ACS Synthetic Biology最新文献

{"title":"Multilevel Optimization of 3-Ketosteroid-9α-Hydroxylase for Enhanced 9α-Hydroxy-4-androstene-3,17-dione Production.","authors":"Jing Tao, Ting Zhang, Kun Jiang, Xiaohui Cheng, Yuting Zhang, Mengfei Long, Guojian Liao","doi":"10.1021/acssynbio.5c00476","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00476","url":null,"abstract":"<p><p>9α-Hydroxylation represents a critical modification step in steroid pharmaceutical synthesis, where 9α-hydroxy-4-androstene-3,17-dione (9α-OH-AD) serves as an important intermediate for producing high-potency steroids, such as dexamethasone. The biosynthesis of 9α-OH-AD is catalyzed by a 3-ketosteroid-9α-hydroxylase (KSH) system comprising Rieske oxygenase (KshA) and ferredoxin reductase (KshB). In this study, we cloned the <i>kshA</i> and <i>kshB</i> genes from <i>Mycobacterium smegmatis</i> mc²155 and optimized their heterologous expression in <i>E. coli</i>, enhancing 9α-OH-AD yield by 4.9-fold. We implemented a 17β-carbonyl reductase (17β-CR)-mediated NADH regeneration system to ensure sufficient cofactor supply, which increased 9α-OH-AD production by 5.8-fold, while computer-aided design yielded the optimal KshA mutant A83V-G186L-A249W with a remarkable 13.8-fold improvement in catalytic efficiency compared to the wild-type. Through biotransformation optimization, the engineered microbial cell factory demonstrated a 17.2 times enhancement over the starting strain at the flask level, with a fed-batch strategy. This enzymatic conversion strategy establishes an eco-efficient platform for the sustainable synthesis of steroid therapeutics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145204976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Vivo DNA Assembly in <i>Yarrowia lipolytica</i>.","authors":"Wei Jiang, Ioannis Georgiadis, Tommaso Fumagalli, Shengbao Wang, Christina Vasileiou, Jonathan Dahlin, Irina Borodina","doi":"10.1021/acssynbio.5c00296","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00296","url":null,"abstract":"<p><p>The oleaginous yeast <i>Yarrowia lipolytica</i> is an important platform organism for biotechnology applications. In this study, we established an <i>in vivo</i> DNA assembly system leveraging CRISPR-Cas9 for efficient genomic integration of multiple DNA fragments into the genome of <i>Y. lipolytica</i>. Using the green fluorescent protein mNeonGreen as a model, we demonstrated 53% correct assembly of three DNA fragments with homology arms as short as 50 bp. The system was further validated by constructing 2-3 step biosynthetic pathways for pigments betaxanthin and betanin. To improve the homologous recombination efficiency of <i>Y. lipolytica</i>, we expressed <i>S. cerevisiae RAD52</i> (<i>ScRAD52</i>) or a Cas9-hBrex27 fusion protein. While <i>ScRAD52</i> expression impaired growth, the <i>cas9-hBrex27</i> fusion enhanced integration efficiency, particularly for multifragment pathway assemblies. The <i>in vivo</i> assembly method simplifies pathway construction and gene overexpression in <i>Y. lipolytica</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145197397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Integrated Nucleic Acid Sequence-Based Amplification (NASBA) and CRISPR-Cas13a-Based Platform for Accurate and Sensitive Detection of Cucumber Mosaic Virus.","authors":"Herma A Demissie, Subha Das, Jeremy R Thompson, Julius B Lucks","doi":"10.1021/acssynbio.5c00406","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00406","url":null,"abstract":"<p><p>Cucumber mosaic virus (CMV) is a highly prevalent ssRNA viral crop pathogen that contributes to substantial losses in agricultural productivity worldwide. The first step in managing the impact of this pathogen is an accurate and timely diagnosis. However, current sensing strategies are hampered by several limitations, including insufficient sensitivity, off-target effects, and the need for complex instrumentation. To address these challenges, we refined a highly specific and sensitive system that pairs nucleic acid sequence-based amplification (NASBA) with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a to selectively amplify and detect crop pathogens. To configure this system for CMV biosensing, we first screened guide RNAs and successfully validated designs that detect attomolar concentrations of purified CMV fragments. We then developed a simplified reaction assembly workflow toward optimizing the system for downstream point-of-use utility. Using this workflow, we demonstrated minimal matrix effects when detecting purified CMV fragments in a range of plant lysate backgrounds and showed high test specificity to CMV in the presence of common nontarget viral crop pathogens. We also showed that the NASBA-Cas13a system effectively detects the viral target in infected plant samples, as validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Finally, we optimized the system for lyophilization and long-term storage, toward preparing it for point-of-use settings. This work expands the suite of CMV diagnostic tools, offering a sensitive, specific, and user-friendly biosensing strategy. Through modular design, this assay has the potential to be reconfigured for the detection of a range of crop viruses, enhancing viral surveillance and improving infection management.