ACS Synthetic Biology最新文献

{"title":"Sustainable Production of Cyanidin-3-<i>O</i>-galactoside by Metabolic Engineered <i>Escherichia coli</i> from Catechin.","authors":"Zhen Zong, Lianghua Xie, Jiaqi Fu, Zhongyang Liu, Wen-Wen Zhou, Wei Chen","doi":"10.1021/acssynbio.5c00094","DOIUrl":"10.1021/acssynbio.5c00094","url":null,"abstract":"<p><p>Cyanidin-3-<i>O</i>-galactoside (C3Ga), a natural pigment, has various beneficial biological activities and is widely used as a food colorant. However, traditional plant extraction methods are time-consuming and unsustainable. Rapid and sustainable synthesis of C3Ga by engineered microorganisms offers a promising alternative to traditional plant-based methods and deserves to be explored. In this study, the bioproduction of C3Ga by <i>Escherichia coli</i> was achieved for the first time. The biosynthetic pathway of C3Ga from (+)-catechin was constructed by introducing anthocyanidin synthase (ANS) and UDP-galactose:cyanidin galactosyltransferase. Some strategies, including enhancement of the UDP-galactose biosynthesis pathway, identification of efficient ANS, overexpression of the C3Ga transporter, and modulation of multigene expression, were subsequently used to drive the metabolic flux toward C3Ga production. Next, the two-stage process for C3Ga production was optimized to mitigate limitations for further metabolic engineering. Combined with the knockout of β-phosphoglucomutase (<i>ycjU</i>), a newly identified competitive pathway for UDP-galactose, the production of C3Ga finally reached 217.9 mg/L. The strategies used in this study could be applied to the biosynthesis of other anthocyanins and galactosylated natural products.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2634-2643"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144315473","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":"Novel FNR-Dependent Oxygen-Responsive Promoters in <i>Escherichia coli</i>: Design, Characterization, and Metabolic Engineering Applications.","authors":"Sen Yang, Wen-Yue Tong, Chao-Hao Guo, Nan Shi, Xiao-Yun Liu, Ming Kang","doi":"10.1021/acssynbio.5c00215","DOIUrl":"10.1021/acssynbio.5c00215","url":null,"abstract":"<p><p>Promoters responsive to changes in cultivation conditions are essential tools for dynamic metabolic engineering. Oxygen-responsive promoters, in particular, exhibit significant application potential in oxygen-limited fermentation processes. However, currently reported oxygen-dependent promoters exhibit limited dynamic ranges, and notably, there remains a lack of research on oxygen-responsive negatively regulated promoters. In this study, we designed and characterized a series of dissolved oxygen-responsive promoters in <i>Escherichia coli</i> under the regulation of the transcription factor fumarate-nitrate reduction (FNR). Anaerobically activated promoters were constructed by inserting FNR binding site (FBS) upstream of inducible core promoters, while anaerobically repressed promoters were developed by inserting FBS downstream of or flanking constitutive promoters. The most effective anaerobically activated promoters showed 24-138-fold higher activity under anaerobic conditions compared to aerobic conditions. Under anaerobic conditions, promoters with DNA looping-mediated anaerobic repression maintained only 8-17% of the activity observed under aerobic conditions. These promoters were specifically regulated by FNR, as confirmed by tests in a DH5α Δ<i>fnr</i> strain, and responded rapidly to oxygen depletion (within 30 min). The utility of these genetic tools was demonstrated by applying them to enhance pyruvate production in <i>E. coli</i>. An engineered strain with anaerobic-repressed <i>aceE</i> and anaerobic-activated <i>atpAGD</i> genes produced 5.76 g/L pyruvate at 55.7% yield in shake flask fermentations. This study offers an expanded toolbox of oxygen-responsive promoters that enable precise gene regulation based on dissolved oxygen levels, providing novel genetic strategies for developing efficient two-stage fermentation processes with separated growth and production phases.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2832-2844"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144339687","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":"Microbial Dynamic Regulatory Tools: Design, Applications, and Prospects.","authors":"Haibin Qin, Junping Zhou, Aiping Pang, Lianggang Huang, Zhiqiang Liu, Yuguo Zheng","doi":"10.1021/acssynbio.5c00219","DOIUrl":"10.1021/acssynbio.5c00219","url":null,"abstract":"<p><p>Establishing efficient microbial cell factories for the production of functional nutraceuticals, pharmaceuticals, biofuels, and chemical products requires precise regulation to adapt key enzymes and pathway modules. Dynamic regulatory strategies are a promising and effective approach to achieve balanced cell growth and metabolite production. Dynamic regulatory tools, as the executors of regulatory strategies, usually require rationally designed modification strategies to provide libraries of tools with reliable quality. Here, typical dynamic regulatory tools at the DNA level (transcriptional level), the RNA level (post-transcriptional and translational level), and the protein level (post-translational) are presented. The regulatory mechanisms and design modification strategies of each tool are highlighted. Subsequently, strategies for applying regulatory tools to construct dynamic regulatory networks of metabolic pathways are summarized. Finally, the limitations of current dynamic regulatory tools are discussed and future trends are outlooked.