Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome

IF 14.7 1区 综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES
Hugh D. Goold, Heinrich Kroukamp, Paige E. Erpf, Yu Zhao, Philip Kelso, Julie Calame, John J. B. Timmins, Elizabeth L. I. Wightman, Kai Peng, Alexander C. Carpenter, Briardo Llorente, Carmen Hawthorne, Samuel Clay, Niël van Wyk, Elizabeth L. Daniel, Fergus Harrison, Felix Meier, Robert D. Willows, Yizhi Cai, Roy S. K. Walker, Xin Xu, Monica I. Espinosa, Giovanni Stracquadanio, Joel S. Bader, Leslie A. Mitchell, Jef D. Boeke, Thomas C. Williams, Ian T. Paulsen, Isak S. Pretorius
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Abstract

The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5’ UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.

Abstract Image

903kb合成酿酒酵母染色体synXVI的构建与迭代再设计
Sc2.0全球联盟于2006年开始设计和构建基于酿酒酵母基因组的合成基因组,包括16条合成染色体和一条新的自然tRNA新染色体。本文介绍了Sc2.0项目中902994 bp合成酿酒酵母染色体synXVI的组装与调试。应用CRISPR D-BUGS协议鉴定出缺陷位点,并对其进行修饰,使其在37˚C下以甘油作为唯一碳源生长时,能够促进产孢并恢复野生型生长。插入可疑开放阅读框下游的LoxPsym位点影响了最佳生长所需基因的5 ' UTR,并被确定为生长缺陷的系统性原因。基于Sc2.0缺陷和synXVI分析的经验教训,对synXVI染色体进行了计算机再设计,可作为未来合成酵母基因组设计的蓝图。synXVI的芯片重新设计包括减少PCR标签频率,修改块和超大块末端,以及调整loxPsym位点和TAA终止密码子分配给可疑的orf。这一重新设计为Sc2.0策略在非酵母生物中的应用提供了路线图。
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来源期刊
Nature Communications
Nature Communications Biological Science Disciplines-
CiteScore
24.90
自引率
2.40%
发文量
6928
审稿时长
3.7 months
期刊介绍: Nature Communications, an open-access journal, publishes high-quality research spanning all areas of the natural sciences. Papers featured in the journal showcase significant advances relevant to specialists in each respective field. With a 2-year impact factor of 16.6 (2022) and a median time of 8 days from submission to the first editorial decision, Nature Communications is committed to rapid dissemination of research findings. As a multidisciplinary journal, it welcomes contributions from biological, health, physical, chemical, Earth, social, mathematical, applied, and engineering sciences, aiming to highlight important breakthroughs within each domain.
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