How to make a minimal genome for synthetic minimal cell

As a key focus of synthetic biology, building a minimal artificial cell has given rise to many discussions. A synthetic minimal cell will provide an appropriate chassis to integrate functional synthetic parts, devices and systems with functions that cannot generally be found in nature. The design and construction of a functional minimal genome is a key step while building such a cell/chassis since all the cell functions can be traced back to the genome. Kinds of approaches, based on bioinformatics and molecular biology, have been developed and proceeded to derive essential genes and minimal gene sets for the synthetic minimal genome. Experiments about streamlining genomes of model bacteria revealed genome reduction led to unanticipated beneficial properties, such as high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Recent achievements in chemical synthesis technology for large DNA segments together with the rapid development of the whole-genome sequencing, have transferred synthesis of genes to assembly of the whole genomes based on oligonucleotides, and thus created strong preconditions for synthesis of artificial minimal genome. Here in this article, we review briefly the history and current state of research in this field and summarize the main methods for making a minimal genome. We also discuss the impacts of minimized genome on metabolism and regulation of artificial cell.     

On the road to synthetic life: the minimal cell and genome-scale engineering

Synthetic biology employs rational engineering principles to build biological systems from the libraries of standard, well characterized biological parts. Biological systems designed and built by synthetic biologists fulfill a plethora of useful purposes, ranging from better healthcare and energy production to biomanufacturing. Recent advancements in the synthesis, assembly and “booting-up” of synthetic genomes and in low and high-throughput genome engineering have paved the way for engineering on the genome-wide scale. One of the key goals of genome engineering is the construction of minimal genomes consisting solely of essential genes (genes indispensable for survival of living organisms). Besides serving as a toolbox to understand the universal principles of life, the cell encoded by minimal genome could be used to build a stringently controlled “cell factory” with a desired phenotype. This review provides an update on recent advances in the genome-scale engineering with particular emphasis on the engineering of minimal genomes. Furthermore, it presents an ongoing discussion to the scientific community for better suitability of minimal or robust cells for industrial applications.     

微生物基因组简化的研究进展

微生物基因组简化是合成生物学研究热点之一。基因组的适度精简可使细胞代谢途径得以优化,改善细胞对底物、能量的利用效率,大大提高细胞生理性能的预测性和可控性。基因组简化细胞将为生物技术的应用提供理想的底盘细胞。同顾了构建基因组简化细胞的研究策略、研究方法及一些模式生物相关研究进展,总结了基因组简化研究所面临的问题及解决办法,对基因组减小化研究发展趋势前景进行了展望。     

Toward minimal bacterial cells: evolution vs. design

Recent technical and conceptual advances in the biological sciences opened the possibility of the construction of newly designed cells. In this paper we review the state of the art of cell engineering in the context of genome research, paying particular attention to what we can learn on naturally reduced genomes from either symbiotic or free living bacteria. Different minimal hypothetically viable cells can be defined on the basis of several computational and experimental approaches. Projects aiming at simplifying living cells converge with efforts to make synthetic genomes for minimal cells. The panorama of this particular view of synthetic biology lead us to consider the use of defined minimal cells to be applied in biomedical, bioremediation, or bioenergy application by taking advantage of existing naturally minimized cells.     

Cell-free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab-on-chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell-free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell-cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell-free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.     

DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis

The availability of sequences of entire genomes has dramatically increased the number of protein targets, many of which will need to be overexpressed in cells other than the original source of DNA. Gene synthesis often provides a fast and economically efficient approach. The synthetic gene can be optimized for expression and constructed for easy mutational manipulation without regard to the parent genome. Yet design and construction of synthetic genes, especially those coding for large proteins, can be a slow, difficult and confusing process. We have written a computer program that automates the design of oligonucleotides for gene synthesis. Our program requires simple input information, i.e. amino acid sequence of the target protein and melting temperature (needed for the gene assembly) of synthetic oligonucleotides. The program outputs a series of oligonucleotide sequences with codons optimized for expression in an organism of choice. Those oligonucleotides are characterized by highly homogeneous melting temperatures and a minimized tendency for hairpin formation. With the help of this program and a two-step PCR method, we have successfully constructed numerous synthetic genes, ranging from 139 to 1042 bp. The approach presented here simplifies the production of proteins from a wide variety of organisms for genomics-based studies.     

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.     

非天然氨基酸细胞工厂的构建与应用

非天然氨基酸在医药、农药、材料等领域得到广泛应用,其绿色、高效合成越来越受到关注.近年来,随着合成生物学的快速发展,微生物细胞工厂为非天然氨基酸的制造提供了重要手段.文中从合成途径的重构、关键酶的设计改造及与前体的协同调控、竞争性旁路途径的敲除、辅因子循环系统的构建等方面介绍了 一系列非天然氨基酸细胞工厂构建与应用的研...     

Construction of a Prophage-Free Variant of Corynebacterium glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology

The activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. The genome of the industrial amino acid producer Corynebacterium glutamicum ATCC 13032 contains three prophages (CGP1, CGP2, and CGP3) of so far unknown functionality. Several phage genes are regularly expressed, and the large prophage CGP3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. Here, we present the construction of MB001, a prophage-free variant of C. glutamicum ATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of a C. glutamicum chassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology.     