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2418-2432"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144482542","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":"The Framework for Genetic Engineering in Desulfovibrionaceae.","authors":"Luciana Almeida, Elena Fajardo-Ruiz, Sophie Brameyer, Kirsten Jung","doi":"10.1021/acssynbio.5c00351","DOIUrl":"10.1021/acssynbio.5c00351","url":null,"abstract":"<p><p>Desulfovibrionaceae are sulfate-reducing bacteria that are ubiquitous in anoxic environments and are also found in the human gastrointestinal tract. Their numbers are low in a healthy gut, but various intestinal and extraintestinal diseases are associated with a massive proliferation of <i>Desulfovibrio</i> spp., leading to an increase in toxic hydrogen sulfide, the product of dissimilatory sulfate respiration. The physiological and signaling capabilities of intestinal <i>Desulfovibrio</i> spp. are virtually unknown. To explore them, an efficient platform for targeted genetic engineering of <i>Desulfovibrio</i> spp. is required. In this review, we provide an overview of the available genetic parts and techniques for establishing efficient and precise genetic manipulation systems in Desulfovibrionaceae and discuss the challenges.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2445-2454"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144525403","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":"Establishment of a Visible Reporter System in <i>Zymomonas mobilis</i> through Random Mutagenesis and Rational Design of a Chromoprotein eforRed.","authors":"Xinyu Yang, Ning Yang, Siqi Wu, Shuche He, Wei Jiang, WeiXuan Zhong, Jun Du, Guimin Zhang, Xia Wang, Shihui Yang","doi":"10.1021/acssynbio.4c00872","DOIUrl":"10.1021/acssynbio.4c00872","url":null,"abstract":"<p><p>The lack of reporter-gene systems that can measure the activities of biological parts quantitatively and qualitatively impedes the development of robust cell factories to meet the needs of fast-growing biomanufacturing. Chromoproteins are homologues of fluorescent proteins, which are good reporter-gene candidates for their special absorption of natural light, making them visible to the naked eye without additional instrumentation. In this study, a fluorescent chromoprotein eforRed was selected to establish the visible reporter system in <i>Zymomonas mobilis</i>, a non-model ethanologenic Gram-negative bacterium with many excellent industrial properties to be developed as a biorefinery chassis for lignocellulosic biochemical production. Coupled with random error-prone PCR and protein rational design, the spectral characteristics of the eforRed chromoprotein were enhanced, particularly the fluorescence intensity. Mechanistic studies revealed that substitutions at the amino acid residues K201 and T24 situated on the surface region of eforRed, especially the double-mutant K201H-T24V, could optimize the protein optical characteristics by concurrently increasing intrinsic fluorescence and elevating protein expression levels. Finally, a visible reporter-gene system was successfully established in <i>Z. mobilis</i> by expressing the mutant eforRed^K201H-T24V under a strong promoter like P<i>gap</i>-4S or P<i>T7</i> that gave colonies a visible red color for phenotypic screening and retained high fluorescence intensity for quantitative analysis. This study thus not only constructed a robust visible reporter-gene system in the non-model bacterium <i>Z. mobilis</i> but also helped expand the applications of chromoproteins in biotechnology and synthetic biology, especially in non-model microorganisms.