Synthetic biology (Oxford, England)最新文献

{"title":"Automated design and implementation of a NOR gate in Pseudomonas putida.","authors":"Huseyin Tas,&nbsp;Lewis Grozinger,&nbsp;Angel Goñi-Moreno,&nbsp;Victor de Lorenzo","doi":"10.1093/synbio/ysab024","DOIUrl":"https://doi.org/10.1093/synbio/ysab024","url":null,"abstract":"<p><p>Boolean NOR gates have been widely implemented in <i>Escherichia coli</i> as transcriptional regulatory devices for building complex genetic circuits. Yet, their portability to other bacterial hosts/chassis is generally hampered by frequent changes in the parameters of the INPUT/OUTPUT response functions brought about by new genetic and biochemical contexts. Here, we have used the circuit design tool CELLO for assembling a NOR gate in the soil bacterium and the metabolic engineering platform <i>Pseudomonas putida</i> with components tailored for <i>E. coli.</i> To this end, we capitalized on the functional parameters of 20 genetic inverters for each host and the resulting compatibility between NOT pairs. Moreover, we added to the gate library three inducible promoters that are specific to <i>P. putida</i>, thus expanding cross-platform assembly options. While the number of potential connectable inverters decreased drastically when moving the library from <i>E. coli</i> to <i>P. putida</i>, the CELLO software was still able to find an effective NOR gate in the new chassis. The automated generation of the corresponding DNA sequence and <i>in vivo</i> experimental verification accredited that some genetic modules initially optimized for <i>E. coli</i> can indeed be reused to deliver NOR logic in <i>P. putida</i> as well. Furthermore, the results highlight the value of creating host-specific collections of well-characterized regulatory inverters for the quick assembly of genetic circuits to meet complex specifications.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab024"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ff/5e/ysab024.PMC8546601.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39839147","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":"Synthetic biological toggle circuits that respond within seconds and teach us new biology.","authors":"Sonja Billerbeck","doi":"10.1093/synbio/ysab027","DOIUrl":"https://doi.org/10.1093/synbio/ysab027","url":null,"abstract":"Imagine it would take several minutes or even hours for your light bulb to turn on after you hit the switch—not very useful for many daily (and nightly) activities. Light switches are made from the so-called toggle switches; basic and widely used electrical components that provide binary on–off control over electrical circuits, allowing quick decisionmaking and memory. Synthetic biologists have built analogues genetic toggle switches but until now those only responded in time-ranges of minutes to hours, limiting their use in applications that require real-time action. Mishra et al. have recently built a biological bistable toggle switch in yeast that responds within seconds by mimicking nature’s way of rapid response generation (1). Synthetic biologists envision to control cellular behavior by engineering biology in analogy to electrical circuits. Implementing a synthetic biological toggle switch was thus one of the early achievements of the field (2). While, over the last two decades, synthetic biologists have mastered to build toggle switches that respond to various inputs—chemicals, light or temperature—and show high switching robustness (3); one challenge remained: timing! Existing circuits act slow as they rely on transcription and translation for signal progression, resulting in significant delays between input-sensing and toggling into the corresponding response state. Not only Synthetic Biologists but nature itself controls important decisions—such as cell cycle progression, embryonal development or induced cell death—via bistable toggle switches. But nature knows how to act fast: rapid responses are not mediated by genetics but via post-translational protein modifications, such as phosphorylations. Although phosphor-regulation has long been known, it was difficult to engineer and concert into designed behavior. Mishra et al. overcame this hurdle by developing (phosphorylation-required interaction and mediated effect (PRIME) that harnesses the modularity of natural phosphate regulators and allows us to build chimeric proteins that can be combined to ‘phosphor-in phosphorout’ gates. One gate consists of two chimeric proteins that interact in a phosphate-dependent manner: once the upstream protein partner gets activated by a trigger, it binds to and activates or deactivates (phosphorylates or de-phosphorylates) its downstream partner. The downstreampartner then acts as the activator within the next PRIME gate. Using the PRIME gates, the authors build a network of logic gates resulting in a new-to-nature toggle network architecture that could be switched from one state to the other by two different chemical inputs, sorbitol and isopentenyl adenine—two chemicals for which receptors were readily available. As the first test read-out of the system, they used a green fluorescent