Synthetic biology (Oxford, England)最新文献

{"title":"Engineering <i>Escherichia coli</i> towards <i>de novo</i> production of gatekeeper (2<i>S</i>)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol.","authors":"Mark S Dunstan, Christopher J Robinson, Adrian J Jervis, Cunyu Yan, Pablo Carbonell, Katherine A Hollywood, Andrew Currin, Neil Swainston, Rosalind Le Feuvre, Jason Micklefield, Jean-Loup Faulon, Rainer Breitling, Nicholas Turner, Eriko Takano, Nigel S Scrutton","doi":"10.1093/synbio/ysaa012","DOIUrl":"10.1093/synbio/ysaa012","url":null,"abstract":"<p><p>Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2<i>S</i>)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of <i>Escherichia coli</i> production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of <i>in vivo</i> production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa012"},"PeriodicalIF":0.0,"publicationDate":"2020-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysaa012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38615936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DNA-BOT: a low-cost, automated DNA assembly platform for synthetic biology.","authors":"Marko Storch,&nbsp;Matthew C Haines,&nbsp;Geoff S Baldwin","doi":"10.1093/synbio/ysaa010","DOIUrl":"https://doi.org/10.1093/synbio/ysaa010","url":null,"abstract":"<p><p>Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process is now receiving serious attention. Automation will enable larger builds using less researcher time, while increasing the accessible design space. However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT). For this purpose, we developed an open-source software package and demonstrated the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, evaluating the promoter, ribosome binding site and gene order design space for a three-gene operon. All 88 constructs were assembled with high accuracy, at a consumables cost of $1.50-$5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT, making it accessible for most labs and democratizing automated DNA assembly.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa010"},"PeriodicalIF":0.0,"publicationDate":"2020-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysaa010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"BEAMS: a workforce development program to bridge the gap between biologists and material scientists.","authors":"Marilyn S Lee,&nbsp;Matthew W Lux,&nbsp;Jared B DeCoste","doi":"10.1093/synbio/ysaa009","DOIUrl":"https://doi.org/10.1093/synbio/ysaa009","url":null,"abstract":"<p><p>To maximize innovation in materials science and synthetic biology, it is critical to master interdisciplinary understanding and communication within an organization. Programming aimed at this juncture has the potential to bring members of the workforce together to frame new networks and spark collaboration. In this article, we recognize the potential synergy between materials and synthetic biology research and describe our approach to this challenge as a case study. A workforce development program was devised consisting of a lecture series, laboratory demonstrations and a hands-on laboratory competition to produce a bacterial cellulose material with the highest tensile strength. This program, combined with support for infrastructure and research, resulted in a significant return on investment with new externally funded synthetic biology for materials programs for our organization. The learning elements described here may be adapted by other institutions for a variety of settings and goals.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa009"},"PeriodicalIF":0.0,"publicationDate":"2020-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysaa009","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38658944","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure determines function-the role of topology in the functionality of gene circuits.","authors":"Daniel Bojar","doi":"10.1093/synbio/ysaa008","DOIUrl":"https://doi.org/10.1093/synbio/ysaa008","url":null,"abstract":"As synthetic biologists, we sometimes forget the toggle switch and the self-activating switch, the foundational advances that launched the entire field of synthetic biology a mere two decades ago. As the first in a long line of increasingly sophisticated gene circuits with applications in biocomputing and biomedical therapies, these combinations of genetic parts in the humble bacterium Escherichia coli demonstrated that biology can—in principle—be programmed. In a recent study in the journal Nature Chemical Biology (1), Zhang et al. from the group of XiaoJun Tian at Arizona State University revisited the toggle switch and the self-activating switch, demonstrating the differential impact of cell division and growth on the function of these circuits, which could make some circuit designs unviable in the environment of dilution or growth characterizing many applications. As the output of the self-activating switch activates its own transcription, it should exhibit a stable ON-state beyond a certain inducer threshold. Yet what Zhang et al. discovered was that, once these green fluorescent protein (GFP)-positive, ONstate bacteria are diluted, the formerly stable ON-state disintegrates and the bacteria were suddenly indistinguishable from those that were never stimulated in the first place. Theory and their mathematical models, however, predicted that these once-ON-bacteria would remain ON, even after dilution. The supposedly stable memory of the self-activating switch was broken by a simple dilution. The hidden variable that accounted for the circuit’s memory lapse was growth. It has been recently appreciated that gene circuits place a metabolic burden on cells and therefore inhibit growth (2), while Zhang et al. additionally discovered that growth inhibited the functionality of their gene circuit. This resulted in a seesaw dynamic after diluting cells into medium rich with inducer: first, GFP fluorescence crashed, and then, after cell growth subsided, GFP resumed production. Factoring in the interfacing of growth and gene circuit into their models indeed resolved any unexplained differences