ACS Synthetic Biology最新文献

{"title":"Engineering Chromatin Regulation of Xylose Utilization in Budding Yeast <i>Saccharomyces cerevisiae</i> for Efficient Bioconversion.","authors":"Wei-Bin Wang, Rui-Qi Tang, Bing Yuan, Yue Wang, Guo-Dong Liu, Dong-Min Li, Hong-Jia Zhang, Xin-Qing Zhao, Feng-Wu Bai","doi":"10.1021/acssynbio.4c00730","DOIUrl":"10.1021/acssynbio.4c00730","url":null,"abstract":"<p><p>Utilization of xylose as a renewable carbon source has received constant interest. Considering that the structure and state of eukaryotic chromatin are inextricably intertwined, it is significant to explore chromatin regulation for engineering xylose metabolism in yeast. Here, we show that two chromatin remodelers, namely, Swr1 and Isw1, affect xylose utilization in recombinant budding yeast<i>Saccharomyces cerevisiae</i>. Overexpressing <i>SWR1</i> showed the highest increase in xylose utilization, up to 29.3%, compared to that of the parent strain. Furthermore, comparative transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed significantly different changes of gene expression by elevated expression of Swr1 and Isw1. Reduced histone H2A.Z occupancy in two key carbon-metabolism regulators of Mig2 and Sip2 was further observed in the engineered yeast. Further tests showed improved xylose utilization of the engineered yeast in the presence of corncob hydrolysate. Our results suggest that chromatin regulators are critical genetic elements in recombinant <i>S. cerevisiae</i> for engineering xylose metabolism.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"794-803"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143595769","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":"Improving <i>Bacillus subtilis</i> as Biological Chassis Performance by the CRISPR Genetic Toolkit.","authors":"Xianhai Cao, Xiaojuan Wang, Ruirui Chen, Lu Chen, Yang Liu, Meng Wang","doi":"10.1021/acssynbio.4c00844","DOIUrl":"10.1021/acssynbio.4c00844","url":null,"abstract":"<p><p><i>Bacillus subtilis</i> is the model Gram-positive and industrial chassis bacterium; it has blossomed as a robust and promising host for enzyme, biochemical, or bioflocculant production. However, synthetic biology and metabolic engineering technologies of <i>B. subtilis</i> have lagged behind the most widely used industrial chassis <i>Saccharomyces cerevisiae</i> and <i>Escherichia coli</i>. CRISPR (an acronym for <b>c</b>lustered <b>r</b>egularly <b>i</b>nterspaced <b>s</b>hort <b>p</b>alindromic <b>r</b>epeats) enables efficient, site-specific, and programmable DNA cleavage, which has revolutionized the manner of genome editing. In 2016, CRISPR technology was first introduced into <i>B. subtilis</i> and has been intensely upgraded since then. In this Review, we discuss recently developed key additions to CRISPR toolkit design in <i>B. subtilis</i> with gene editing, transcriptional regulation, and enzyme modulation. Second, advances in the <i>B. subtilis</i> chassis of efficient biochemicals and proteins with CRISPR engineering are discussed. Finally, we conclude with perspectives on the challenges and opportunities of CRISPR-based biotechnology in <i>B. subtilis</i>, wishing that <i>B. subtilis</i> can be comparable to traditional industrial microorganisms such as <i>E. coli</i> and <i>S. cerevisiae</i> someday soon.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"677-688"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143555304","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":"Liposome-Encapsulated <i>Escherichia coli</i> Lysates to Reconstitute Intracellular Macromolecular Crowding Effects.","authors":"Milara S Kalacheva, Nuno R da Silva, Arnold J Boersma","doi":"10.1021/acssynbio.4c00824","DOIUrl":"10.1021/acssynbio.4c00824","url":null,"abstract":"<p><p>Intracellular macromolecular crowding impacts biomacromolecule behavior, including oligomerization, phase separation, and diffusion. However, understanding crowding effects in cells is challenging as cells respond and adapt to perturbations. Therefore, replicating in-cell crowding in liposomes would provide a good alternative to studying the consequences of macromolecular crowding. Here, we achieve physiological macromolecular crowding levels using <i>Escherichia coli</i> lysates in liposomes, as verified with a macromolecular crowding sensor. We shrink liposomes with a gradient-wise osmotic upshift to reach the high macromolecular crowding effects. We see that lysate induces higher macromolecular crowding than BSA at the same mg/mL, showing the need to use lysates to replicate in-cell behavior. We study the consequences of small cosolutes on macromolecular crowding and see that sugars and ATP modulate the lysate macromolecular crowding, implying they would also affect macromolecular crowding in cells. These artificial cells display the same crowding as <i>E. coli</i> at 220-300 mg/mL lysate and the same crowding as HEK293T at 50-100 mg/mL lysate. Hence, these artificial cells are a platform for obtaining information on physiologically relevant macromolecular crowding effects in a controlled environment.