Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.     

Synthetic biology: a new approach to study biological pattern formation

The principles and molecular mechanisms underlying biological pattern formation are difficult to elucidate in most cases due to the overwhelming physiologic complexity associated with the natural context. The understanding of a particular mechanism, not to speak of underlying universal principles, is difficult due to the diversity and uncertainty of the biological systems. Although current genetic and biochemical approaches have greatly advanced our understanding of pattern formation, the progress mainly relies on experimental phenotypes obtained from time-consuming studies of gain or loss of function mutants. It is prevailingly considered that synthetic biology will come to the application age, but more importantly synthetic biology can be used to understand the life. Using periodic stripe pattern formation as a paradigm, we discuss how to apply synthetic biology in understanding biological pattern formation and hereafter foster the applications like tissue engineering.     

Computer-aided design of biological circuits using TinkerCell

Synthetic biology is an engineering discipline that builds on modeling practices from systems biology and wet-lab techniques from genetic engineering. As synthetic biology advances, efficient procedures will be developed that will allow a synthetic biologist to design, analyze, and build biological networks. In this idealized pipeline, computer-aided design (CAD) is a necessary component. The role of a CAD application would be to allow efficient transition from a general design to a final product. TinkerCell is a design tool for serving this purpose in synthetic biology. In TinkerCell, users build biological networks using biological parts and modules. The network can be analyzed using one of several functions provided by TinkerCell or custom programs from third-party sources. Since best practices for modeling and constructing synthetic biology networks have not yet been established, TinkerCell is designed as a flexible and extensible application that can adjust itself to changes in the field.     

The dynamic systems approach to control and regulation of intracellular networks

Systems theory and cell biology have enjoyed a long relationship that has received renewed interest in recent years in the context of systems biology. The term 'systems' in systems biology comes from systems theory or dynamic systems theory: systems biology is defined through the application of systems- and signal-oriented approaches for an understanding of inter- and intra-cellular dynamic processes. The aim of the present text is to review the systems and control perspective of dynamic systems. The biologist's conceptual framework for representing the variables of a biochemical reaction network, and for describing their relationships, are pathway maps. A principal goal of systems biology is to turn these static maps into dynamic models, which can provide insight into the temporal evolution of biochemical reaction networks. Towards this end, we review the case for differential equation models as a 'natural' representation of causal entailment in pathways. Block-diagrams, commonly used in the engineering sciences, are introduced and compared to pathway maps. The stimulus-response representation of a molecular system is a necessary condition for an understanding of dynamic interactions among the components that make up a pathway. Using simple examples, we show how biochemical reactions are modelled in the dynamic systems framework and visualized using block-diagrams.     

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。     

应用基因组工程构建新型大肠杆菌细胞工厂

基因组工程（genome engineering）是指为了实现某一目标对复制系统进行有意地、广泛地遗传修饰。大肠杆菌作为常用细胞工厂之一,在生物制造领域应用广泛,已成为合成生物学的主要研究对象。应用基因组工程改造大肠杆菌可以进一步拓展其应用范围。介绍了基因组工程最新技术发展,如基因组整合、染色体进化、多元自动化基因组工程、可寻迹多元重组工程等,及其在改造大肠杆菌提高其生产能力、稳定性及环境耐受能力等方面的研究进展。     

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。     

A synthetic pathway for the production of 2-hydroxyisovaleric acid in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Synthetic biology, encompassing the design and construction of novel artificial biological pathways and organisms and the redesign of existing natural biological systems, is rapidly expanding the number of applications for which biological systems can play an integral role. In the context of chemical production, the combination of synthetic biology and metabolic engineering approaches continues to unlock the ability to biologically produce novel and complex molecules from a variety of feedstocks. Here, we utilize a synthetic approach to design and build a pathway to produce 2-hydroxyisovaleric acid in Escherichia coli and demonstrate how pathway design can be supplemented with metabolic engineering approaches to improve pathway performance from various carbon sources. Drawing inspiration from the native pathway for the synthesis of the 5-carbon amino acid l-valine, we exploit the decarboxylative condensation of two molecules of pyruvate, with subsequent reduction and dehydration reactions enabling the synthesis of 2-hydroxyisovaleric acid. Key to our approach was the utilization of an acetolactate synthase which minimized kinetic and regulatory constraints to ensure sufficient flux entering the pathway. Critical host modifications enabling maximum product synthesis from either glycerol or glucose were then examined, with the varying degree of reduction of these carbons sources playing a major role in the required host background. Through these engineering efforts, the designed pathway produced 6.2 g/L 2-hydroxyisovaleric acid from glycerol at 58% of maximum theoretical yield and 7.8 g/L 2-hydroxyisovaleric acid from glucose at 73% of maximum theoretical yield. These results demonstrate how the combination of synthetic biology and metabolic engineering approaches can facilitate bio-based chemical production.     

The Corynebacterium glutamicum genome: features and impacts on biotechnological processes

Corynebacterium glutamicum has played a principal role in the progress of the amino acid fermentation industry. The complete genome sequence of the representative wild-type strain of C. glutamicum, ATCC 13032, has been determined and analyzed to improve our understanding of the molecular biology and physiology of this organism, and to advance the development of more efficient production strains. Genome annotation has helped in elucidation of the gene repertoire defining a desired pathway, which is accelerating pathway engineering. Post genome technologies such as DNA arrays and proteomics are currently undergoing rapid development in C. glutamicum. Such progress has already exposed new regulatory networks and functions that had so far been unidentified in this microbe. The next goal of these studies is to integrate the fruits of genomics into strain development technology. A novel methodology that merges genomics with classical strain improvement has been developed and applied for the reconstruction of classically derived production strains. How can traditional fermentation benefit from the C. glutamicum genomic data? The path from genomics to biotechnological processes is presented.     

