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.     

Bacterial quorum sensing: circuits and applications

Bacterial quorum sensing (QS) systems are cell density—dependent regulatory networks that coordinate bacterial behavioural changes from single cellular organisms at low cell densities to multicellular types when their population density reaches a threshold level. At this stage, bacteria produce and perceive small diffusible signal molecules, termed autoinducers in order to mediate gene expression. This often results in phenotypic shifts, like planktonic to biofilm or non-virulent to virulent. In this way, they regulate varied physiological processes by adjusting gene expression in concert with their population size. In this review we give a synopsis of QS mediated cell–cell communication in bacteria. The first part focuses on QS circuits of some Gram-negative and Gram-positive bacteria. Thereafter, attention is drawn on the recent applications of QS in development of synthetic biology modules, for studying the principles of pattern formation, engineering bi-directional communication system and building artificial communication networks. Further, the role of QS in solving the problem of biofouling is also discussed.     

Applications of quorum sensing in biotechnology

Many unicellular microorganisms use small signaling molecules to determine their local concentration. The processes involved in the production and recognition of these signals are collectively known as quorum sensing (QS). This form of cell–cell communication is used by unicellular microorganisms to co-ordinate their activities, which allows them to function as multi-cellular systems. Recently, several groups have demonstrated artificial intra-species and inter-species communication through synthetic circuits which incorporate components of bacterial QS systems. Engineered QS-based circuits have a wide range of applications such as production of biochemicals, tissue engineering, and mixed-species fermentations. They are also highly useful in designing microbial biosensors to identify bacterial species present in the environment and within living organisms. In this review, we first provide an overview of bacterial QS systems and the mechanisms developed by bacteria and higher organisms to obstruct QS communications. Next, we describe the different ways in which researchers have designed QS-based circuits and their applications in biotechnology. Finally, disruption of quorum sensing is discussed as a viable strategy for preventing the formation of harmful biofilms in membrane bioreactors and marine transportation.     

历久弥新：进化中的代谢工程

20世纪90年代,Bailey及Stephanopoulos等提出了经典代谢工程的理念,旨在利用DNA重组技术对代谢网络进行改造,以达到细胞性能改善,目标产物增加的目的。自代谢工程诞生以来的30年,生命科学蓬勃发展,基因组学、系统生物学、合成生物学等新学科不断涌现,为代谢工程的发展注入了新的内涵与活力。经典代谢工程研究已进入到前所未有的系统代谢工程阶段。组学技术、基因组代谢模型、元件组装、回路设计、动态控制、基因组编辑等合成生物学工具与策略的应用,大大提升了复杂代谢的设计与合成能力;机器学习的介入以及进化工程与代谢工程的结合,为系统代谢工程的未来开辟了新的方向。文中对过去30年代谢工程的发展趋势作了梳理,介绍了代谢工程在发展中不断创新的理论与方法及其应用。     

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.     

In Vivo Programmed Gene Expression Based on Artificial Quorum Networks

The quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of the Vibrio fischeri luxI-luxR quorum sensing system. In order to achieve in vivo programmed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter P_araBAD, into the QS system. In vitro expression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density. In vivo expression assays confirmed that the araQS system presented an in vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applications in vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuated Edwardsiella tarda strain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expression in vivo and might have potential uses, including, but not limited to, bacterial vector vaccines.     

Modular electron transfer circuits for synthetic biology: insulation of an engineered biohydrogen pathway

Electron transfer is central to a wide range of essential metabolic pathways, from photosynthesis to fermentation. The evolutionary diversity and conservation of proteins that transfer electrons makes these pathways a valuable platform for engineered metabolic circuits in synthetic biology. Rational engineering of electron transfer pathways containing hydrogenases has the potential to lead to industrial scale production of hydrogen as an alternative source of clean fuel and experimental assays for understanding the complex interactions of multiple electron transfer proteins in vivo. We designed and implemented a synthetic hydrogen metabolism circuit in Escherichia coli that creates an electron transfer pathway both orthogonal to and integrated within existing metabolism. The design of such modular electron transfer circuits allows for facile characterization of in vivo system parameters with applications toward further engineering for alternative energy production.     