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145197452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Model-Based Optimization of a qCRISPRi Circuit for Dynamic Control of Metabolic Pathways.","authors":"Sai Akhil Golla, Mona Abo-Hashesh, Dev Gupta, Yilan Liu, Radhakrishnan Mahadevan","doi":"10.1021/acssynbio.5c00095","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00095","url":null,"abstract":"<p><p>Metabolic engineering enables sustainable chemical production but often imposes metabolic burdens that reduce cellular viability and productivity. Dynamic control strategies, such as quorum sensing (QS)-based circuits, can mitigate these effects by autonomously regulating gene expression in response to cell density. In this study, we investigated a QS-regulated CRISPR interference (qCRISPRi) circuit for the dynamic control of metabolic pathways, focusing on the role of leaky expression and regulator stringency. Using a combination of mathematical modeling and experiments, we evaluated how promoter leakiness and LuxR stringency influence key switching characteristics including maximum gene expression, switching density, fold repression, and transition time. Our results show that high leaky expression of dCas9 reduces switching density and represses GFP prematurely, whereas a high-stringency LuxR variant enhances switching precision by reducing leakiness and enabling sharper transitions. These model predictions were validated experimentally in <i>E. coli</i>, confirming that LuxR stringency improves dynamic circuit performance. Together, this work provides a quantitative framework for optimizing QS-based regulatory systems and offers generalizable design insights for implementing dynamic control in metabolic engineering.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145190459","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Decade of SBOL Visual: Growing Adoption of a Diagram Standard for Engineering Biology.","authors":"Lukas Buecherl, Felipe X Buson, Georgie Hau Sørensen, Carolus Vitalis, Erik Kubaczka, Gonzalo Vidal, Bryan Bartley, Yan-Kay Ho, Göksel Mısırlı, Thomas E Gorochowski, Jacob Beal, Chris J Myers, Prashant Vaidyanathan","doi":"10.1021/acssynbio.5c00417","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00417","url":null,"abstract":"<p><p>Standards play a crucial role in ensuring consistency, interoperability, and efficiency of communication across various disciplines. In the field of synthetic biology, the Synthetic Biology Open Language (SBOL) Visual standard was introduced in 2013 to establish a structured framework for visually representing genetic designs. Over the past decade, SBOL Visual has evolved from a simple set of 21 glyphs into a comprehensive diagrammatic language for biological designs. This perspective reflects on the first ten years of SBOL Visual, tracing its evolution from inception to version 3.0. We examine the standard's adoption over time, highlighting its growing use in scientific publications, the development of supporting visualization tools, and ongoing efforts to enhance clarity and accessibility in communicating genetic design information. While trends in adoption show steady increases, achieving full compliance and use of best practices will require additional efforts. Looking ahead, the continued refinement of SBOL Visual and broader community engagement will be essential to ensuring its long-term value as the field of synthetic biology develops.