基因组设计与合成：从复写到理性设计

doi:10.12211/2096-8208.2020-051

摘要/Abstract

摘要：

基因组合成相关技术的进步推动人工基因组合成能力不断取得突破，合成基因组学成为了近年来的研究热点，逐步完成了病毒、原核生物基因组的全合成，真核生物基因组设计合成也取得了阶段性突破。在基因组合成的研究过程中，基因组设计的原则不断拓展，基因组设计的尺度由病毒及噬菌体基因组成功复写发展到蕈状支原体JCVI-syn3.0基因组大幅度简化，在真核生物酿酒酵母基因组合成中探索多种基因组设计原则。本文主要综述了人工基因组设计的相关进展，主要内容包括：①人工基因组密码子的改变：外源基因密码子的优化，密码子丰度转变原则的探究，大肠杆菌中密码子的删除；②人工标签的添加及应用：利用同义密码子替换在合成型基因组中添加标签以区分合成型与野生型基因组；③人工位点的添加：酿酒酵母合成型染色体重组系统的开发，优化及应用；④基因组简化方面的研究；⑤对基因组理性设计、基因组简化规律挖掘等进行了展望。

关键词: 合成基因组学, 基因组设计, 密码子删除, 合成型标签, SCRaMbLE, 基因组简化

Abstract:

With the advancement of related technologies for gene assembly, artificial genome assembly capabilities have made breakthroughs continuously, and synthetic genomics has been developed as a research hotspot in recent years. The chemical synthesis of genomes for several viruses and prokaryotes have been completed gradually, and the synthesis of eukaryotic genomes has also been explored. In the study of synthetic genomics, researchers continue to explore principles for genome design and increase its scale and depth, from the successful replication of virus and bacteriophage genomes, the substantial simplification in the Mycoplasma mycoides JCVI-syn3.0 genome to exploring multiple genome design principles in the eukaryotic Saccharomyces cerevisiae. This article summarizes the related progress of artificial genome design, with its main contents focused on: the change of artificial genome codons, the addition of artificial tags, the insertion of artificial sites and the research of genome simplification. In 2016, Church's group used synonymous codons to replace seven codons within the entire genome of E. coli, but the E. coli strain could not survive. In 2019, Chin's research group completed the synthesis of E. coli containing 61 codons using the method of complete genome synthesis, and the E. coli strain can survive normally. Four "watermark" sequences were introduced into the synthetic M. mycoides genome, and a large number of PCR tags were introduced into the synthetic Saccharomyces cerevisiae genome to distinguish between synthetic and wild-type genomes. A highlight of the synthetic yeast chromosome design in the Sc2.0 is the insertion of a reverse symmetric artificial site-loxPsym sequence after the stop codon of each non-essential gene. As a result, a SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution) system that can rapidly perform genome rearrangement including deletion, duplication, inversion and translocation was formed in the synthetic yeast. The system has been continuously improved in applications, gradually making it an effective mean to optimize the host, increase the yield and enhance stress tolerance of the strain. In addition, the prospect of the rational design of genomes and rules for genome simplification is also discussed.

Key words: synthetic genomics, genome design, codon deletion, synthetic tag, SCRaMbLE, genome simplification

中图分类号:

汪君仪, 武晓乐, 曹月阳, 李炳志. 基因组设计与合成：从复写到理性设计[J]. 合成生物学, 2021, 2(2): 247-255.

Junyi WANG, Xiaole WU, Yueyang CAO, Bingzhi LI. Genome design and synthesis: from replication to rational design[J]. Synthetic Biology Journal, 2021, 2(2): 247-255.

图/表 2

图1 密码子删除相关研究进展[16,18]（2016年Church团队在大肠杆菌全基因组范围内使用同义密码子系统地替换了7个密码子，90%以上的基因仍具有功能[16] ；2019年Chin团队重新编码了大肠杆菌的18 214个密码子，创建了使用61个密码子的生物[18]）

Fig. 1 Research progress on codons deletion[16,18](In 2016, seven codons were replaced with synonymous alternatives the entire genome of E. coli by Church's group, more than 90% of the genes retained functionality[16]; In 2019, Chin's group recoded 18 214 codons to create a strain with a 61-codon genome [18])

图2 基因组简化的研究进展[19,50-51][(a)利用Tn5转座子的衍生物将大肠杆菌K-12的基因组缩减200 kb[50]；(b)利用LATOUR删除法将粟酒裂殖酵母基因组缩减657.3 kb[51]；(c)利用全基因组设计和化学合成的方法简化支原体JCVI-syn1.0(1079 kb)，通过设计、合成、测试三个步骤的循环，保留了必需基因和准必需基因，得到支原体JCVI-syn3.0（531 kb,473个基因）[19]]

Fig. 2 Advances in genome simplification[19,50-51][(a) Simplification of the E. coli K-12 genome. The deletion procedure has reduced the genome at an average of 200 kb by using specialized transposons (Tn5 derivatives) to create deletions in the E. coli K-12 chromosome[50]. (b) Simplification of the Schizosaccharomyces pombe genome. Researchers have reduced the genome of S. pombe by 657.3 kb using a large-scale gene deletion method called LATOUR[51]. (c) Simplification and chemical synthesis of M. mycoides genome. Using whole-genome design and complete chemical synthesis, researchers have minimized the 1079-kilobase pair synthetic genome of M. mycoides JCVI-syn1.0. Three cycles of design, synthesis, and testing, with the retention of essential and quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes)[19]]

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