合成生物学与代谢工程

随着DNA重组技术的日趋成熟,代谢工程的理论和应用已经得到了迅速发展。合成生物学是近年来蓬勃发展的一门新兴学科,在许多领域都具有重要的应用。以下从改造细胞代谢的关键因子、代谢途径的调节和宿主细胞与代谢途径构建的关系等方面详细讨论了合成生物学的最新进展和合成生物学在代谢工程领域的应用。     

The promise of synthetic biology

DNA synthesis has become one of the technological bases of a new concept in biology: synthetic biology. The vision of synthetic biology is a systematic, hierarchical design of artificial, biology-inspired systems using robust, standardized, and well-characterized building blocks. The design concept and examples from four fields of application (genetic circuits, protein design, platform technologies, and pathway engineering) are discussed, which demonstrate the usefulness and the promises of synthetic biology. The vision of synthetic biology is to develop complex systems by simplified solutions using available material and knowledge. Synthetic biology also opens a door toward new biomaterials that do not occur in nature.     

Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression

Quorum sensing (QS) regulates many natural phenotypes (e.q. virulence, biofilm formation, antibiotic resistance), and its components, when incorporated into synthetic genetic circuits, enable user-directed phenotypes. We created a library of Escherichia coli lsr operon promoters using error-prone PCR (ePCR) and selected for promoters that provided E. coli with higher tetracycline resistance over the native promoter when placed upstream of the tet(C) gene. Among the fourteen clones identified, we found several mutations in the binding sites of QS repressor, LsrR. Using site-directed mutagenesis we restored all p-lsrR-box sites to the native sequence in order to maintain LsrR repression of the promoter, preserving the other mutations for analysis. Two promoter variants, EP01rec and EP14rec, were discovered exhibiting enhanced protein expression. In turn, these variants retained their ability to exhibit the LsrR-mediated QS switching activity. Their sequences suggest regulatory linkage between CytR (CRP repressor) and LsrR. These promoters improve upon the native system and exhibit advantages over synthetic QS promoters previously reported. Incorporation of these promoters will facilitate future applications of QS-regulation in synthetic biology and metabolic engineering.     

A revolutionary tool: CRISPR technology plays an important role in construction of intelligentized gene circuits

With the development of synthetic biology, synthetic gene circuits have shown great applied potential in medicine, biology, and as commodity chemicals. An ultimate challenge in the construction of gene circuits is the lack of effective, programmable, secure and sequence‐specific gene editing tools. The clustered regularly interspaced short palindromic repeat (CRISPR) system, a CRISPR‐associated RNA‐guided endonuclease Cas9 (CRISPR‐associated protein 9)‐targeted genome editing tool, has recently been applied in engineering gene circuits for its unique properties‐operability, high efficiency and programmability. The traditional single‐targeted therapy cannot effectively distinguish tumour cells from normal cells, and gene therapy for single targets has poor anti‐tumour effects, which severely limits the application of gene therapy. Currently, the design of gene circuits using tumour‐specific targets based on CRISPR/Cas systems provides a new way for precision cancer therapy. Hence, the application of intelligentized gene circuits based on CRISPR technology effectively guarantees the safety, efficiency and specificity of cancer therapy. Here, we assessed the use of synthetic gene circuits and if the CRISPR system could be used, especially artificial switch‐inducible Cas9, to more effectively target and treat tumour cells. Moreover, we also discussed recent advances, prospectives and underlying challenges in CRISPR‐based gene circuit development.     

Towards systems metabolic engineering of microorganisms for amino acid production

Microorganisms capable of efficient production of amino acids have traditionally been developed by random mutation and selection method, which might cause unwanted physiological changes in cellular metabolism. Rational genome-wide metabolic engineering based on systems and synthetic biology tools, which is termed 'systems metabolic engineering', is rising as an alternative to overcome these problems. Recently, several amino acid producers have been successfully developed by systems metabolic engineering, where the metabolic engineering procedures were performed within a systems biology framework, and entire metabolic networks, including complex regulatory circuits, were engineered in an integrated manner. Here we review the current status of systems metabolic engineering successfully applied for developing amino acid producing strains and discuss future prospects.     

