Synthetic modular systems--reverse engineering of signal transduction

During the last decades, biology has decomposed cellular systems into genetic, functional and molecular networks. It has become evident that these networks consist of components with specific functions (e.g., proteins and genes). This has generated a considerable amount of knowledge and hypotheses concerning cellular organization. The idea discussed here is to test the extent of this knowledge by reconstructing, or reverse engineering, new synthetic biological systems from known components. We will discuss how integration of computational methods with proteomics and engineering concepts might lead us to a deeper and more abstract understanding of signal transduction systems. Designing and successfully introducing synthetic proteins into cellular pathways would provide us with a powerful research tool with many applications, such as development of biosensors, protein drugs and rewiring of biological pathways.     

Opportunities for yeast metabolic engineering: Lessons from synthetic biology

Constant progress in genetic engineering has given rise to a number of promising areas of research that facilitated the expansion of industrial biotechnology. The field of metabolic engineering, which utilizes genetic tools to manipulate microbial metabolism to enhance the production of compounds of interest, has had a particularly strong impact by providing new platforms for chemical production. Recent developments in synthetic biology promise to expand the metabolic engineering toolbox further by creating novel biological components for pathway design. The present review addresses some of the recent advances in synthetic biology and how these have the potential to affect metabolic engineering in the yeast Saccharomyces cerevisiae. While S. cerevisiae for years has been a robust industrial organism and the target of multiple metabolic engineering trials, its potential for synthetic biology has remained relatively unexplored and further research in this field could strongly contribute to industrial biotechnology. This review also addresses are general considerations for pathway design, ranging from individual components to regulatory systems, overall pathway considerations and whole-organism engineering, with an emphasis on potential contributions of synthetic biology to these areas. Some examples of applications for yeast synthetic biology and metabolic engineering are also discussed.     

合成生物学及其在生物技术中的应用进展

合成生物学是一门21世纪生物学的新兴学科,它着眼生物科学与工程科学的结合,把生物系统当作工程系统"从下往上"进行处理,由"单元"(unit)到"部件"(device)再到"系统"(system)来设计,修改和组装细胞构件及生物系统.合成生物学是分子和细胞生物学、进化系统学、生物化学、信息学、数学、计算机和工程等多学科交叉的产物.目前研究应用包括两个主要方面:一是通过对现有的、天然存在的生物系统进行重新设计和改造,修改已存在的生物系统,使该系统增添新的功能.二是通过设计和构建新的生物零件、组件和系统,创造自然界中尚不存在的人工生命系统.合成生物学作为一门建立在基因组方法之上的学科,主要强调对创造人工生命形态的计算生物学与实验生物学的协同整合.必须强调的是,用来构建生命系统新结构、产生新功能所使用的组件单元既可以是基因、核酸等生物组件,也可以是化学的、机械的和物理的元件.本文跟踪合成生物学研究及应用,对其在DNA水平编程、分子修饰、代谢途径、调控网络和工业生物技术等方面的进展进行综述.     

Protein and RNA engineering to customize microbial molecular reporting

Nature takes advantage of the malleability of protein and RNA sequence and structure to employ these macromolecules as molecular reporters whose conformation and functional roles depend on the presence of a specific ligand (an "effector" molecule). By following nature's example, ligand-responsive proteins and RNA molecules are now routinely engineered and incorporated into customized molecular reporting systems (biosensors). Microbial small-molecule biosensors and endogenous molecular reporters based on these sensing components find a variety of applications that include high-throughput screening of biosynthesis libraries, environmental monitoring, and novel gene regulation in synthetic biology. Here, we review recent advances in engineering small-molecule recognition by proteins and RNA and in coupling in vivo ligand binding to reporter-gene expression or to allosteric activation of a protein conferring a detectable phenotype. Emphasis is placed on microbial screening systems that serve as molecular reporters and facilitate engineering the ligand-binding component to recognize new molecules.     

Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways

Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.     

Structure-driven protein engineering for production of valuable natural products

Proteins are the most frequently used biocatalysts, and their structures determine their functions. Modifying the functions of proteins on the basis of their structures lies at the heart of protein engineering, opening a new horizon for metabolic engineering by efficiently generating stable enzymes. Many attempts at classical metabolic engineering have focused on improving specific metabolic fluxes and producing more valuable natural products by increasing gene expression levels and enzyme concentrations. However, most naturally occurring enzymes show limitations, and such limitations have hindered practical applications. Here we review recent advances in protein engineering in synthetic biology, chemoenzymatic synthesis, and plant metabolic engineering and describe opportunities for designing and constructing novel enzymes or proteins with desirable properties to obtain more active natural products.     

