Predicting inter-species cross-talk in two-component signalling systems

Phosphosignalling pathways are an attractive option for the synthetic biologist looking for a wide repertoire of modular components from which to build. We demonstrate that two-component systems can be used in synthetic biology. However, their potential is limited by the fact that host cells contain many of their own phosphosignalling pathways and these may interact with, and cross-talk to, the introduced synthetic components. In this paper we also demonstrate a simple bioinformatic tool that can help predict whether interspecies cross-talk between introduced and native two-component signalling pathways will occur and show both in vitro and in vivo that the predicted interactions do take place. The ability to predict potential cross-talk prior to designing and constructing novel pathways or choosing a host organism is essential for the promise that phosphosignalling components hold for synthetic biology to be realised.     

Screening of natural phenazine producers for electroactivity in bioelectrochemical systems

Mediated extracellular electron transfer (EET) might be a great vehicle to connect microbial bioprocesses with electrochemical control in stirred-tank bioreactors. However, mediated electron transfer to date is not only much less efficient but also much less studied than microbial direct electron transfer to an anode. For example, despite the widespread capacity of pseudomonads to produce phenazine natural products, only Pseudomonas aeruginosa has been studied for its use of phenazines in bioelectrochemical applications. To provide a deeper understanding of the ecological potential for the bioelectrochemical exploitation of phenazines, we here investigated the potential electroactivity of over 100 putative diverse native phenazine producers and the performance within bioelectrochemical systems. Five species from the genera Pseudomonas, Streptomyces, Nocardiopsis, Brevibacterium and Burkholderia were identified as new electroactive bacteria. Electron discharge to the anode and electric current production correlated with the phenazine synthesis of Pseudomonas chlororaphis subsp. aurantiaca. Phenazine-1-carboxylic acid was the dominant molecule with a concentration of 86.1 μg/ml mediating an anodic current of 15.1 μA/cm². On the other hand, Nocardiopsis chromatogenes used a wider range of phenazines at low concentrations and likely yet-unknown redox compounds to mediate EET, achieving an anodic current of 9.5 μA/cm². Elucidating the energetic and metabolic usage of phenazines in these and other species might contribute to improving electron discharge and respiration. In the long run, this may enhance oxygen-limited bioproduction of value-added compounds based on mediated EET mechanisms.     

Vibrio natriegens: an ultrafast-growing marine bacterium as emerging synthetic biology chassis

The marine bacterium Vibrio natriegens is the fastest-growing non-pathogenic bacterium known to date and is gaining more and more attention as an alternative chassis organism to Escherichia coli. A recent wave of synthetic biology efforts has focused on the establishment of molecular biology tools in this fascinating organism, now enabling exciting applications – from speeding up our everyday laboratory routines to increasing the pace of biotechnological production cycles. In this review, we seek to give a broad overview on the literature on V. natriegens, spanning all the way from its initial isolation to its latest applications. We discuss its natural ecological niche and interactions with other organisms, unveil some of its extraordinary traits, review its genomic organization and give insight into its diverse metabolism – key physiological insights required to further develop this organism into a synthetic biology chassis. By providing a comprehensive overview on the established genetic tools, methods and applications we highlight the current possibilities of this organism, but also identify some of the gaps that could drive future lines of research, hopefully stimulating the growth of the V. natriegens research community.     

非常规酵母的分子遗传学及合成生物学研究进展

先进的合成生物学技术与传统的分子遗传学技术的结合更有助于实现酵母底盘细胞的快速改造和优化。酵母合成生物学研究最早开始于常规酵母——酿酒酵母(Saccharomyces cerevisiae),近些年来又迅速扩展至一些非常规酵母,包括巴斯德毕赤酵母(Pichiapastoris)、解脂耶氏酵母(Yarrowialipolytica)、乳酸克鲁维酵母(Kluyveromyces lactis)和多形汉逊酵母(Hansenula polymorpha)等。借助合成生物学技术与工具,目前科学家们已经成功开发出了能够高效生产生物材料、生物燃料、生物基化学品、蛋白质制剂、食品添加剂和药物等工业产品的重组非常规酵母工程菌株。本文系统总结了合成生物学工具(主要是基因组编辑工具)、合成生物学组件(主要是启动子和终止子)和相关分子遗传学方法在上述非常规酵母系统(底盘细胞)中的最新研究进展和应用情况,并讨论了其他合成生物学技术在这些非常规酵母表达系统中的潜在适用性和应用前景。这为研究人员利用合成生物学方法在这一新型非模式微生物底盘细胞中设计和构建各种高附加值工业产品的异源合成模块并最终实现目标化合物的高效生物合成提供了科学的理论指导。     

Network methods for describing sample relationships in genomic datasets: application to Huntington’s disease

Background

We consider the possibility of engineering metabolic pathways in a chassis organism in order to synthesize novel target compounds that are heterologous to the chassis. For this purpose, we model metabolic networks through hypergraphs where reactions are represented by hyperarcs. Each hyperarc represents an enzyme-catalyzed reaction that transforms set of substrates compounds into product compounds. We follow a retrosynthetic approach in order to search in the metabolic space (hypergraphs) for pathways (hyperpaths) linking the target compounds to a source set of compounds.