protein that could be toggled between localization in the cytosol or the nucleus. Eventually, the authors showed to control a complex cellular function, yeast bud format","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab027"},"PeriodicalIF":0.0,"publicationDate":"2021-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8434798/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39436531","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":"Erratum to: A MATLAB toolbox for modeling genetic circuits in cell-free systems.","authors":"Vipul Singhal,&nbsp;Zoltan A Tuza,&nbsp;Zachary Z Sun,&nbsp;Richard M Murray","doi":"10.1093/synbio/ysab016","DOIUrl":"https://doi.org/10.1093/synbio/ysab016","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.1093/synbio/ysab007.].</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab016"},"PeriodicalIF":0.0,"publicationDate":"2021-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/68/7d/ysab016.PMC8379372.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39341295","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":"High-yield 'one-pot' biosynthesis of raspberry ketone, a high-value fine chemical.","authors":"Simon J Moore,&nbsp;Tommaso Tosi,&nbsp;David Bell,&nbsp;Yonek B Hleba,&nbsp;Karen M Polizzi,&nbsp;Paul S Freemont","doi":"10.1093/synbio/ysab021","DOIUrl":"https://doi.org/10.1093/synbio/ysab021","url":null,"abstract":"<p><p>Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction from a yield of ∼1-4 mg kg^-1 of fruit. Due to toxicity, microbial production provides only low yields of up to 5-100 mg L^-1. Herein, we report an efficient cell-free strategy to probe into a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one, into raspberry ketone at up to 100% conversion. As part of this strategy, it is essential to recycle inexpensive cofactors. Specifically, the final enzyme step in the pathway is catalyzed by raspberry ketone/zingerone synthase (RZS1), an NADPH-dependent double bond reductase. To relax cofactor specificity towards NADH, the preferred cofactor for cell-free biosynthesis, we identify a variant (G191D) with strong activity with NADH. We implement the RZS1 G191D variant within a 'one-pot' cell-free reaction to produce raspberry ketone at high-yield (61 mg L^-1), which provides an alternative route to traditional microbial production. In conclusion, our cell-free strategy complements the growing interest in engineering synthetic enzyme cascades towards industrially relevant value-added chemicals.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab021"},"PeriodicalIF":0.0,"publicationDate":"2021-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8546603/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39825394","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":"Enzyme engineering and <i>in vivo</i> testing of a formate reduction pathway.","authors":"Jue Wang,&nbsp;Karl Anderson,&nbsp;Ellen Yang,&nbsp;Lian He,&nbsp;Mary E Lidstrom","doi":"10.1093/synbio/ysab020","DOIUrl":"https://doi.org/10.1093/synbio/ysab020","url":null,"abstract":"<p><p>Formate is an attractive feedstock for sustainable microbial production of fuels and chemicals, but its potential is limited by the lack of efficient assimilation pathways. The reduction of formate to formaldehyde would allow efficient downstream assimilation, but no efficient enzymes are known for this transformation. To develop a 2-step formate reduction pathway, we screened natural variants of acyl-CoA synthetase (ACS) and acylating aldehyde dehydrogenase (ACDH) for activity on one-carbon substrates and identified active and highly expressed homologs of both enzymes. We then performed directed evolution, increasing ACDH-specific activity by 2.5-fold and ACS lysate activity by 5-fold. To test for the <i>in vivo</i> activity of our pathway, we expressed it in a methylotroph which can natively assimilate formaldehyde. Although the enzymes were active in cell extracts, we could not detect formate assimilation into biomass, indicating that further improvement will be required for formatotrophy. Our work provides a foundation for further development of a versatile pathway for formate assimilation.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab020"},"PeriodicalIF":0.0,"publicationDate":"2021-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8511477/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39519298","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":"Rational gRNA design based on transcription factor binding data.","authors":"David Bergenholm,&nbsp;Yasaman Dabirian,&nbsp;Raphael Ferreira,&nbsp;Verena Siewers,&nbsp;Florian David,&nbsp;Jens Nielsen","doi":"10.1093/synbio/ysab014","DOIUrl":"https://doi.org/10.1093/synbio/ysab014","url":null,"abstract":"<p><p>The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a standard tool in many genome engineering endeavors. The endonuclease-deficient version of Cas9 (dCas9) is also a powerful programmable tool for gene regulation. In this study, we made use of <i>Saccharomyces cerevisiae</i> transcription factor (TF) binding data to obtain a better understanding of the interplay between TF binding and binding of dCas9 fused to an activator domain, VPR. More specifically, we targeted dCas9-VPR toward binding sites of Gcr1-Gcr2 and Tye7 present in several promoters of genes encoding enzymes engaged in the central carbon metabolism. From our data, we observed an upregulation of gene expression when dCas9-VPR was targeted next to a TF binding motif, whereas a downregulation or no change was observed when dCas9 was bound on a TF motif. This suggests a steric competition between dCas9 and the specific TF. Integrating TF binding data, therefore, proved to be useful for designing guide RNAs for CRISPR interference or CRISPR activation applications.