Plant Metabolic Modeling: Achieving New Insight into Metabolism and Metabolic Engineering

Models are used to represent aspects of the real world for specific purposes, and mathematical models have opened up new approaches in studying the behavior and complexity of biological systems. However, modeling is often time-consuming and requires significant computational resources for data development, data analysis, and simulation. Computational modeling has been successfully applied as an aid for metabolic engineering in microorganisms. But such model-based approaches have only recently been extended to plant metabolic engineering, mainly due to greater pathway complexity in plants and their highly compartmentalized cellular structure. Recent progress in plant systems biology and bioinformatics has begun to disentangle this complexity and facilitate the creation of efficient plant metabolic models. This review highlights several aspects of plant metabolic modeling in the context of understanding, predicting and modifying complex plant metabolism. We discuss opportunities for engineering photosynthetic carbon metabolism, sucrose synthesis, and the tricarboxylic acid cycle in leaves and oil synthesis in seeds and the application of metabolic modeling to the study of plant acclimation to the environment. The aim of the review is to offer a current perspective for plant biologists without requiring specialized knowledge of bioinformatics or systems biology.     

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.     

Toward computational systems biology

The development and successful application of high-throughput technologies are transforming biological research. The large quantities of data being generated by these technologies have led to the emergence of systems biology, which emphasizes large-scale, parallel characterization of biological systems and integration of fragmentary information into a coherent whole. Complementing the reductionist approach that has dominated biology for the last century, mathematical modeling is becoming a powerful tool to achieve an integrated understanding of complex biological systems and to guide experimental efforts of engineering biological systems for practical applications. Here I give an overview of current mainstream approaches in modeling biological systems, highlight specific applications of modeling in various settings, and point out future research opportunities and challenges.     

Synthetic tissue biology: tissue engineering meets synthetic biology

We propose the term "synthetic tissue biology" to describe the use of engineered tissues to form biological systems with metazoan-like complexity. The increasing maturity of tissue engineering is beginning to render this goal attainable. As in other synthetic biology approaches, the perspective is bottom-up; here, the premise is that complex functional phenotypes (on par with those in whole metazoan organisms) can be effected by engineering biology at the tissue level. To be successful, current efforts to understand and engineer multicellular systems must continue, and new efforts to integrate different tissues into a coherent structure will need to emerge. The fruits of this research may include improved understanding of how tissue systems can be integrated, as well as useful biomedical technologies not traditionally considered in tissue engineering, such as autonomous devices, sensors, and manufacturing.     

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.     

Recent advances in producing food additive L-malate: Chassis,substrate, pathway,fermentation regulation and application

In addition to being an important intermediate in the TCA cycle, L-malate is also widely used in the chemical and beverage industries. Due to the resulting high demand, numerous studies investigated chemical methods to synthesize L-malate from petrochemical resources, but such approaches are hampered by complex downstream processing and environmental pollution. Accordingly, there is an urgent need to develop microbial methods for environmentally-friendly and economical L-malate biosynthesis. The rapid progress and understanding of DNA manipulation, cell physiology, and cell metabolism can improve industrial L-malate biosynthesis by applying intelligent biochemical strategies and advanced synthetic biology tools. In this paper, we mainly focused on biotechnological approaches for enhancing L-malate synthesis, encompassing the microbial chassis, substrate utilization, synthesis pathway, fermentation regulation, and industrial application. This review emphasizes the application of novel metabolic engineering strategies and synthetic biology tools combined with a deep understanding of microbial physiology to improve industrial L-malate biosynthesis in the future.     

2013合成生物学专刊序言

合成生物学目前在全球得到迅猛发展。在此专刊中,综述了一些相关技术在合成生物学领域的进展,其中有:链霉菌无痕敲除方法、基因合成技术、DNA组装新方法、最小化基因组的方法及分析、合成生物系统的组合优化。也讨论了应用合成生物学策略优化光合蓝细菌底盘、产溶剂梭菌分子遗传操作技术、蛋白质预算(Protein budget)作为合成生物学的成本标尺。最后,用几个例子说明了合成生物学的应用,包括复杂天然产物合成人工生物系统的设计与构建、微生物木糖代谢途径改造制备生物基化学品以及构建酿酒酵母工程菌合成香紫苏醇。     

Biology coming full circle: Joining the whole and the parts

The new cover of Experimental Biology and Medicine features the hermeneutic circle of biology, a concept we have adapted from the hermeneutic principle that one understands the whole only in terms of each part and the parts only in terms of the whole. Our hermeneutic circle summarizes the course of experimental biology through 2500 years of the achievements of reductionist research (understanding the parts), which culminates in our ability to rapidly sequence the genome. Rather than returning along the same path in a constructionist approach that simply builds upon this knowledge, but in reverse, an alternative is to close the circle with synthetic constructions that seek to integrate the full complexity of biological and physiological systems (understanding the whole), of which organs-on-chips are one example. This closing of the circle cannot be a comprehensively accurate representation of biology, but it can be a synthetic one that effectively defines particular biological subsystems. The illustration of the hermeneutic circle of biology is also intended to suggest both the multiple cycles that may be required to reach such a synthesis and the expansion of the circle in an outward spiral as knowledge increases. Our commentary explains the symbolism of the new cover in a philosophical and scientific discussion.