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"901-908"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934223/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143466556","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":"Enhanced Integration of Single-Cell Multi-Omics Data Using Graph Attention Networks.","authors":"Xingyu Liao, Yanyan Li, Shuangyi Li, Long Wen, Xingyi Li, Bin Yu","doi":"10.1021/acssynbio.4c00864","DOIUrl":"10.1021/acssynbio.4c00864","url":null,"abstract":"<p><p>The continuous advancement of single-cell multimodal omics (scMulti-omics) technologies offers unprecedented opportunities to measure various modalities, including RNA expression, protein abundance, gene perturbation, DNA methylation, and chromatin accessibility at single-cell resolution. These advances hold significant potential for breakthroughs by integrating diverse omics modalities. However, the data generated from different omics layers often face challenges due to high dimensionality, heterogeneity, and sparsity, which can adversely impact the accuracy and efficiency of data integration analyses. To address these challenges, we propose a high-precision analysis method called scMGAT (single-cell multiomics data analysis based on multihead graph attention networks). This method effectively coordinates reliable information across multiomics data sets using a multihead attention mechanism, allowing for better management of the heterogeneous characteristics inherent in scMulti-omics data. We evaluated scMGAT's performance on eight sets of real scMulti-omics data, including samples from both human and mouse. The experimental results demonstrate that scMGAT significantly enhances the quality of multiomics data and improves the accuracy of cell-type annotation compared to state-of-the-art methods. scMGAT is now freely accessible at https://github.com/Xingyu-Liao/scMGAT.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"931-942"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143070726","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":"Multi-Layer Autocatalytic Feedback Enables Integral Control Amidst Resource Competition and Across Scales","authors":"Armin M. Zand,&nbsp;Stanislav Anastassov,&nbsp;Timothy Frei and Mustafa Khammash*,&nbsp;","doi":"10.1021/acssynbio.4c0057510.1021/acssynbio.4c00575","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00575https://doi.org/10.1021/acssynbio.4c00575","url":null,"abstract":"<p >Integral feedback control strategies have proven effective in regulating protein expression in unpredictable cellular environments. These strategies, grounded in model-based designs and control theory, have advanced synthetic biology applications. Autocatalytic integral feedback controllers, utilizing positive autoregulation for integral action, are one class of simplest architectures to design integrators. This class of controllers offers unique features, such as robustness against dilution effects and cellular growth, as well as the potential for synthetic realizations across different biological scales, owing to their similarity to self-regenerative behaviors widely observed in nature. Despite this, their potential has not yet been fully exploited. One key reason, we discuss, is that their effectiveness is often hindered by resource competition and context-dependent couplings. This study addresses these challenges using a multilayer feedback strategy. Our designs enabled population-level integral feedback and multicellular integrators, where the control function emerges as a property of coordinated interactions distributed across different cell populations coexisting in a multicellular consortium. We provide a generalized mathematical framework for modeling resource competition in complex genetic networks, supporting the design of intracellular control circuits. The use of our proposed multilayer autocatalytic controllers is examined in two typical control tasks that pose significant relevance to synthetic biology applications: concentration regulation and ratiometric control. We define a ratiometric control task and solve it using a variant of our controller. The effectiveness of our controller motifs is demonstrated through a range of application examples, from precise regulation of gene expression and gene ratios in embedded designs to population growth and coculture composition control in multicellular designs within engineered microbial ecosystems. These findings offer a versatile approach to achieving robust adaptation and homeostasis from subcellular to multicellular scales.