Patho-biotechnology: using bad bugs to do good things

Pathogenic bacteria have evolved sophisticated strategies to overcome host defences, to interact with the immune system and to interfere with essential host systems. We coin the term 'patho-biotechnology' to describe the exploitation of these valuable traits in biotechnology, medicine and food. This approach shows promise for the development of novel vaccine and drug delivery systems, as well as for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications. The genetic tractability of Listeria monocytogenes, the availability of the complete genome sequence of this intracellular pathogen, its ability to cope with stress, and its ability to traverse the gastrointestinal tract and induce a strong cellular immune response make L. monocytogenes an ideal model organism for demonstrating the patho-biotechnology concept.     

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.     

Synthetic biology in plastids

Plastids (chloroplasts) harbor a small gene‐dense genome that is amenable to genetic manipulation by transformation. During 1 billion years of evolution from the cyanobacterial endosymbiont to present‐day chloroplasts, the plastid genome has undergone a dramatic size reduction, mainly as a result of gene losses and the large‐scale transfer of genes to the nuclear genome. Thus the plastid genome can be regarded as a naturally evolved miniature genome, the gradual size reduction and compaction of which has provided a blueprint for the design of minimum genomes. Furthermore, because of the largely prokaryotic genome structure and gene expression machinery, the high transgene expression levels attainable in transgenic chloroplasts and the very low production costs in plant systems, the chloroplast lends itself to synthetic biology applications that are directed towards the efficient synthesis of green chemicals, biopharmaceuticals and other metabolites of commercial interest. This review describes recent progress with the engineering of plastid genomes with large constructs of foreign or synthetic DNA, and highlights the potential of the chloroplast as a model system in bottom‐up and top‐down synthetic biology approaches.     

A systems-level approach for metabolic engineering of yeast cell factories

The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories.     

Robust model matching design methodology for a stochastic synthetic gene network

Synthetic biology has shown its potential and promising applications in the last decade. However, many synthetic gene networks cannot work properly and maintain their desired behaviors due to intrinsic parameter variations and extrinsic disturbances. In this study, the intrinsic parameter uncertainties and external disturbances are modeled in a non-linear stochastic gene network to mimic the real environment in the host cell. Then a non-linear stochastic robust matching design methodology is introduced to withstand the intrinsic parameter fluctuations and to attenuate the extrinsic disturbances in order to achieve a desired reference matching purpose. To avoid solving the Hamilton-Jacobi inequality (HJI) in the non-linear stochastic robust matching design, global linearization technique is used to simplify the design procedure by solving a set of linear matrix inequalities (LMIs). As a result, the proposed matching design methodology of the robust synthetic gene network can be efficiently designed with the help of LMI toolbox in Matlab. Finally, two in silico design examples of the robust synthetic gene network are given to illustrate the design procedure and to confirm the robust model matching performance to achieve the desired behavior in spite of stochastic parameter fluctuations and environmental disturbances in the host cell.     

Synthesizing artificial cells from giant unilamellar vesicles: State‐of‐the art in the development of microfluidic technology

Microfluidic technology – the manipulation of fluids at micrometer scales – has revolutionized many areas of synthetic biology. The bottom‐up synthesis of “minimal” cell models has traditionally suffered from poor control of assembly conditions. Giant unilamellar vesicles (GUVs) are good models of living cells on account of their size and unilamellar membrane structure. In recent years, a number of microfluidic approaches for constructing GUVs has emerged. These provide control over traditionally elusive parameters of vesicular structure, such as size, lamellarity, membrane composition, and internal contents. They also address sophisticated cellular functions such as division and protein synthesis. Microfluidic techniques for GUV synthesis can broadly be categorized as continuous‐flow based approaches and droplet‐based approaches. This review presents the state‐of‐the‐art of microfluidic technology, a robust platform for recapitulating complex cellular structure and function in synthetic models of biological cells.     

Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria

The advances in synthetic biology bring exciting new opportunities to reprogram microorganisms with novel functionalities for environmental applications. For real-world applications, a genetic tool that enables genetic engineering in a stably genomic inherited manner is greatly desired. In this work, we design a novel genetic device for rapid and efficient genome engineering based on the i ntron-encoded homing-endonuclease empowered genome editing (iEditing). The iEditing device enables rapid and efficient genome engineering in Shewanella oneidensis MR-1, the representative strain of the electroactive bacteria group. Moreover, combining with the Red or RecET recombination system, the genome-editing efficiency was greatly improved, up to approximately 100%. Significantly, the iEditing device itself is eliminated simultaneously when genome editing occurs, thereby requiring no follow-up to remove the encoding system. Then, we develop a new extracellular electron transfer (EET) engineering strategy by programming the parallel EET systems to enhance versatile EET. The engineered strains exhibit sufficiently enhanced electron output and pollutant reduction ability. Furthermore, this device has demonstrated its great potential to be extended for genome editing in other important microbes. This work provides a useful and efficient tool for the rapid generation of synthetic microorganisms for various environmental applications.     

Electrogenetic signaling and information propagation for controlling microbial consortia via programmed lysis

To probe signal propagation and genetic actuation in microbial consortia, we have coopted the components of both redox and quorum sensing (QS) signaling into a communication network for guiding composition by “programming” cell lysis. Here, we use an electrode to generate hydrogen peroxide as a redox cue that determines consortia composition. The oxidative stress regulon of Escherichia coli, OxyR, is employed to receive and transform this signal into a QS signal that coordinates the lysis of a subpopulation of cells. We examine a suite of information transfer modalities including “monoculture” and “transmitter-receiver” models, as well as a series of genetic circuits that introduce time-delays for altering information relay, thereby expanding design space. A simple mathematical model aids in developing communication schemes that accommodate the transient nature of redox signals and the “collective” attributes of QS signals. We suggest this platform methodology will be useful in understanding and controlling synthetic microbial consortia for a variety of applications, including biomanufacturing and biocontainment.