Results

To select the best pathways to engineer, we have developed an objective function that computes the cost of inserting a heterologous pathway in a given chassis organism. In order to find minimum-cost pathways, we propose in this paper two methods based on steady state analysis and network topology that are to the best of our knowledge, the first to enumerate all possible heterologous pathways linking a target compounds to a source set of compounds. In the context of metabolic engineering, the source set is composed of all naturally produced chassis compounds (endogenuous chassis metabolites) and the target set can be any compound of the chemical space. We also provide an algorithm for identifying precursors which can be supplied to the growth media in order to increase the number of ways to synthesize specific target compounds.

Conclusions

We find the topological approach to be faster by several orders of magnitude than the steady state approach. Yet both methods are generally scalable in time with the number of pathways in the metabolic network. Therefore this work provides a powerful tool for pathway enumeration with direct application to biosynthetic pathway design.     

Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology

The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced.     

Electrochemically applied potentials induce growth and metabolic shift changes in the hyperthermophilic bacterium Thermotoga maritima MSB8

Bioelectrochemical systems are an attractive technology for regulating microbial activity. The effect of an applied potential on hydrolysis of starch in Thermotoga maritima as a model bacterium was investigated in this study. A cathodic potential (?0.6 and ?0.8 V) induced 5-h earlier growth initiation of T. maritima with starch as the polymeric substrate than that without electrochemical regulation. Moreover, metabolic patterns of starch consumption were altered by the cathodic potential. While acetate, H², and CO₂ were the major products of starch consumption in the control experiment without electrolysis, lactate accumulation was detected rather than decreased acetate and H₂ levels in the bioelectrochemical system experiments with the cathodic potential. These results indicate that the applied potential could control microbial activities related to the hydrolysis of polymeric organic substances and shift carbon and electron flux to a lactate-producing reaction in T. maritima.     

Evolutionary engineering of Corynebacterium glutamicum

A unique feature of biotechnology is that we can harness the power of evolution to improve process performance. Rational engineering of microbial strains has led to the establishment of a variety of successful bioprocesses, but it is hampered by the overwhelming complexity of biological systems. Evolutionary engineering represents a straightforward approach for fitness‐linked phenotypes (e.g., growth or stress tolerance) and is successfully applied to select for strains with improved properties for particular industrial applications. In recent years, synthetic evolution strategies have enabled selection for increased small molecule production by linking metabolic productivity to growth as a selectable trait. This review summarizes the evolutionary engineering strategies performed with the industrial platform organism Corynebacterium glutamicum. An increasing number of recent studies highlight the potential of adaptive laboratory evolution (ALE) to improve growth or stress resistance, implement the utilization of alternative carbon sources, or improve small molecule production. Advances in next‐generation sequencing and automation technologies will foster the application of ALE strategies to streamline microbial strains for bioproduction and enhance our understanding of biological systems.     

Inducible gene expression systems inLactococcus lactis

Lactococcus lactis is industrially important microorganism used in many dairy fermentations. Numerous genes and gene expression signals from this organism have now been identified and characterized. Recently, several naturally occurring, inducible gene-expression systems have also been described inL. lactis. The main features of these systems can be exploited to design genetically engineered expression cassettes for controlled production of various proteins and enzymes. Novel gene-expression systems inLactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.     

Electrochemical control of redox potential affects methanogenesis of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus

To investigate the precise effect of the redox potential on the methanogenesis of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus by using an electrochemical redox controlling system without adding oxidizing or reducing agents. A bioelectrochemical system was applied to control the redox conditions in culture and to measure the methane‐producing activity of M. thermautotrophicus at a constant potential from +0·2 to ?0·8 V (vs Ag/AgCl). Methane production and growth of M. thermautotrophicus were 1·6 and 3·5 times increased at ?0·8 V, compared with control experiments without electrolysis, respectively, while methanogenesis was suppressed between +0·2 and ?0·2 V. A clear relationship between an electrochemically regulated redox potential and methanogenesis was revealed.

Significance and Impact of the Study

A novel bioelectrochemical method can activate the methanogenesis of M. thermautotrophicus by controlling the redox potential in culture conditions at ?0·8 V, which is a difficult potential to achieve by conventional methods (e.g. by adding reducing agents). This study provides useful insights for the application of a bioelectrochemical system in industrial processes involving methanogens, such as in anaerobic digesters.     

An efficient CRISPR/Cas9-based genome editing system for alkaliphilic Bacillus sp. N16-5 and application in engineering xylose utilization for D-lactic acid production

Alkaliphiles are considered more suitable chassis than traditional neutrophiles due to their excellent resistance to microbial contamination. Alkaliphilic Bacillus sp. N16-5, an industrially interesting strain with great potential for the production of lactic acid and alkaline polysaccharide hydrolases, can only be engineered genetically by the laborious and time-consuming homologous recombination. In this study, we reported the successful development of a CRISPR/Cas9-based genome editing system with high efficiency for single-gene deletion, large gene fragment deletion and exogenous DNA chromosomal insertion. Moreover, based on a catalytically dead variant of Cas9 (dCas9), we also developed a CRISPRi system to efficiently regulate gene expression. Finally, this efficient genome editing system was successfully applied to engineer the xylose metabolic pathway for the efficient bioproduction of D -lactic acid. Compared with the wild-type Bacillus sp. N16-5, the final engineered strain with XylR deletion and AraE overexpression achieved 34.3% and 27.7% increases in xylose consumption and D -lactic acid production respectively. To our knowledge, this is the first report on the development and application of CRISPR/Cas9-based genome editing system in alkaliphilic Bacillus, and this study will significantly facilitate functional genomic studies and genome manipulation in alkaliphilic Bacillus, laying a foundation for the development of more robust microbial chassis.     

