酿酒酵母染色体设计与合成研究进展

随着合成生物学的蓬勃发展,基因组学的研究正在由读取基因组信息拓展到以编写基因组信息为主的合成基因组学时代。2009年,由Jef D. Boeke教授提出的人工合成酵母基因组计划(Sc2.0)旨在合成世界上首个真核生物基因组。在美、中、英、法、澳大利亚、新加坡等多国科学家的努力下,目前已经完成1/3的酵母染色体的人工合成。本文从合成基因组学领域的发展历程出发,介绍了Sc2.0计划中酿酒酵母(Saccharomyces cerevisiae)染色体设计与合成的最新进展,包括酿酒酵母9号染色体右臂、3号染色体、2号染色体、5号染色体、6号染色体、10号染色体和12号染色体的设计与合成过程,阐述了其各自的合成策略以及生物学意义,以期为合成基因组学的深入开展提供借鉴与参考。     

qPCRTag Analysis - A High Throughput,Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII¹, and one synthetic chromosome arm, synIXR², have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.     

Rewriting the blueprint of life by synthetic genomics and genome engineering

Advances in DNA synthesis and assembly methods over the past decade have made it possible to construct genome-size fragments from oligonucleotides. Early work focused on synthesis of small viral genomes, followed by hierarchical synthesis of wild-type bacterial genomes and subsequently on transplantation of synthesized bacterial genomes into closely related recipient strains. More recently, a synthetic designer version of yeast Saccharomyces cerevisiae chromosome III has been generated, with numerous changes from the wild-type sequence without having an impact on cell fitness and phenotype, suggesting plasticity of the yeast genome. A project to generate the first synthetic yeast genome - the Sc2.0 Project - is currently underway.     

合成基因组学：设计与合成的艺术

随着基因组相关技术(测序、编辑、合成等)和知识(功能基因组学)的日益成熟,合成基因组学在本世纪迎得了发展的契机。病毒、原核生物的全基因组相继被化学合成并支持生命的存活,第1个真核生物合成基因组计划已经完成过半,人类基因组编写计划提上日程。在基因组合成的实践过程中,研究者们不断探索对基因组进行重编和设计所应遵循的规则,提高从头合成、组装和替换基因组的技术手段。合成基因组在工业、环境、健康和基础研究领域有着广阔的应用前景,同时也带来了相应的伦理问题。结合在Sc2.0计划中的基因组合成研究和近期合成基因组学所取得的重大进展,本文综述了基因组设计和合成相关的科学、技术和伦理内容,并探讨了未来发展所面对的挑战。作为合成生物学最重要的领域之一,合成基因组学方兴未艾。     

Synthetic genome engineering forging new frontiers for wine yeast

Over the past 15 years, the seismic shifts caused by the convergence of biomolecular, chemical, physical, mathematical, and computational sciences alongside cutting-edge developments in information technology and engineering have erupted into a new field of scientific endeavor dubbed Synthetic Biology. Recent rapid advances in high-throughput DNA sequencing and DNA synthesis techniques are enabling the design and construction of new biological parts (genes), devices (gene networks) and modules (biosynthetic pathways), and the redesign of biological systems (cells and organisms) for useful purposes. In 2014, the budding yeast Saccharomyces cerevisiae became the first eukaryotic cell to be equipped with a fully functional synthetic chromosome. This was achieved following the synthesis of the first viral (poliovirus in 2002 and bacteriophage Phi-X174 in 2003) and bacterial (Mycoplasma genitalium in 2008 and Mycoplasma mycoides in 2010) genomes, and less than two decades after revealing the full genome sequence of a laboratory (S288c in 1996) and wine (AWRI1631 in 2008) yeast strain. A large international project – the Synthetic Yeast Genome (Sc2.0) Project – is now underway to synthesize all 16 chromosomes (～12?Mb carrying ～6000 genes) of the sequenced S288c laboratory strain by 2018. If successful, S. cerevisiae will become the first eukaryote to cross the horizon of in silico design of complex cells through de novo synthesis, reshuffling, and editing of genomes. In the meantime, yeasts are being used as cell factories for the semi-synthetic production of high-value compounds, such as the potent antimalarial artemisinin, and food ingredients, such as resveratrol, vanillin, stevia, nootkatone, and saffron. As a continuum of previously genetically engineered industrially important yeast strains, precision genome engineering is bound to also impact the study and development of wine yeast strains supercharged with synthetic DNA. The first taste of what the future holds is the de novo production of the raspberry ketone aroma compound, 4-[4-hydroxyphenyl]butan-2-one, in a wine yeast strain (AWRI1631), which was recently achieved via metabolic pathway engineering and synthetic enzyme fusion. A peek over the horizon is revealing that the future of “Wine Yeast 2.0” is already here. Therefore, this article seeks to help prepare the wine industry – an industry rich in history and tradition on the one hand, and innovation on the other – for the inevitable intersection of the ancient art practiced by winemakers and the inventive science of pioneering “synthetic genomicists”. It would be prudent to proactively engage all stakeholders – researchers, industry practitioners, policymakers, regulators, commentators, and consumers – in a meaningful dialog about the potential challenges and opportunities emanating from Synthetic Biology. To capitalize on the new vistas of synthetic yeast genomics, this paper presents wine yeast research in a fresh context, raises important questions and proposes new directions.     

