Synthetic biology (Oxford, England)最新文献

{"title":"Arch enemy no more: designing the first synthetic globular all-beta proteins with beta-arches.","authors":"Daniel Bojar","doi":"10.1093/synbio/ysz002","DOIUrl":"https://doi.org/10.1093/synbio/ysz002","url":null,"abstract":"De novo protein design, taking its first steps at the turn of the century (1), aims to create novel proteins without an existing protein scaffold wholly from foundational structural principles and simulations. These designed proteins often have sequences and functions that are unlike anything found in nature and can even exhibit completely novel protein folds. The wholesale design of new proteins thus requires an exquisite understanding of their 3D structure. Thanks to their modular properties, proteins consisting solely of alpha-helices were the first to yield to protein design de novo. In a recent publication in the journal Nature Structural & Molecular Biology, David Baker’s team at the University of Washington made a foray into uncharted territory and for the first time succeeded in designing a globular all-beta protein de novo (2). To understand their success, it is paramount to understand why it is so difficult to design globular proteins consisting only of beta-sheets. In contrast to amino acid residues in alphahelices, which establish most of their contacts with residues nearby, residues in beta-sheets frequently interact with residues that are farther apart on the sequence, making them harder to design. For example, beta-arches are loops that connect two beta-strands that do not form a continuous beta-sheet. In this publication, the authors discovered and utilized structural principles of all-beta proteins to facilitate their design process. These principles for instance constrained the geometry of beta-arches by assigning amino acid frequencies as well as the orientation of their side chains to specific types of beta-arches. Led by researchers Enrique Marcos and Tamuka M. Chidyausiku, the team used the protein modeling software Rosetta to construct candidates for globular all-beta proteins with differing lengths of beta-strands and beta-arches. Choosing 19 of these final jellyroll protein structure candidates consisting of eight antiparallel beta-strands for experimental characterization, they recombinantly expressed them in E. coli. Obtaining an nuclear magnetic resonance (NMR) spectroscopy structure of one of these expressed designer proteins, the authors could demonstrate that the actual protein structure closely resembled their de novo design. While proteins consisting of beta-strands connected by tight loops (effectively forming a flowing carpet of paired beta-strands) have been attempted before, the de novo design of beta-arches is definitely a novelty. This characteristic allows all-beta proteins to fold into globular proteins, in contrast to the previous elongated designs, and is needed for complex proteins such as antibodies. Why is all of this important? In addition to a profound conceptual leap in our ability to design proteins de novo, this work furthers our structural understanding of all-beta proteins. Many important proteins, such as the nucleosome-chaperone nucleoplasmin and many viral capsid proteins, contain jellyroll ","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz002"},"PeriodicalIF":0.0,"publicationDate":"2019-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38537364","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":"Developing a graduate training program in Synthetic Biology: SynBioCDT.","authors":"Idil Cazimoglu,&nbsp;Alexander P S Darlington,&nbsp;Aurelija Grigonyte,&nbsp;Charlotte E G Hoskin,&nbsp;Juntai Liu,&nbsp;Robert Oppenheimer,&nbsp;Jesús A Siller-Farfán,&nbsp;Claire Grierson,&nbsp;Antonis Papachristodoulou","doi":"10.1093/synbio/ysz006","DOIUrl":"https://doi.org/10.1093/synbio/ysz006","url":null,"abstract":"<p><p>This article presents the experience of a team of students and academics in developing a post-graduate training program in the new field of Synthetic Biology. Our Centre for Doctoral Training in Synthetic Biology (SynBioCDT) is an initiative funded by the United Kingdom's Research Councils of Engineering and Physical Sciences (EPSRC), and Biotechnology and Biological Sciences (BBSRC). SynBioCDT is a collaboration between the Universities of Oxford, Bristol and Warwick, and has been successfully running since 2014, training 78 students in this field. In this work, we discuss the organization of the taught, research and career development training. We also address the challenges faced when offering an interdisciplinary program. The article concludes with future directions to continue the development of the SynBioCDT.