in circuit memory. Interestingly, dilution into conditioned rather than fresh medium, thus inhibiting rapid growth, did preserve the memory of the self-activating switch. Naturally, Zhang et al. investigated whether this growth feedback also affected other circuit architectures, such as the toggle switch that can be used to switch between two stable states. Yet, overall, the toggle switch seemed to exhibit a much broader resistance to memory loss through growth effects, as long as the two constituting transcription factors operated on a similar timescale. Dilution into fresh or conditioned medium led to nearly the same output in terms of GFP fluorescence, demonstrating the perseverance of memory. The authors also note the crucial difference between transcriptional activation (self-activating switch) and inhibition (toggle switch), as the former seemed much more se","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa008"},"PeriodicalIF":0.0,"publicationDate":"2020-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysaa008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dual UTR-A novel 5' untranslated region design for synthetic biology applications.","authors":"Simone Balzer Le,&nbsp;Ingerid Onsager,&nbsp;Jon Andreas Lorentzen,&nbsp;Rahmi Lale","doi":"10.1093/synbio/ysaa006","DOIUrl":"https://doi.org/10.1093/synbio/ysaa006","url":null,"abstract":"<p><p>Bacterial 5' untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5' UTR, dual UTR, utilizing the transcriptional and translational characteristics of 5' UTRs in a single expression cassette. The dual UTR consists of two 5' UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling <i>de novo</i> translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in <i>Escherichia coli</i> and <i>Pseudomonas putida</i> using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximization of protein production.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa006"},"PeriodicalIF":0.0,"publicationDate":"2020-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysaa006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genomics-enabled analysis of specialized metabolism in bioenergy crops: current progress and challenges.","authors":"Kira Tiedge, Andrew Muchlinski, Philipp Zerbe","doi":"10.1093/synbio/ysaa005","DOIUrl":"10.1093/synbio/ysaa005","url":null,"abstract":"<p><p>Plants produce a staggering diversity of specialized small molecule metabolites that play vital roles in mediating environmental interactions and stress adaptation. This chemical diversity derives from dynamic biosynthetic pathway networks that are often species-specific and operate under tight spatiotemporal and environmental control. A growing divide between demand and environmental challenges in food and bioenergy crop production has intensified research on these complex metabolite networks and their contribution to crop fitness. High-throughput omics technologies provide access to ever-increasing data resources for investigating plant metabolism. However, the efficiency of using such system-wide data to decode the gene and enzyme functions controlling specialized metabolism has remained limited; due largely to the recalcitrance of many plants to genetic approaches and the lack of 'user-friendly' biochemical tools for studying the diverse enzyme classes involved in specialized metabolism. With emphasis on terpenoid metabolism in the bioenergy crop switchgrass as an example, this review aims to illustrate current advances and challenges in the application of DNA synthesis and synthetic biology tools for accelerating the functional discovery of genes, enzymes and pathways in plant specialized metabolism. These technologies have accelerated knowledge development on the biosynthesis and physiological roles of diverse metabolite networks across many ecologically and economically important plant species and can provide resources for application to precision breeding and natural product metabolic engineering.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa005"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445794/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436592","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Universal loop assembly: open, efficient and cross-kingdom DNA fabrication.","authors":"Bernardo Pollak, Tamara Matute, Isaac Nuñez, Ariel Cerda, Constanza Lopez, Valentina Vargas, Anton Kan, Vincent Bielinski, Peter von Dassow, Chris L Dupont, Fernán Federici","doi":"10.1093/synbio/ysaa001","DOIUrl":"10.1093/synbio/ysaa001","url":null,"abstract":"<p><p>Standardized type IIS DNA assembly methods are becoming essential for biological engineering and research. These methods are becoming widespread and more accessible due to the proposition of a 'common syntax' that enables higher interoperability between DNA libraries. Currently, Golden Gate (GG)-based assembly systems, originally implemented in host-specific vectors, are being made compatible with multiple organisms. We have recently developed the GG-based Loop assembly system for plants, which uses a small library and an intuitive strategy for hierarchical fabrication of large DNA constructs (>30 kb). Here, we describe 'universal Loop' (uLoop) assembly, a system based on Loop assembly for use in potentially any organism of choice. This design permits the use of a compact number of plasmids (two sets of four odd and even vectors), which are utilized repeatedly in alternating steps. The elements required for transformation/maintenance in target organisms are also assembled as standardized parts, enabling customization of host-specific plasmids. Decoupling of the Loop assembly logic from the host-specific propagation elements enables universal DNA assembly that retains high efficiency regardless of the final host. As a proof-of-concept, we show the engineering of multigene expression vectors in diatoms<i>,</i> yeast, plants and bacteria. These resources are available through the OpenMTA for unrestricted sharing and open access.