Recent advances in plasmid-based tools for establishing novel microbial chassis

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with “build-your-own” microbial host.     

Electrochemically active biofilms: facts and fiction. A review

This review examines the electrochemical techniques used to study extracellular electron transfer in the electrochemically active biofilms that are used in microbial fuel cells and other bioelectrochemical systems. Electrochemically active biofilms are defined as biofilms that exchange electrons with conductive surfaces: electrodes. Following the electrochemical conventions, and recognizing that electrodes can be considered reactants in these bioelectrochemical processes, biofilms that deliver electrons to the biofilm electrode are called anodic, ie electrode-reducing, biofilms, while biofilms that accept electrons from the biofilm electrode are called cathodic, ie electrode-oxidizing, biofilms. How to grow these electrochemically active biofilms in bioelectrochemical systems is discussed and also the critical choices made in the experimental setup that affect the experimental results. The reactor configurations used in bioelectrochemical systems research are also described and the authors demonstrate how to use selected voltammetric techniques to study extracellular electron transfer in bioelectrochemical systems. Finally, some critical concerns with the proposed electron transfer mechanisms in bioelectrochemical systems are addressed together with the prospects of bioelectrochemical systems as energy-converting and energy-harvesting devices.     

Electrochemically active biofilms: facts and fiction. A review

This review examines the electrochemical techniques used to study extracellular electron transfer in the electrochemically active biofilms that are used in microbial fuel cells and other bioelectrochemical systems. Electrochemically active biofilms are defined as biofilms that exchange electrons with conductive surfaces: electrodes. Following the electrochemical conventions, and recognizing that electrodes can be considered reactants in these bioelectrochemical processes, biofilms that deliver electrons to the biofilm electrode are called anodic, ie electrode-reducing, biofilms, while biofilms that accept electrons from the biofilm electrode are called cathodic, ie electrode-oxidizing, biofilms. How to grow these electrochemically active biofilms in bioelectrochemical systems is discussed and also the critical choices made in the experimental setup that affect the experimental results. The reactor configurations used in bioelectrochemical systems research are also described and the authors demonstrate how to use selected voltammetric techniques to study extracellular electron transfer in bioelectrochemical systems. Finally, some critical concerns with the proposed electron transfer mechanisms in bioelectrochemical systems are addressed together with the prospects of bioelectrochemical systems as energy-converting and energy-harvesting devices.     

In vivo,in vitro and in silico: an open space for the development of microbe-based applications of synthetic biology

Living systems are studied using three complementary approaches: living cells, cell-free systems and computer-mediated modelling. Progresses in understanding, allowing researchers to create novel chassis and industrial processes rest on a cycle that combines in vivo, in vitro and in silico studies. This design–build–test–learn iteration loop cycle between experiments and analyses combines together physiology, genetics, biochemistry and bioinformatics in a way that keeps going forward. Because computer-aided approaches are not directly constrained by the material nature of the entities of interest, we illustrate here how this virtuous cycle allows researchers to explore chemistry which is foreign to that present in extant life, from whole chassis to novel metabolic cycles. Particular emphasis is placed on the importance of evolution.     

Synthetic biology in multicellular organisms: Opportunities in nematodes

Synthetic biology has mainly focused on introducing new or altered functionality in single cell systems: primarily bacteria, yeast, or mammalian cells. Here, we describe the extension of synthetic biology to nematodes, in particular the well-studied model organism Caenorhabditis elegans, as a convenient platform for developing applications in a multicellular setting. We review transgenesis techniques for nematodes, as well as the application of synthetic biology principles to construct nematode gene switches and genetic devices to control motility. Finally, we discuss potential applications of engineered nematodes.     

Building the blueprint of life

With recent breakthroughs in experimental microbiology making it possible to synthesize and implant an entire genome to create a living cell, the challenge of constructing a working blueprint for the first truly minimal synthetic organism is more important than ever. Here we review the significant progress made in the design and creation of a minimal organism. We discuss how comparative genomes, gene essentiality data, naturally small genomes, and metabolic modeling are all being applied to produce a catalogue of the biological functions essential for life. We compare the minimal gene sets from three published sources with functions identified in 13 existing gene essentiality datasets. We examine how genome-scale metabolic models have been applied to design a minimal metabolism for growth in simple and complex media. Additionally, we survey the progress of efforts to construct a minimal organism, either through implementation of combinatorial deletions in Bacillus subtilis and Escherichia coli or through the synthesis and implantation of synthetic genomes.