Yarimizu Tohru, Nakamura Mikiko, Hoshida Hisashi, Akada Rinji
Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai 2-16-1, Ube 755-8611, Japan.
Innovation Center, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan.
J Biosci Bioeng. 2015 Mar;119(3):251-9. doi: 10.1016/j.jbiosc.2014.08.006. Epub 2014 Sep 6.
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.
由于合成的基因常常含有突变，因此需要无错误基因合成的方法。通过在酵母中克隆与选择标记融合的PCR组装寡核苷酸片段，我们开发了一种在基因合成中筛选准确克隆的新方法。作为一个模型案例，合成了来自高斯海萤（GLuc）的555bp荧光素酶基因，使其包含酵母优化密码子（以下称为yGLuc）。经过标准的PCR介导的寡核苷酸组装后，许多克隆没有显示出荧光素酶活性。在这些克隆中，大多数含有随机定位的核苷酸缺失，这些缺失产生移码并导致提前终止。为了排除提前终止的克隆，将合成的yGLuc基因框内克隆，与URA3编码序列融合，URA3编码序列在马克斯克鲁维酵母中作为选择标记。预期Ura（+）转化选择将消除具有移码错误的克隆。结果表明，框内标记选择将活性yGLuc基因的频率提高到了79%。我们使用这种策略合成了来自米根霉的编码葡糖淀粉酶的1812bp基因。七个Ura（+）克隆中有五个表现出淀粉酶活性。在功能克隆中，一个含有正确的序列，四个含有核苷酸变化的序列，这表明框内选择经常产生功能突变体。马克斯克鲁维酵母非同源末端连接介导的基因合成克隆方法将对合成生物学研究有用。