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz006"},"PeriodicalIF":0.0,"publicationDate":"2019-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38439139","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":"An educational module to explore CRISPR technologies with a cell-free transcription-translation system.","authors":"Daphne Collias,&nbsp;Ryan Marshall,&nbsp;Scott P Collins,&nbsp;Chase L Beisel,&nbsp;Vincent Noireaux","doi":"10.1093/synbio/ysz005","DOIUrl":"https://doi.org/10.1093/synbio/ysz005","url":null,"abstract":"<p><p>Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing tools. The resulting CRISPR technologies have driven innovations for treating genetic diseases and eradicating human pests while raising societal questions about gene editing in human germline cells as well as crop plants. Bringing CRISPR into the classroom therefore offers a means to expose students to cutting edge technologies and to promote discussions about ethical questions at the intersection of science and society. However, working with these technologies in a classroom setting has been difficult because typical experiments rely on cellular systems such as bacteria or mammalian cells. We recently reported the use of an <i>E. coli</i> cell-free transcription-translation (TXTL) system that simplifies the demonstration and testing of CRISPR technologies with shorter experiments and limited equipment. Here, we describe three educational modules intended to expose undergraduate students to CRISPR technologies using TXTL. The three sequential modules comprise (i) designing the RNAs that guide DNA targeting, (ii) measuring DNA cleavage activity in TXTL and (iii) testing how mutations to the targeting sequence or RNA backbone impact DNA binding and cleavage. The modules include detailed protocols, questions for group discussions or individual evaluation, and lecture slides to introduce CRISPR and TXTL. We expect these modules to allow students to experience the power and promise of CRISPR technologies in the classroom and to engage with their instructor and peers about the opportunities and potential risks for society.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz005"},"PeriodicalIF":0.0,"publicationDate":"2019-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38439138","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":"Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes.","authors":"Julia Stifel,&nbsp;Maike Spöring,&nbsp;Jörg Steffen Hartig","doi":"10.1093/synbio/ysy022","DOIUrl":"https://doi.org/10.1093/synbio/ysy022","url":null,"abstract":"<p><p>Artificial riboswitches based on ribozymes serve as versatile tools for ligand-dependent gene expression regulation. Advantages of these so-called aptazymes are their modular architecture and the comparably little coding space they require. A variety of aptamer-ribozyme combinations were constructed in the past 20 years and the resulting aptazymes were applied in diverse contexts in prokaryotic and eukaryotic systems. Most <i>in vivo</i> functional aptazymes are OFF-switches, while ON-switches are more advantageous regarding potential applications in e.g. gene therapy vectors. We developed new ON-switching aptazymes in the model organism <i>Escherichia coli</i> and in mammalian cell culture using the intensely studied guanine-sensing <i>xpt</i> aptamer. Utilizing a high-throughput screening based on fluorescence-activated cell sorting in bacteria we identified up to 9.2-fold ON-switches and OFF-switches with a dynamic range up to 32.7-fold. For constructing ON-switches in HeLa cells, we used a rational design approach based on existing tetracycline-sensitive ON-switches. We discovered that communication modules responding to tetracycline are also functional in the context of guanine aptazymes, demonstrating a high degree of modularity. Here, guanine-responsive ON-switches with a four-fold dynamic range were designed. Summarizing, we introduce a series of novel guanine-dependent ribozyme switches operative in bacteria and human cell culture that significantly broaden the existing toolbox.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysy022"},"PeriodicalIF":0.0,"publicationDate":"2019-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysy022","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38537362","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":"Degrading an enzyme to increase its product: a novel approach to decoupling biosynthesis and growth.","authors":"Ciarán L Kelly","doi":"10.1093/synbio/ysz001","DOIUrl":"https://doi.org/10.1093/synbio/ysz001","url":null,"abstract":"In biomanufacturing, it is often desirable to uncouple enzyme levels and activities from bacterial growth rates. When an enzyme-encoding gene that is used to produce a target chemical is constitutively expressed, production of the chemical of choice is limited, as most of the carbon is diverted into biomass production. Many different approaches have been used to tackle this problem. Most commonly, gene expression is turned on at specific phases in bacterial growth to control the timing of protein production. A recent article in ACS Synthetic Biology introduces ‘FENIX’, a novel, post-translational system for uncoupling biosynthesis from growth. While most uncoupling strategies rely on an ‘on switch’ to delay the induction of gene expression, the unique FENIX system is an ‘off switch’ that stops the