Genetic and metabolic engineering of microorganisms for the development of new flavor compounds from terpenic substrates

Throughout human history, natural products have been the basis for the discovery and development of therapeutics, cosmetic and food compounds used in industry. Many compounds found in natural organisms are rather difficult to chemically synthesize and to extract in large amounts, and in this respect, genetic and metabolic engineering are playing an increasingly important role in the production of these compounds, such as new terpenes and terpenoids, which may potentially be used to create aromas in industry. Terpenes belong to the largest class of natural compounds, are produced by all living organisms and play a fundamental role in human nutrition, cosmetics and medicine. Recent advances in systems biology and synthetic biology are allowing us to perform metabolic engineering at the whole-cell level, thus enabling the optimal design of microorganisms for the efficient production of drugs, cosmetic and food additives. This review describes the recent advances made in the genetic and metabolic engineering of the terpenes pathway with a particular focus on systems biotechnology.     

RNA synthetic biology

RNA molecules play important and diverse regulatory roles in the cell by virtue of their interaction with other nucleic acids, proteins and small molecules. Inspired by this natural versatility, researchers have engineered RNA molecules with new biological functions. In the last two years efforts in synthetic biology have produced novel, synthetic RNA components capable of regulating gene expression in vivo largely in bacteria and yeast, setting the stage for scalable and programmable cellular behavior. Immediate challenges for this emerging field include determining how computational and directed-evolution techniques can be implemented to increase the complexity of engineered RNA systems, as well as determining how such systems can be broadly extended to mammalian systems. Further challenges include designing RNA molecules to be sensors of intracellular and environmental stimuli, probes to explore the behavior of biological networks and components of engineered cellular control systems.     

Molecular diversity--the toolbox for synthetic gene switches and networks

The rapid development of synthetic biology is a paradigm of how the molecular diversity of naturally occurring gene control components can be used to design synthetic control devices and gene networks that provide precisely programmed transgene expression dynamics in space and time. Here we offer an overview on recent advances in the modular design of trigger-inducible mammalian expression devices that are either responsive by exogenous stimuli such as chemicals and physical cues or controlled by endogenous metabolites driving prosthetic circuits to treat metabolic disorders in a self-sufficient manner. Compatible genetic switches can also be assembled to synthetic gene networks that show highly complex expression dynamics such as temporally resolved band-detect functions or oscillating transgene expression profiles. The ongoing metagenomic discovery and characterization of the unexplored sequence space is constantly increasing the molecular diversity in fundamental control components that fuels the further development of synthetic biology.     

天然产物类药物的合成生物学研究

结构复杂多样的天然产物是现代药物的重要组成部分和新药发现的重要源泉。建立在基因工程及代谢工程、合成化学、基因组学、系统生物学等学科基础上的合成生物学研究对于结构复杂的天然产物类药物研究有特殊的意义。核心是通过在发酵友好、高效的微生物中设计、构建目标化合物的生物合成途径,经系统地调控和优化由重组微生物发酵生产来源稀缺的天然产物类药物或前体。该方法是不远的将来解决来源、成本与环境、资源协调问题最好的途径之一,也是解决海洋天然产物或特殊生境微生物药物面临的如何持续供应化合物这一个瓶颈问题的最佳选择。该文将对天然产物类药物合成生物学研究涉及的主要策略和重要进展进行阐述。     

Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

基因组装技术在合成生物学中的应用

基因组装技术是合成生物学领域近年来发展起来的新型技术。它基于大规模基因组数据分析,发现新型的或隐藏的生物活性物质合成基因簇。利用基因组装技术,可提高或激活沉默的生物合成基因簇在微生物中的表达,从而合成潜在的、有价值的生物活性物质。本文旨在阐明最新的体内和体外基因组装技术的设计原理、关键策略及其应用。基因组装技术是合成生物学、代谢工程和功能基因组学研究的重要工具,对生物活性物质的高效生产及合成具有重要意义。     

生物元件的挖掘、改造与标准化

生物元件是合成生物学中的三大基本要素之一,是合成生物学的基石。现阶段,生物元件的挖掘、鉴定和改造仍然是合成生物学领域的重要研究方向之一。合成生物学与基因工程和代谢工程最显著的差别在于能够将大量的生物元件进行快速、随意的组装,而实现这一目标的前提是将生物元件标准化。目前,已经有大量基因组被解析,通过这些基因组数据库的注释与功能验证,并借助于各种生物信息学软件预测启动子、终止子、操纵了、转录因子和转录因子结合位点、核糖体结合位点以及蛋白质编码区等部件,为合成生物学提供丰富的生物元件信息资源。随着元基因组技术的兴起,大量未培养微生物中的基因和基因簇信息被解析,使得我们可以从占自然界中实际存在微生物总数99％的未知微生物中挖掘更多的生物元件。另外,生物元件可以从自然界分离出来,也可以对天然生物元件进行修饰、重组和改造后得到新的元件。酵母是异源蛋白表达的通用宿主和生物基产品生产的细胞工厂,但其本身可用的启动子非常有限,近年来各国学者在酵母启动子改造和文库构建方面做了很多工作,该文也将概述酵母启动子改造和在合成生物生物学研究领域中的应用方面的研究进展。     