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145184244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A DNA Part Library for Reliable Engineering of the Emerging Model Nematode Symbiotic Bacterium <i>Xenorhabdus griffiniae</i> HGB2511.","authors":"Elin M Larsson, Olivia Y Wang, Richard M Murray","doi":"10.1021/acssynbio.5c00414","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00414","url":null,"abstract":"<p><p><i>Xenorhabdus griffiniae</i> is a bacterium that lives inside the intestine of the entomopathogenic nematode <i>Steinernema hermaphroditum</i> and partners with the nematode to infect and kill insect larvae in soil. The construction of gene circuits, such as reporters, in <i>X. griffiniae</i> would provide tools to study and better understand the symbiotic relationship it has with its host. However, because <i>X. griffiniae</i> is not a model organism, information about gene circuit construction in <i>X. griffiniae</i> is limited. We developed and characterized a DNA part library similar to the CIDAR MoClo extension library for <i>E. coli</i> to allow more efficient construction of genetic circuits in <i>X. griffiniae</i>. TurboRFP expressing strains with different constitutive Anderson promoters and different ribosome binding sites (RBS) were constructed to quantify promoter and RBS strengths in <i>X. griffiniae</i>. Furthermore, two fluorescent proteins sfGFP and sfYFP as well as the bioluminescent <i>luxCDABE</i> operon were added to the part library and successfully expressed in <i>X. griffiniae</i>. We then used the characterized parts of the cell to build and characterize IPTG inducible constructs.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145147100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Programmable Biointerfaces and Adaptive Functionality in Next-Generation Green Nanomaterials.","authors":"Navid Rabiee","doi":"10.1021/acssynbio.5c00620","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00620","url":null,"abstract":"<p><p>Recent advances in green nanomaterials have primarily focused on mitigating toxicity through passive approaches, yet emerging technologies suggest a transformative paradigm shift toward programmable nanomaterials with dynamic biointerfaces. This Review explores how convergent innovations in synthetic biology, DNA nanotechnology, artificial intelligence, and advanced manufacturing are creating unprecedented opportunities for developing nanomaterials with context-responsive functionality. Integration of cell-free synthetic biology enables nanomaterials with genetic-circuit-driven responses to biological cues, allowing expression of bioactive compounds precisely when and where needed. DNA nanotechnology provides molecular-level programmability through stimuli-responsive structures that can perform logical operations based on complex biological inputs. Advanced machine learning approaches are revolutionizing predictive design by identifying nonintuitive correlations between green synthesis parameters and programmable functionalities. Metabolic engineering approaches utilizing engineered microbial systems offer unprecedented control over nanomaterial synthesis with reduced batch-to-batch variability, while 4D bioprinting enables macroscale assemblies with nanoscale programmable elements distributed in precise spatiotemporal arrangements. These converging technologies are enabling the development of autonomous theranostic systems with closed-loop functionality, capable of sensing biological parameters, processing this information through molecular computing, and adjusting therapeutic activity accordingly. This evolution represents a fundamental reconceptualization of biocompatibility from a static property to a dynamic, programmable characteristic, potentially yielding nanomaterials that behave more like sophisticated biological entities than traditional therapeutic agents. While significant challenges remain in stability, sensitivity, and manufacturing scalability, this emerging paradigm promises transformative advances in precision nanomedicine through self-regulating, patient-responsive therapeutic systems.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145147081","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>De Novo</i> Production of Xanthohumol by a Metabolically Engineered <i>Escherichia coli</i>.","authors":"Daniela Gomes, Joana Santos, Armando Venâncio, Joana L Rodrigues, Nigel S Scrutton, Ligia R Rodrigues","doi":"10.1021/acssynbio.5c00221","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00221","url":null,"abstract":"<p><p>Xanthohumol is a prenylflavonoid from hops with relevant bioactivities. Microbial production has emerged as a sustainable and potentially economic solution to produce it. Herein, we constructed a pathway for the <i>de novo</i> production of xanthohumol in <i>Escherichia coli</i>. Since the xanthohumol pathway depends on the availability of dimethylallyl pyrophosphate (DMAPP) and <i>S-</i>adenosylmethionine (SAM), SAM synthase (<i>metK</i>) was integrated into the genome of <i>E. coli</i> strains with previously engineered DMAPP pathways. Eleven prenyltransferases (PT) and the <i>O</i>-methyltransferase (OMT) from <i>Humulus lupulus</i> (<i>Hl</i>OMT1) were