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab014"},"PeriodicalIF":0.0,"publicationDate":"2021-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/3d/cc/ysab014.PMC8546606.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39825389","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":"Building Biofoundry India: challenges and path forward.","authors":"Binay Panda,&nbsp;Pawan K Dhar","doi":"10.1093/synbio/ysab015","DOIUrl":"https://doi.org/10.1093/synbio/ysab015","url":null,"abstract":"<p><p>Biofoundry is a place where biomanufacturing meets automation. The highly modular structure of a biofoundry helps accelerate the design-build-test-learn workflow to deliver products fast and in a streamlined fashion. In this perspective, we describe our efforts to build Biofoundry India, where we see the facility add a substantial value in supporting research, innovation and entrepreneurship. We describe three key areas of our focus, harnessing the potential of non-expressing parts of the sequenced genomes, using deep learning in pathway reconstruction and synthesising enzymes and metabolites. Toward the end, we describe specific challenges in building such facility in India and the path to mitigate some of those working with the other biofoundries worldwide.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab015"},"PeriodicalIF":0.0,"publicationDate":"2021-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ff/dc/ysab015.PMC8546612.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39825390","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":"Pathway engineering for high-yield production of lutein in Escherichia coli.","authors":"Miho Takemura,&nbsp;Akiko Kubo,&nbsp;Asuka Watanabe,&nbsp;Hanayo Sakuno,&nbsp;Yuka Minobe,&nbsp;Takehiko Sahara,&nbsp;Masahiro Murata,&nbsp;Michihiro Araki,&nbsp;Hisashi Harada,&nbsp;Yoshinobu Terada,&nbsp;Katsuro Yaoi,&nbsp;Kohji Ohdan,&nbsp;Norihiko Misawa","doi":"10.1093/synbio/ysab012","DOIUrl":"https://doi.org/10.1093/synbio/ysab012","url":null,"abstract":"<p><p>Lutein is an industrially important carotenoid pigment, which is essential for photoprotection and photosynthesis in plants. Lutein is crucial for maintaining human health due to its protective ability from ocular diseases. However, its pathway engineering research has scarcely been performed for microbial production using heterologous hosts, such as <i>Escherichia coli</i>, since the engineering of multiple genes is required. These genes, which include tricky key carotenoid biosynthesis genes typically derived from plants, encode two sorts of cyclases (lycopene ε- and β-cyclase) and cytochrome P450 CYP97C. In this study, upstream genes effective for the increase in carotenoid amounts, such as isopentenyl diphosphate isomerase (<i>IDI</i>) gene, were integrated into the <i>E. coli</i> JM101 (DE3) genome. The most efficient set of the key genes (<i>MpLCYe, MpLCYb</i> and <i>MpCYP97C</i>) was selected from among the corresponding genes derived from various plant (or bacterial) species using <i>E. coli</i> that had accumulated carotenoid substrates. Furthermore, to optimize the production of lutein in <i>E. coli</i>, we introduced several sorts of plasmids that contained some of the multiple genes into the genome-inserted strain and compared lutein productivity. Finally, we achieved 11 mg/l as lutein yield using a mini jar. Here, the high-yield production of lutein was successfully performed using <i>E. coli</i> through approaches of pathway engineering. The findings obtained here should be a base reference for substantial lutein production with microorganisms in the future.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab012"},"PeriodicalIF":0.0,"publicationDate":"2021-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysab012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39573081","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 computational walk to the hidden peaks of protein performance.","authors":"Sonja Billerbeck","doi":"10.1093/synbio/ysab011","DOIUrl":"https://doi.org/10.1093/synbio/ysab011","url":null,"abstract":"Spiders use them to catch their prey, plants rely on them to fix carbon and mammals need them for eye vision—proteins. Proteins play critical roles in nature, and not surprisingly, synthetic biologists heavily rely on their functional diversity to build new therapeutics (1), catalysts (2) and materials (3). But natural proteins are rarely optimal for their envisioned human uses. They rather