Reconstruction of methanol and formate metabolic pathway in non-native host for biosynthesis of chemicals and biofuels

One-carbon feedstock such as methanol and formate has attracted much attention as carbon substrate of industrial biotechnology for production of value-added chemicals and biofuels. Productivity improvement of natural one-carbon metabolic pathways in native hosts such as methanotrophs is somewhat difficult due to inefficient genetic tools and low specific growth rate. As an alternative, metabolic engineering can create new and efficient metabolic pathways of one-carbon substrate that can be readily transferred to non-native hosts. In this paper, recent progresses in protein and metabolic engineering for creation of methanol and formate-utilizing synthetic pathways based on RuMP cycle and formolase are reviewed. Perspectives on one-carbon metabolic pathway engineering in non-native host are also discussed.     

Synthetic regulatory tools for microbial engineering

Microbial engineering requires accurate information about cellular metabolic networks and a set of molecular tools that can be predictably applied to the efficient redesign of such networks. Recent advances in the field of metabolic engineering and synthetic biology, particularly the development of molecular tools for synthetic regulation in the static and dynamic control of gene expression, have increased our ability to efficiently balance the expression of genes in various biological systems. It would accelerate the creation of synthetic pathways and genetic programs capable of adapting to environmental changes in real time to perform the programmed cellular behavior. In this paper, we review current developments in the field of synthetic regulatory tools for static and dynamic control of microbial gene expression.     

Quorum Sensing System Used as a Tool in Metabolic Engineering

Quorum sensing (QS) is a ubiquitous cell–cell communication mechanism in microbes that coordinates population‐level cell behaviors, such as biofilm production, virulence, swarming motility, and bacterial persistence. Efforts to engineer QS systems to take part in metabolic network regulation represent a promising strategy for synthetic biology and pathway engineering. Recently, design, construction, and implementation of QS circuits for programmed control of bacterial phenotypes and metabolic pathways have gained much attention, but have not been reviewed recently. In this article, the architectural organizations and genetic contributions of the naturally occurring QS components to understand the mechanisms are summarized. Then, the most recent progress in application of QS toolkits to develop synthetic networks for novel cell behaviors creation and metabolic pathway engineering is highlighted. The current challenges in large‐scale application of these QS circuits in synthetic biology and metabolic engineering fields are discussed and future perspectives for further engineering efforts are provided.     

Optimization of industrial microorganisms: recent advances in synthetic dynamic regulators

Production of biochemicals by industrial fermentation using microorganisms requires maintaining cellular production capacity, because maximal productivity is economically important. High-productivity microbial strains can be developed using static engineering, but these may not maintain maximal productivity throughout the culture period as culture conditions and cell states change dynamically. Additionally, economic reasons limit heterologous protein expression using inducible promoters to prevent metabolic burden for commodity chemical and biofuel production. Recently, synthetic and systems biology has been used to design genetic circuits, precisely controlling gene expression or influencing genetic behavior toward a desired phenotype. Development of dynamic regulators can maintain cellular phenotype in a maximum production state in response to factors including cell concentration, oxygen, temperature, pH, and metabolites. Herein, we introduce dynamic regulators of industrial microorganism optimization and discuss metabolic flux fine control by dynamic regulators in response to metabolites or extracellular stimuli, robust production systems, and auto-induction systems using quorum sensing.     

Engineering and applications of genetic circuits

In the field of synthetic biology, recent genetic engineering efforts have enabled the construction of novel genetic circuits with diverse functionalities and unique activation mechanisms. Because of these advances, artificial genetic networks are becoming increasingly complex, and are demonstrating more robust behaviors with reduced crosstalk between defined modules. These properties have allowed for the identification of a growing set of design principles that govern genetic networks, and led to an increased number of applications for genetic circuits in the fields of metabolic engineering and biomedical engineering. Such progress indicates that synthetic biology is rapidly evolving into an integrated engineering practice that uses rational and combinatorial design of synthetic gene networks to solve complex problems in biology, medicine, and human health.     

Synthetic biology evolves

Synthetic biology is advancing rapidly as biologists, physicists and engineers are combining their efforts to understand and program cell function. By characterizing isolated genetic components or modules, experimentalists have paved the way for more quantitative analyses of genetic networks. A recent paper presents a method of computational, or in silico, evolution in which a set of components can evolve into networks that display desired behaviors. An integrated approach that includes a strategy of in silico design by evolution, together with efforts exploiting directed evolution in vivo, is likely to be the next step in the evolution of synthetic biology.