The Saccharomyces cerevisiae SCRaMbLE system and genome minimization

We have recently reported the first partially synthetic eukaryotic genome. Saccharomyces cerevisiae chromosomes synIXR and semi-synVIL are fully synthetic versions of the right arm of chromosome IX and the telomeric segment of the left arm of chromosome VI, respectively, and represent the beginning of the synthetic yeast genome project, Sc2.0, that progressively replaces native yeast DNA with synthetic sequences. We have designed synthetic chromosome sequences according to principles specifying a wild-type phenotype, highly stable genome, and maintenance of genetic flexibility. Although other synthetic genome projects exist, the Sc2.0 approach is unique in that we have implemented design specifications predicted to generate a wild-type phenotype until induction of "SCRaMbLE," an inducible evolution system that generates significant genetic diversity. Here we further explore the significance of Sc2.0 and show how SCRaMbLE can serve as a genome minimization tool.     

合成型酵母基因组重排技术

基因组的结构变异是生物体表型进化的重要驱动力之一。设计与合成酵母基因组为人工基因组结构变异提供了新途径。人工合成酿酒酵母基因组(Sc2.0)通过系统性地引入重排元件,赋予了基因组柔性可变的功能,可诱导产生 DNA 片段的删除、反转、复制、移位等基因组结构变异。合成型酵母基因组重排技术可实现菌株性状的快速进化,并且为研究基因组结构变异与表型变化间的关系提供了一种快速、全新的方法。综述了合成型酵母基因组重排技术的研究热点和技术进展,并展示了其在创新菌种中的应用价值。     

A Personalist Ontological Approach to Synthetic Biology

Although synthetic biology is a promising discipline, it also raises serious ethical questions that must be addressed in order to prevent unwanted consequences and to ensure that its progress leads toward the good of all. Questions arise about the role of this discipline in a possible redefinition of the concept of life and its creation. With regard to the products of synthetic biology, the moral status that they should be given as well as the ethically correct way to behave towards them are not clear. Moreover, risks that could result from a misuse of this technology or from an accidental release of synthetic organisms into the environment cannot be ignored; concerns about biosecurity and biosafety appear. Here we discuss these and other questions from a personalist ontological framework, which defends human life as an essential value and proposes a set of principles to ensure the safeguarding of this and other values that are based on it.     

Synthetic biology - the state of play

Just over two years ago there was an article in Nature entitled "Five Hard Truths for Synthetic Biology". Since then, the field has moved on considerably. A number of economic commentators have shown that synthetic biology very significant industrial potential. This paper addresses key issues in relation to the state of play regarding synthetic biology. It first considers the current background to synthetic biology, whether it is a legitimate field and how it relates to foundational biological sciences. The fact that synthetic biology is a translational field is discussed and placed in the context of the industrial translation process. An important aspect of synthetic biology is platform technology, this topic is also discussed in some detail. Finally, examples of application areas are described.     