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa001"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052795/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37729142","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Permutational analysis of <i>Saccharomyces cerevisiae</i> regulatory elements.","authors":"Namrita Dhillon,&nbsp;Robert Shelansky,&nbsp;Brent Townshend,&nbsp;Miten Jain,&nbsp;Hinrich Boeger,&nbsp;Drew Endy,&nbsp;Rohinton Kamakaka","doi":"10.1093/synbio/ysaa007","DOIUrl":"https://doi.org/10.1093/synbio/ysaa007","url":null,"abstract":"<p><p>Gene expression in <i>Saccharomyces cerevisiae</i> is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin factors has led to the delineation of various modular regulatory elements-enhancers (upstream activating sequences), core promoters, 5' untranslated regions (5' UTRs) and transcription terminators/3' untranslated regions (3' UTRs). However, only a few of these elements have been tested in combinations with other elements and the functional interactions between the different modular regulatory elements remain under explored. We describe a simple and rapid approach to build a combinatorial library of regulatory elements and have used this library to study 26 different enhancers, core promoters, 5' UTRs and transcription terminators/3' UTRs to estimate the contribution of individual regulatory parts in gene expression. Our combinatorial analysis shows that while enhancers initiate gene expression, core promoters modulate the levels of enhancer-mediated expression and can positively or negatively affect expression from even the strongest enhancers. Principal component analysis (PCA) indicates that enhancer and promoter function can be explained by a single principal component while UTR function involves multiple functional components. The PCA also highlights outliers and suggest differences in mechanisms of regulation by individual elements. Our data also identify numerous regulatory cassettes composed of different individual regulatory elements that exhibit equivalent gene expression levels. These data thus provide a catalog of elements that could in future be used in the design of synthetic regulatory circuits.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa007"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysaa007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38247428","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biosensor-based enzyme engineering approach applied to psicose biosynthesis.","authors":"Jeremy Armetta, Rose Berthome, Antonin Cros, Celine Pophillat, Bruno Maria Colombo, Amir Pandi, Ioana Grigoras","doi":"10.1093/synbio/ysz028","DOIUrl":"10.1093/synbio/ysz028","url":null,"abstract":"<p><p>Bioproduction of chemical compounds is of great interest for modern industries, as it reduces their production costs and ecological impact. With the use of synthetic biology, metabolic engineering and enzyme engineering tools, the yield of production can be improved to reach mass production and cost-effectiveness expectations. In this study, we explore the bioproduction of D-psicose, also known as D-allulose, a rare non-toxic sugar and a sweetener present in nature in low amounts. D-psicose has interesting properties and seemingly the ability to fight against obesity and type 2 diabetes. We developed a biosensor-based enzyme screening approach as a tool for enzyme selection that we benchmarked with the <i>Clostridium cellulolyticum</i> D-psicose 3-epimerase for the production of D-psicose from D-fructose. For this purpose, we constructed and characterized seven psicose responsive biosensors based on previously uncharacterized transcription factors and either their predicted promoters or an engineered promoter. In order to standardize our system, we created the Universal Biosensor Chassis, a construct with a highly modular architecture that allows rapid engineering of any transcription factor-based biosensor. Among the seven biosensors, we chose the one displaying the most linear behavior and the highest increase in fluorescence fold change. Next, we generated a library of D-psicose 3-epimerase mutants by error-prone PCR and screened it using the biosensor to select gain of function enzyme mutants, thus demonstrating the framework's efficiency.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz028"},"PeriodicalIF":0.0,"publicationDate":"2019-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445875/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436590","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid generation of sequence-diverse terminator libraries and their parameterization using quantitative Term-Seq.","authors":"Andrew J Hudson,&nbsp;Hans-Joachim Wieden","doi":"10.1093/synbio/ysz026","DOIUrl":"https://doi.org/10.1093/synbio/ysz026","url":null,"abstract":"<p><p>Synthetic biology and the rational design and construction of biological devices require vast numbers of characterized biological parts, as well as reliable design tools to build increasingly complex, multigene architectures. Design principles for intrinsic terminators have been established; however, additional sequence-structure studies are needed to refine parameters for termination-based genetic devices. We report a rapid single-pot method to generate libraries of thousands of randomized bidirectional intrinsic terminators and a modified quantitative Term-Seq (qTerm-Seq) method to simultaneously identify terminator sequences and measure their termination efficiencies (TEs). Using qTerm-Seq, we characterize hundreds of additional strong terminators (TE > 90%) with some terminators reducing transcription read-through by up to 1000-fold in <i>Escherichia coli</i>. Our terminator library and qTerm-Seq pipeline constitute a flexible platform enabling identification of terminator parts that can achieve transcription termination not only over a desired range but also to investigate their sequence-structure features, including for specific genetic and application contexts beyond the common <i>in vivo</i> systems such as <i>E. coli</i>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz026"},"PeriodicalIF":0.0,"publicationDate":"2019-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz026","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}