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2548-2558"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144537392","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":"Enhanced 1,4-Butanediol de Novo Synthesis in <i>Yarrowia lipolytica</i> by Incorporating Dynamic Regulation of Thiamine.","authors":"Hongwei Guo, Yaqing Zeng, Mengqi Zhu, Tianqiu Huang, Weigao Wang, Catherine Madzak, Jun Zhao, Xiaohui Sun, Hongwen Chen, Guo Chen","doi":"10.1021/acssynbio.5c00015","DOIUrl":"10.1021/acssynbio.5c00015","url":null,"abstract":"<p><p>1,4-Butanediol (1,4-BDO), one of the most important platform diols, has been widely utilized as a comonomer to manufacture several million tons of polymers every year. Microbial synthesis of the non-natural 1,4-BDO is extremely challenging. Although several artificial routes have been proposed and applied in <i>Escherichia coli</i>, among them, the branching metabolic flux from α-ketoglutarate (α-KG) to the artificial route was considered the most thermodynamically efficient solution. Establishing a 1,4-BDO synthesis route in <i>Yarrowia lipolytica</i>, a native α-KG and succinate hyperproducer, should have high potential. A CoA-dependent 1,4-BDO synthesis route was introduced into <i>Y. lipolytica</i>, followed by the investigation of rate-limiting steps, optimization of the thiamine dosage, improvement of the precursor and cofactor availability, and deletion of the competition step. It was illustrated that 1,4-BDO synthesis was susceptible to metabolic throughput of the GABA-succinate shunt and NADPH availability. Also, there was a trade-off between cellular growth and 1,4-BDO synthesis. A combinational strategy, in which hereditary dynamic regulation of thiamine was incorporated into those positive metabolic modifications, was implemented, and 6217.1 mg·L^-1 1,4-BDO was achieved under the batch fermentation model. This work also demonstrated the high potential and capacity for biosynthesis of non-nature chemicals from TCA intermediates in the yeast cells, and provided a novel clue to rebalance the metabolic flux between the heterologous pathway and central metabolism.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2572-2583"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144558397","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 Integrative Strategy Enhancing Nanobody Thermostability via CDR Grafting, In Silico Mutagenesis Screening, and Multiplex Evaluation.","authors":"Weijie Ou, Jing Dan, Xuzhen Guo, Qiong Liu, Lei Tan","doi":"10.1021/acssynbio.5c00112","DOIUrl":"10.1021/acssynbio.5c00112","url":null,"abstract":"<p><p>Nanobodies are transformative tools in biomedical research and therapy due to their structural advantages and exceptional stability. However, their intrinsic stability varies significantly, while existing stabilization strategies often face various limitations. Here, we report a computational-experimental integrative approach that combines complementarity-determining region (CDR) grafting with virtual mutagenesis for stabilization. Using A4.2m as the framework region donor and Nb20, a SARS-CoV-2 spike protein-targeting nanobody, as the CDR source, Nb20-4.2m was engineered and demonstrated a 10 °C enhancement in melting temperature (<i>T</i>_m) and 55% improvement of refolding efficiency. Subsequently, through a computational pipeline, experimental validations, and a combination of mutations, the final construct was yielded with 68 °C <i>T</i>_m and >82% refolding efficiency. A molecular dynamics simulation indicated that the stability enhancement originates from optimized intramolecular hydrogen bonding networks. With a higher efficiency than conventional methods, this approach offered a paradigm shift in engineering and established a versatile platform for nanobody optimization to fit broad applications in clinics and industry.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2690-2702"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144558395","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":"Establishing a Malonyl-CoA Biosensor for the Two Model Cyanobacteria <i>Synechocystis</i> sp. PCC 6803 and <i>Synechococcus elongatus</i> PCC 7942.","authors":"Ivana Cengic, Elton P Hudson","doi":"10.1021/acssynbio.5c00320","DOIUrl":"10.1021/acssynbio.5c00320","url":null,"abstract":"<p><p>Malonyl-CoA, produced by the first committed step of fatty acid biosynthesis, is a precursor for many valuable bioproducts, making it an important metabolic engineering target. Here, we establish a malonyl-CoA biosensor for the model cyanobacteria <i>Synechocystis</i> sp. PCC 6803 and <i>Synechococcus elongatus</i> PCC 7942. The developed biosensor utilizes FapR, a malonyl-CoA-regulated transcriptional repressor from <i>Bacillus subtilis</i>, and novel FapR-regulated and cyanobacteria-compatible hybrid promoters for expressing Yfp, the biosensor output reporter. A l-rhamnose-inducible promoter <i>P</i>_{<i>rhaBAD</i>}, characterized in combination with ribosome binding sites of varied strengths, was evaluated for titratable FapR expression. Additionally, the placement and quantity of the FapR-recognized operator within the hybrid promoter was evaluated for its effect on biosensor performance. The optimal operator placement was found to differ for the biosensor variants that achieved maximum reporter expression in the two considered model cyanobacteria. Overall, this biosensor provides new opportunities for further development of cyanobacterial cell factories.