Engineering antibiotic production and overcoming bacterial resistance

Progress in DNA technology, analytical methods and computational tools is leading to new developments in synthetic biology and metabolic engineering, enabling new ways to produce molecules of industrial and therapeutic interest. Here, we review recent progress in both antibiotic production and strategies to counteract bacterial resistance to antibiotics. Advances in sequencing and cloning are increasingly enabling the characterization of antibiotic biosynthesis pathways, and new systematic methods for de novo biosynthetic pathway prediction are allowing the exploration of the metabolic chemical space beyond metabolic engineering. Moreover, we survey the computer-assisted design of modular assembly lines in polyketide synthases and non-ribosomal peptide synthases for the development of tailor-made antibiotics. Nowadays, production of novel antibiotic can be tranferred into any chosen chassis by optimizing a host factory through specific strain modifications. These advances in metabolic engineering and synthetic biology are leading to novel strategies for engineering antimicrobial agents with desired specificities.     

基于图论和约束优化的异源代谢途径设计方法及应用

构建高产高附加值产品的微生物细胞工厂是代谢工程的研究目标之一,设计高效的产品合成途径是实现这一目标的重要方式.不同微生物底盘因其代谢能力有所差异,故而可以利用的底物和生产的产品范围有限.为了扩大其生产能力,需要进行代谢途径从无到有的设计.传统代谢工程基于经验进行异源途径设计的方式低效且无法确保结果的全面性,而系统生物学...     

From unicellular properties to multicellular behavior: bacteria quorum sensing circuitry and applications

Cell-cell communication and coordinated population-based behavior among single cell organisms have gained considerable attention in the recent years. The ability to send, receive, and process information allows unicellular organisms to act as multicellular entities and increases their chances of survival in complex environments. Quorum sensing (QS), a density-dependent cell-signaling mechanism, is one way by which bacteria 'talk' to one another. QS is commonly associated with adverse health effects such as biofilm formation, bacteria pathogenicity, and virulence. But there exists great potential to harness QS circuitry and its properties for other applications, enabling even wider societal impact. Interesting avenues are envisioned for the detection of chemicals and pathogens, the navigation of interspecies communication, the synchronization and control of cell phenotype, and the creation of novel materials based on synthetic biology. In this review, we first highlight the recent discoveries of the molecular underpinnings of QS function, with emphasis on the formation of biofilms. We then discuss how researchers have used QS circuitry to their advantage to build synthetic networks, rewire native metabolic pathways, and engineer cells for a variety of applications.     

In vivo biosensors: mechanisms,development, and applications

In vivo biosensors can recognize and respond to specific cellular stimuli. In recent years, biosensors have been increasingly used in metabolic engineering and synthetic biology, because they can be implemented in synthetic circuits to control the expression of reporter genes in response to specific cellular stimuli, such as a certain metabolite or a change in pH. There are many types of natural sensing devices, which can be generally divided into two main categories: protein-based and nucleic acid-based. Both can be obtained either by directly mining from natural genetic components or by engineering the existing genetic components for novel specificity or improved characteristics. A wide range of new technologies have enabled rapid engineering and discovery of new biosensors, which are paving the way for a new era of biotechnological progress. Here, we review recent advances in the design, optimization, and applications of in vivo biosensors in the field of metabolic engineering and synthetic biology.     

Quorum sensing-based metabolic engineering of the precursor supply in Streptomyces coelicolor to improve heterologous production of neoaureothin

Streptomyces are important industrial bacteria that produce pharmaceutically valuable polyketides. However, mass production on an industrial scale is limited by low productivity, which can be overcome through metabolic engineering and the synthetic biology of the host strain. Recently, the introduction of an auto-inducible expression system depending on microbial physiological state has been suggested as an important tool for the industrial-scale production of polyketides. In this study, titer improvement by enhancing the pool of CoA-derived precursors required for polyketide production was driven in a quorum sensing (QS)-dependent manner. A self-sustaining and inducer-independent regulatory system, named the QS-based metabolic engineering of precursor pool (QMP) system, was constructed, wherein the expression of genes involved in precursor biosynthesis was regulated by the QS-responsive promoter, scbAp. The QMP system was applied for neoaureothin production in a heterologous host, Streptomyces coelicolor M1152, and productivity increased by up to 4-fold. In particular, the engineered hyperproducers produced high levels of neoaureothin without adversely affecting cell growth. Overall, this study showed that self-regulated metabolic engineering mediated by QS has the potential to engineer strains for polyketide titer improvement.