Synthetic Genomics and Synthetic Biology Applications Between Hopes and Concerns

New organisms and biological systems designed to satisfy human needs are among the aims of synthetic genomics and synthetic biology. Synthetic biology seeks to model and construct biological components, functions and organisms that do not exist in nature or to redesign existing biological systems to perform new functions. Synthetic genomics, on the other hand, encompasses technologies for the generation of chemically-synthesized whole genomes or larger parts of genomes, allowing to simultaneously engineer a myriad of changes to the genetic material of organisms. Engineering complex functions or new organisms in synthetic biology are thus progressively becoming dependent on and converging with synthetic genomics. While applications from both areas have been predicted to offer great benefits by making possible new drugs, renewable chemicals or clean energy, they have also given rise to concerns about new safety, environmental and socio-economic risks – stirring an increasingly polarizing debate. Here we intend to provide an overview on recent progress in biomedical and biotechnological applications of synthetic genomics and synthetic biology as well as on arguments and evidence related to their possible benefits, risks and governance implications.     

Synthetic biology: ethical ramifications 2009

During 2007 and 2008 synthetic biology moved from the manifesto stage to research programs. As of 2009, synthetic biology is ramifying; to ramify means to produce differentiated trajectories from previous determinations. From its inception, most of the players in synthetic biology agreed on the need for (a) rationalized design and construction of new biological parts, devices, and systems as well as (b) the re-design of natural biological systems for specified purposes, and that (c) the versatility of designed biological systems makes them suitable to address such challenges as renewable energy, the production of inexpensive drugs, and environmental remediation, as well as providing a catalyst for further growth of biotechnology. What is understood by these goals, however, is diverse. Those assorted understandings are currently contributing to different ramifications of synthetic biology. The Berkeley Human Practices Lab, led by Paul Rabinow, is currently devoting its efforts to documenting and analyzing these ramifications as they emerge.     

合成生物学应用产品开发现状与趋势

目的：从产品开发角度分析全球合成生物学发展现状和趋势。方法：在伍德罗·威尔逊国际学者中心的合成生物学产品和应用清单（synthetic biology products and applications inventory）的数据基础上,对全球合成生物学产品的开发状态、市场应用和发展前景等进行补充检索和分析。结果：至2015年,全球至少已有81家企业（或研究机构）的116种合成生物学产品得到了市场应用开发,主要开发者为美国企业（或研究机构）,产品主要集中于化学和医药领域。结论：合成生物学实现了从生物学分析向生物学合成的范式转移,其产品开发将给一系列的行业带来深刻的变革。     

Synthetic promoter libraries for Corynebacterium glutamicum

The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7–59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.     

植物合成生物学领域发展态势的文献计量分析

合成生物学作为一种颠覆性技术可应用于农业领域的创新发展,解决当前农业学科中的瓶颈问题。利用文献计量学方法从领域发表论文的时序数量分布、主题分布等探测当前合成生物学的基本态势。基于领域的主题分布可知,其中植物合成生物学这一主题是稳定存在的且主题规模处于稳定增长趋势。聚焦植物合成生物学这一主题方向,在构建引文网络的基础上利用主路径分析方法从知识流动角度探测植物合成生物学领域重要知识节点,内容涵盖介子油苷生物合成途径,重要催化酶功能解析、转录因子的调控作用,组学方法的应用,利用微生物酵母进行生物物质合成,这些内容表征了合成生物的核心理论技术。     

植物合成生物学领域发展态势的文献计量分析

合成生物学作为一种颠覆性技术可应用于农业领域的创新发展,解决当前农业学科中的瓶颈问题。利用文献计量学方法从领域发表论文的时序数量分布、主题分布等探测当前合成生物学的基本态势。基于领域的主题分布可知,其中植物合成生物学这一主题是稳定存在的且主题规模处于稳定增长趋势。聚焦植物合成生物学这一主题方向,在构建引文网络的基础上利用主路径分析方法从知识流动角度探测植物合成生物学领域重要知识节点,内容涵盖介子油苷生物合成途径,重要催化酶功能解析、转录因子的调控作用,组学方法的应用,利用微生物酵母进行生物物质合成,这些内容表征了合成生物的核心理论技术。     