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2865-2877"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12281612/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144525402","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Extracellular Peptide-Ligand Dimerization Actuator Receptor Design for Reversible and Spatially Dosed 3D Cell-Material Communication.","authors":"Matthias Recktenwald, Ritankar Bhattacharya, Mohammed Mehdi Benmassaoud, James MacAulay, Varun M Chauhan, Leah Davis, Evan Hutt, Peter A Galie, Mary M Staehle, Nichole M Daringer, Robert J Pantazes, Sebastián L Vega","doi":"10.1021/acssynbio.4c00482","DOIUrl":"10.1021/acssynbio.4c00482","url":null,"abstract":"<p><p>Transmembrane receptors that endow mammalian cells with the ability to sense and respond to biomaterial-bound ligands will prove instrumental in bridging the fields of synthetic biology and biomaterials. Materials formed with thiol-norbornene chemistry are amenable to thiol-peptide patterning, and this study reports the rational design of synthetic receptors that reversibly activate cellular responses based on peptide-ligand recognition. This transmembrane receptor platform, termed Extracellular Peptide-ligand Dimerization Actuator (EPDA), consists of stimulatory or inhibitory receptor pairs that come together upon extracellular peptide dimer binding with corresponding monobody receptors. Intracellularly, <i>Stimulatory EPDAs</i> phosphorylate a substrate that merges two protein halves, whereas <i>Inhibitory EPDAs</i> revert split proteins back to their unmerged, inactive state via substrate dephosphorylation. To identify ligand-receptor pairs, over 2000 candidate monobodies were built <i>in silico</i> using PETEI, a novel computational algorithm we developed. The top 30 monobodies based on predicted peptide binding affinity were tested experimentally, and monobodies that induced the highest change in protein merging (green fluorescent protein, GFP) were incorporated in the final EPDA receptor design. In soluble form, stimulatory peptides induce intracellular GFP merging in a time- and concentration-dependent manner, and varying levels of green fluorescence were observed based on stimulatory and inhibitory peptide-ligand dosing. EPDA-programmed cells encapsulated in thiol-norbornene hydrogels patterned with stimulatory and inhibitory domains exhibited 3D activation or deactivation based on their location within peptide-patterned hydrogels. EPDA receptors can recognize a myriad of peptide-ligands bound to 3D materials, can reversibly induce cellular responses beyond fluorescence, and are widely applicable in biological research and regenerative medicine.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2494-2513"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12281622/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142862516","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mass Spectrometric Screening for Improving Enzymatic Conversion of Formaldehyde into C2 and C3 Products.","authors":"Yizhou Luo, Lihao Fu, Jueru Chen, Hongrong Xu, Zeqi Song, Khurshid Jalal, Yongcan Chen, Wenhao Xie, Shujun Tian, Xiaoting Fang, Tong Si, Jianzhi Zhang","doi":"10.1021/acssynbio.5c00151","DOIUrl":"10.1021/acssynbio.5c00151","url":null,"abstract":"<p><p>One-carbon biomanufacturing offers a sustainable route for producing value-added chemicals. Glycolaldehyde synthase (GALS), an engineered enzyme from <i>Pseudomonas putida</i>, catalyzes a key step by condensing formaldehyde (FALD) into carbohydrate molecules, such as glycolaldehyde (GALD) and dihydroxyacetone (DHA). However, its industrial application is limited by low catalytic efficiency and lack of high-throughput screening methods. Here, we developed a mass spectrometry (MS)-based assay for simultaneous detection and quantification of GALD and DHA products from whole-cell FALD biotransformation by GALS-expressing <i>Escherichia coli</i>. Integrating this MS assay with a robotic biofoundry, we created and screened site-directed mutagenesis libraries targeting seven key residues of GALS, achieving a throughput of ∼10 s per sample. Several improved mutants were successfully isolated, including one with a 3.7-fold increase in <i>k</i>_cat for GALD production and another with a 5-fold reduction in <i>K</i>_m for DHA production, compared to the wild type GALS. Molecular dynamics simulations were applied to understand the mutational impact on substrate binding and product specificity. This high-throughput workflow may be extended to engineer other enzymes for one-carbon feedstock utilization.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"2718-2728"},"PeriodicalIF":3.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144309248","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}