-16-1, Ube 755-8611, Japan. Electronic address: hoshida@yamaguchi-u.ac.jp.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai 2-16-1, Ube 755-8611, Japan.
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D033342:Copepoda / Q000235:genetics; D059766:DNA End-Joining Repair; D005819:Genetic Markers / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism; D007716:Kluyveromyces / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012233:Rhizopus / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D005819:Genetic Markers; D008156:Luciferases; D005087:Glucan 1,4-alpha-Glucosidase
keywords: Codon optimization; Gene synthesis; Glucoamylase; Kluyveromyces marxianus; Luciferase; Non-homologous end joining-mediated DNA cloning