Synthetic biology: mapping the scientific landscape

This article uses data from Thomson Reuters Web of Science to map and analyse the scientific landscape for synthetic biology. The article draws on recent advances in data visualisation and analytics with the aim of informing upcoming international policy debates on the governance of synthetic biology by the Subsidiary Body on Scientific, Technical and Technological Advice (SBSTTA) of the United Nations Convention on Biological Diversity. We use mapping techniques to identify how synthetic biology can best be understood and the range of institutions, researchers and funding agencies involved. Debates under the Convention are likely to focus on a possible moratorium on the field release of synthetic organisms, cells or genomes. Based on the empirical evidence we propose that guidance could be provided to funding agencies to respect the letter and spirit of the Convention on Biological Diversity in making research investments. Building on the recommendations of the United States Presidential Commission for the Study of Bioethical Issues we demonstrate that it is possible to promote independent and transparent monitoring of developments in synthetic biology using modern information tools. In particular, public and policy understanding and engagement with synthetic biology can be enhanced through the use of online interactive tools. As a step forward in this process we make existing data on the scientific literature on synthetic biology available in an online interactive workbook so that researchers, policy makers and civil society can explore the data and draw conclusions for themselves.     

Synthetic biology and genetic causation

Synthetic biology research is often described in terms of programming cells through the introduction of synthetic genes. Genetic material is seemingly attributed with a high level of causal responsibility. We discuss genetic causation in synthetic biology and distinguish three gene concepts differing in their assumptions of genetic control. We argue that synthetic biology generally employs a difference-making approach to establishing genetic causes, and that this approach does not commit to a specific notion of genetic program or genetic control. Still, we suggest that a strong program concept of genetic material can be used as a successful heuristic in certain areas of synthetic biology. Its application requires control of causal context, and may stand in need of a modular decomposition of the target system. We relate different modularity concepts to the discussion of genetic causation and point to possible advantages of and important limitations to seeking modularity in synthetic biology systems.     

合成生物学与天然产物开发

天然产物依然是临床用药的重要来源。合成生物学的诞生为天然产物的开发提供了全新的机遇,传统的微生物药物、植物天然产物等研究领域都因合成生物学而获得新生。重点介绍了合成生物学在天然产物开发中的应用,包括新化合物及其生物合成元件的筛选,基于理性设计的天然产物异源生物合成,人工底盘细胞的系统优化等。     

Teaching Synthetic Biology, Bioinformatics and Engineering to Undergraduates: The Interdisciplinary Build-a-Genome Course

A major challenge in undergraduate life science curricula is the continual evaluation and development of courses that reflect the constantly shifting face of contemporary biological research. Synthetic biology offers an excellent framework within which students may participate in cutting-edge interdisciplinary research and is therefore an attractive addition to the undergraduate biology curriculum. This new discipline offers the promise of a deeper understanding of gene function, gene order, and chromosome structure through the de novo synthesis of genetic information, much as synthetic approaches informed organic chemistry. While considerable progress has been achieved in the synthesis of entire viral and prokaryotic genomes, fabrication of eukaryotic genomes requires synthesis on a scale that is orders of magnitude higher. These high-throughput but labor-intensive projects serve as an ideal way to introduce undergraduates to hands-on synthetic biology research. We are pursuing synthesis of Saccharomyces cerevisiae chromosomes in an undergraduate laboratory setting, the Build-a-Genome course, thereby exposing students to the engineering of biology on a genomewide scale while focusing on a limited region of the genome. A synthetic chromosome III sequence was designed, ordered from commercial suppliers in the form of oligonucleotides, and subsequently assembled by students into ~750-bp fragments. Once trained in assembly of such DNA “building blocks” by PCR, the students accomplish high-yield gene synthesis, becoming not only technically proficient but also constructively critical and capable of adapting their protocols as independent researchers. Regular “lab meeting” sessions help prepare them for future roles in laboratory science.