tested. <i>E. coli</i> M-PAR-121:<i>Bl</i>IDI:<i>metK</i>, constructed by integrating <i>metK</i> into the <i>E. coli</i> strain with integration of isopentenyl diphosphate isomerase (IDI) from <i>Bacillus licheniformis</i> (<i>E. coli</i> M-PAR-121:<i>Bl</i>IDI) and expressing CdpC3PT from <i>Neosartorya fischeri</i> and <i>Hl</i>OMT1 in combination with the naringenin chalcone pathway, was the best producer. This strain was able to produce 7.3 mg/L of desmethylxanthohumol and 5.3 mg/L of xanthohumol in the bioreactor, representing the first report of <i>de novo</i> production of xanthohumol in <i>E. coli</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145147070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Computational Pipeline for Targeted Integration and Variable Payload Expression in Bacteriophage Engineering.","authors":"Jonas Fernbach, Emese Hegedis, Martin J Loessner, Samuel Kilcher","doi":"10.1021/acssynbio.5c00450","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00450","url":null,"abstract":"<p><p>Bacteriophages offer a promising alternative to conventional antimicrobials, especially when such treatments fail. While natural phages are viable for therapy, advances in synthetic biology allow precise genome modifications to enhance their therapeutic potential. One approach involves inserting antimicrobial genetic payloads into the phage genome. These are typically placed behind late-expressed genes, such as the major capsid gene (<i>cps</i>). However, phages engineered with toxic payloads often fail to produce viable progeny due to premature host shutdown. To broaden the scope of viable genetic insertion sites, we developed a method to identify intergenic loci with favorable expression profiles using the machine learning-based promoter prediction tool, PhagePromoter. Guided by these predictions, we designed a computationally assisted engineering pipeline for targeted genomic payload integration. We validated this approach by engineering bioluminescent reporter genes into the genome of the strictly lytic <i>Staphylococcus</i> phage K at various predicted loci. Using homologous recombination, we generated three recombinant phages, each carrying the reporter at a distinct genomic location. These engineered phages exhibited expression levels consistent with computational predictions and demonstrated temporal expression patterns corresponding to early, middle, or late gene clusters. Our study highlights the power of combining computational tools with classical genome analysis to streamline phage engineering. This method supports rational design and enables high-throughput, automated phage modification, advancing the development of personalized phage therapy.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145123779","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Polymeric Approach to Designing Semisynthetic Enzymes.","authors":"Shan Wang, Vasco Figueiredo Batista, Henrik Karring, Changzhu Wu","doi":"10.1021/acssynbio.5c00455","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00455","url":null,"abstract":"<p><p>Incorporating synthetic chemicals into natural enzyme scaffolds to create semisynthetic enzymes is a promising strategy for achieving novel enzymatic functions. However, limitations such as low efficiency and a lack of control have hindered their industrial application. In this study, we propose a polymeric approach to designing semisynthetic enzymes by integrating ruthenium-containing polymers with the transaminase (ATA) scaffold via a \"grafting from\" copolymerization method. Initially, we combine noncatalytic proteins with polymers acting as dehydrogenases to convert acetophenone to (<i>R</i>)-1-phenylethanol with 99% conversion and 94% enantiomeric excess (<i>ee</i>). Furthermore, a polymeric semisynthetic ATA, referred to as \"PolySemiATA,\" is created by combining ATA and polymer catalysts using the same methodology. Remarkably, PolySemiATA not only retains the natural catalytic activity of enzymes but also enables an efficient one-pot cascade from (<i>S</i>)-1-phenylethylamine to (<i>R</i>)-1-phenylethanol with 99% conversion and 93% <i>ee</i>. Furthermore, PolySemiATA displays a significant advantage in recycling, surpassing the performance of mixtures composed of ATA and polymer catalysts. This study demonstrates the concept of a polymeric approach for designing semisynthetic enzymes, holding potential for producing high-value chemicals with various enzymes combined with different catalytic modular polymers to meet the demands of advanced synthesis.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145111514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}