protein of interest from being degraded. FENIX combines two different proteindegradation machineries, one that is native to the Escherichia coli host and one that is introduced. A sequence encoding a C-terminal Ssra peptide tag is fused to the gene of interest, which is expressed from a plasmid. This tag results in continuous degradation of the protein of interest by proteases always present in the host organism (ClpXP and ClpAP). Encoded immediately upstream of the Ssra tag is another peptide tag that is recognized by the non-native protease NIa (used in viral polypeptide processing). NIa is encoded on a second plasmid under the control of an inducible promoter. When expression of the gene encoding NIa is induced, cleavage of the polypeptide occurs at the NIa target site, removing the Ssra tag from the rest of the protein. Because the gene of interest was already being expressed, turning off degradation allows for rapid accumulation of the protein. The authors initially used fluorescent proteins to successfully demonstrate that the FENIX system enables both tight control and rapid induction of a protein of interest. They then applied the system to the production of the renewable plastic polymer, polyhydroxybutrate (PHB). Many groups have taken the genes encoding the three enzymes required for PHB production, phaC, phaA and phaB from Cupriavidus necator, and expressed them in E. coli, with limited yields to date. The substrate for PHB production is acetyl-CoA, one of the main hubs for carbon and electron flow in metabolism. The authors hypothesized that by decoupling PhaA enzyme accumulation and activity from the exponential phase of bacterial growth, competition for acetyl-CoA would be reduced and greater rates and overall yields of PHB production could be achieved. They showed that by using the FENIX system to control the levels of PhaA (and constitutively expressing phaB and phaC), growth-independent accumulation of PhaA activity and PHB production was possible. Finally, the authors demonstrated that this system could operate as a post-translational metabolic switch, allowing diversion of carbon and electrons away from acetate production towards PHB ","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz001"},"PeriodicalIF":0.0,"publicationDate":"2019-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38537363","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":"CRISPR-interference-based modulation of mobile genetic elements in bacteria.","authors":"Ákos Nyerges,&nbsp;Balázs Bálint,&nbsp;Judit Cseklye,&nbsp;István Nagy,&nbsp;Csaba Pál,&nbsp;Tamás Fehér","doi":"10.1093/synbio/ysz008","DOIUrl":"https://doi.org/10.1093/synbio/ysz008","url":null,"abstract":"<p><p>Spontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of engineered functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS<i>1</i>, IS<i>3</i>, IS<i>5</i> and IS<i>150</i> at up to 38 chromosomal loci in <i>Escherichia coli</i>, <i>in vivo</i>. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. The fitness cost of multiple IS-knockdown, detectable in flask-and-shaker systems was readily outweighed by the less frequent inactivation of the transgene, as observed in green fluorescent protein (GFP)-overexpression experiments. In addition, global transcriptomics analysis revealed only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multiple <i>E. coli</i> strains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz008"},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37348462","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":"Cell-free biosynthesis of limonene using enzyme-enriched <i>Escherichia coli</i> lysates.","authors":"Quentin M Dudley, Connor J Nash, Michael C Jewett","doi":"10.1093/synbio/ysz003","DOIUrl":"10.1093/synbio/ysz003","url":null,"abstract":"<p><p>Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple <i>Escherichia coli</i> lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native <i>E. coli</i> farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD⁺, ATP and CoA, the system can synthesize 0.66 mM (90.2 mg l^-1) limonene over 24 h, a productivity of 3.8 mg l^-1 h^-1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz003"},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/bb/55/ysz003.PMC6407499.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37058980","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":"Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics.","authors":"Richard Kelwick, Luca Ricci, Soo Mei Chee, David Bell, Alexander J Webb, Paul S Freemont","doi":"10.1093/synbio/ysy016","DOIUrl":"10.1093/synbio/ysy016","url":null,"abstract":"<p><p>The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable. Cell-free metabolic engineering strategies have already been used to optimize several biosynthetic pathways and we envisioned that cell-free strategies could be used for optimizing PHAs biosynthetic pathways. To this end, we developed several <i>Escherichia coli</i> cell-free systems for <i>in vitro</i> prototyping PHAs biosynthetic operons, and also for screening