need to be engineered to enhance their performance. Recently, researchers introduced a machine-learning guided paradigm that can predict which mutations in a protein will enhance function with only 24 functional data sets as input (4). This paradigm could significantly accelerate the engineering of improved proteins for medicine, food, agriculture and industrial applications. The desire to optimize a protein’s function has always been a centerpiece of synthetic biology, and for decades, protein engineers have innovated the capacities of directed evolution (2) and rational protein engineering. One prominent bottleneck for the engineering of proteins is the difficulty in understanding a protein’s so-called fitness landscape. That means to know, which mutationwillmake a protein better, while in fact, mostmutations render a protein dysfunctional. The function of a protein is dictated by its amino acid sequence, and protein scientists picture the relationship between sequence and function of a protein as if it was a rugged landscape with shallow hills and high peaks, separated by valleys (5). Valleys represent sequence variants that are not functional, while the highest peaks represent the most functional mutations. Protein engineers now seek to walk through this landscape—each step being one mutation away from the wild-type sequence—in order to explore if they can find higher peaks of performance in sequence space. As the shape of the landscape is mostly unknown, the walk is random and requires the generation of many sequences and the evaluation of their function. Generating this data is often experimentally difficult or expensive. Most importantly, very distant regions of the landscape, where functional peak performance might hide, are not accessible by this search. Recently, researchers have started to perform this walk through a protein’s sequence space computationally, using deep learning (6). Although several success stories have been reported, each case still relies on a large number of experimental input data. The Church group at Harvard Medical School and the Wyss Institute for Biologically Inspired Engineering now developed a way to mitigate the notorious shortage in experimental data that constrains the engineering of many proteins, by making use of the vast number of publicly available protein sequence data (4, 7). Instead of learning the fitness landscape of an individual protein from experimental data, they first built a deep learning algorithm that extracts the fundamental features of all functional proteins from the &gt;20 million available unlabeled a","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab011"},"PeriodicalIF":0.0,"publicationDate":"2021-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysab011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39573080","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":"Three overlooked key functional classes for building up minimal synthetic cells.","authors":"Antoine Danchin","doi":"10.1093/synbio/ysab010","DOIUrl":"https://doi.org/10.1093/synbio/ysab010","url":null,"abstract":"<p><p>Assembly of minimal genomes revealed many genes encoding unknown functions. Three overlooked functional categories account for some of them. Cells are prone to make errors and age. As a first key function, discrimination between proper and changed entities is indispensable. Discrimination requires management of information, an authentic, yet abstract, currency of reality. For example proteins age, sometimes very fast. The cell must identify, then get rid of old proteins without destroying young ones. Implementing discrimination in cells leads to the second set of functions, usually ignored. Being abstract, information must nevertheless be embodied into material entities, with unavoidable idiosyncratic properties. This brings about novel unmet needs. Hence, the buildup of cells elicits specific but awkward material implementations, 'kludges' that become essential under particular settings, while difficult to identify. Finally, a third functional category characterizes the need for growth, with metabolic implementations allowing the cell to put together the growth of its cytoplasm, membranes, and genome, spanning different spatial dimensions. Solving this metabolic quandary, critical for engineering novel synthetic biology chassis, uncovered an unexpected role for CTP synthetase as the coordinator of nonhomothetic growth. Because a significant number of SynBio constructs aim at creating cell factories we expect that they will be attacked by viruses (it is not by chance that the function of the CRISPR system was identified in industrial settings). Substantiating the role of CTP, natural selection has dealt with this hurdle <i>via</i> synthesis of the antimetabolite 3'-deoxy-3',4'-didehydro-CTP, recruited for antiviral immunity in all domains of life.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab010"},"PeriodicalIF":0.0,"publicationDate":"2021-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysab010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39930099","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}