relevant metabolite recycling enzymes. Furthermore, we customized our cell-free reactions through the addition of whey permeate, an industrial waste that has been previously used to optimize <i>in vivo</i> PHAs production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by approximately 50%. In cell-free transcription-translation prototyping reactions, gas chromatography-mass spectrometry quantification of cell-free 3-hydroxybutyrate (3HB) production revealed differences between the activities of the Native <b>Δ</b>PhaC_C319A (1.18 ± 0.39 µM), C104 <b>Δ</b>PhaC_C319A (4.62 ± 1.31 µM) and C101 <b>Δ</b>PhaC_C319A (2.65 ± 1.27 µM) <i>phaCAB</i> operons that were tested. Interestingly, the most active operon, C104 produced higher levels of PHAs (or PHAs monomers) than the Native <i>phaCAB</i> operon in both <i>in vitro</i> and <i>in vivo</i> assays. Coupled cell-free biotransformation/transcription-translation reactions produced greater yields of 3HB (32.87 ± 6.58 µM), and these reactions were also used to characterize a <i>Clostridium propionicum</i> Acetyl-CoA recycling enzyme. Together, these data demonstrate that cell-free approaches complement <i>in vivo</i> workflows for identifying additional strategies for optimizing PHAs production.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"3 1","pages":"ysy016"},"PeriodicalIF":0.0,"publicationDate":"2018-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysy016","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10659712","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":"A set of experimentally validated, mutually orthogonal primers for combinatorially specifying genetic components.","authors":"Subu K Subramanian,&nbsp;William P Russ,&nbsp;Rama Ranganathan","doi":"10.1093/synbio/ysx008","DOIUrl":"https://doi.org/10.1093/synbio/ysx008","url":null,"abstract":"<p><p>The design and synthesis of novel genes and deoxyribonucleic acid (DNA) sequences is a central technique in synthetic biology. Current methods of high throughput gene synthesis use pooled oligonucleotides obtained from custom-designed DNA microarray chips, and rely on orthogonal (non-interacting) polymerase chain reaction primers to specifically de-multiplex, by amplification, the precise subset of oligonucleotides necessary to assemble a full length gene. The availability of a large validated set of mutually orthogonal primers is therefore a crucial reagent for high-throughput gene synthesis. Here, we present a set of 166 20-nucleotide primers that are experimentally verified to be non-interacting, capable of specifying 13 695 unique genes. These primers represent a valuable resource to the synthetic biology community for specifying genetic components that can be assembled through a scalable and modular architecture.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"3 1","pages":"ysx008"},"PeriodicalIF":0.0,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysx008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10592526","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":"Tailor-made exopolysaccharides-CRISPR-Cas9 mediated genome editing in <i>Paenibacillus polymyxa</i>.","authors":"Marius Rütering,&nbsp;Brady F Cress,&nbsp;Martin Schilling,&nbsp;Broder Rühmann,&nbsp;Mattheos A G Koffas,&nbsp;Volker Sieber,&nbsp;Jochen Schmid","doi":"10.1093/synbio/ysx007","DOIUrl":"https://doi.org/10.1093/synbio/ysx007","url":null,"abstract":"<p><p>Application of state-of-the-art genome editing tools like CRISPR-Cas9 drastically increase the number of undomesticated micro-organisms amenable to highly efficient and rapid genetic engineering. Adaptation of these tools to new bacterial families can open up entirely new possibilities for these organisms to accelerate as biotechnologically relevant microbial factories, also making new products economically competitive. Here, we report the implementation of a CRISPR-Cas9 based vector system in <i>Paenibacillus polymyxa</i>, enabling fast and reliable genome editing in this host. Homology directed repair allows for highly efficient deletions of single genes and large regions as well as insertions. We used the system to investigate the yet undescribed biosynthesis machinery for exopolysaccharide (EPS) production in <i>P. polymyxa</i> DSM 365, enabling assignment of putative roles to several genes involved in EPS biosynthesis. Using this simple gene deletion strategy, we generated EPS variants that differ from the wild-type polymer not only in terms of monomer composition, but also in terms of their rheological behavior. The developed CRISPR-Cas9 mediated engineering approach will significantly contribute to the understanding and utilization of socially and economically relevant <i>Paenibacillus</i> species and extend the polymer portfolio.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"2 1","pages":"ysx007"},"PeriodicalIF":0.0,"publicationDate":"2017-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysx007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38435850","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}