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 4","pages":"1041–1061 1041–1061"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00575","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842478","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":"Heterologous Expression of a Cryptic BGC from <i>Bilophila</i> sp. Provides Access to a Novel Family of Antibacterial Thiazoles.","authors":"Maximilian Hohmann, Denis Iliasov, Martin Larralde, Widya Johannes, Klaus-Peter Janßen, Georg Zeller, Thorsten Mascher, Tobias A M Gulder","doi":"10.1021/acssynbio.5c00042","DOIUrl":"10.1021/acssynbio.5c00042","url":null,"abstract":"<p><p>Human health is greatly influenced by the gut microbiota and microbiota imbalance can lead to the development of diseases. It is widely acknowledged that the interaction of bacteria within competitive ecosystems is influenced by their specialized metabolites, which act, e.g., as antibacterials or siderophores. However, our understanding of the occurrence and impact of such natural products in the human gut microbiome remains very limited. As arylthiazole siderophores are an emerging family of growth-promoting molecules in pathogenic bacteria, we analyzed a metagenomic data set from the human microbiome and thereby identified the <i>bil</i>-BGC, which originates from an uncultured <i>Bilophila</i> strain. Through gene synthesis and BGC assembly, heterologous expression and mutasynthetic experiments, we discovered the arylthiazole natural products bilothiazoles A-F. While established activities of related molecules indicate their involvement in metal-binding and -uptake, which could promote the growth of pathogenic strains, we also found antibiotic activity for some bilothiazoles. This is supported by biosensor-experiments, where bilothiazoles C and E show P_<i>recA</i>-suppressing activity, while bilothiazole F induces P_<i>blaZ</i>, a biosensor characteristic for β-lactam antibiotics. These findings serve as a starting point for investigating the role of bilothiazoles in the pathogenicity of <i>Bilophila</i> species in the gut.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"967-978"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934131/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143497451","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":"Synthetic Genetic Elements Enable Rapid Characterization of Inorganic Carbon Uptake Systems in <i>Cupriavidus necator</i> H16.","authors":"Akira K Nakamura, Emily M Fulk, Christopher W Johnson, Farren J Isaacs","doi":"10.1021/acssynbio.4c00869","DOIUrl":"10.1021/acssynbio.4c00869","url":null,"abstract":"<p><p><i>Cupriavidus necator</i> H16 is a facultative chemolithotroph capable of using CO₂ as a carbon source, making it a promising organism for carbon-negative biomanufacturing of petroleum-based product alternatives. In contrast to model microbes, genetic engineering technologies are limited in <i>C. necator</i>, constraining its utility in basic and applied research. Here, we developed a genome engineering technology to efficiently mobilize, integrate, and express synthetic genetic elements (SGEs) in <i>C. necator</i>. We tested the chromosomal expression of four inducible promoters to optimize an engineered genetic landing pad for tunable gene expression. To demonstrate utility, we employed the SGE system to design, mobilize, and express eight heterologous inorganic carbon uptake pathways in <i>C. necator</i>. We demonstrated all inorganic carbon uptake systems' upregulated intracellular bicarbonate concentrations under heterotrophic conditions. This work establishes the utility of the SGE strategy for expedited integration and tunable expression of heterologous pathways, and enhances intracellular bicarbonate concentrations in <i>C. necator</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"943-953"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934965/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565495","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":"Engineering a New Generation of Gene Editors: Integrating Synthetic Biology and AI Innovations.","authors":"Bing Shao Chia, Yu Fen Samantha Seah, Bolun Wang, Kimberle Shen, Diya Srivastava, Wei Leong Chew","doi":"10.1021/acssynbio.4c00686","DOIUrl":"10.1021/acssynbio.4c00686","url":null,"abstract":"<p><p>CRISPR-Cas technology has revolutionized biology by enabling precise DNA and RNA edits with ease. However, significant challenges remain for translating this technology into clinical applications. Traditional protein engineering methods, such as rational design, mutagenesis screens, and directed evolution, have been used to address issues like low efficacy, specificity, and high immunogenicity. These methods are labor-intensive, time-consuming, and resource-intensive and often require detailed structural knowledge. Recently, computational strategies have emerged as powerful solutions to these limitations. Using artificial intelligence (AI) and machine learning (ML), the discovery and design of novel gene-editing enzymes can be streamlined. AI/ML models predict activity, specificity, and immunogenicity while also enhancing mutagenesis screens and directed evolution. These approaches not only accelerate rational design but also create new opportunities for developing safer and more efficient genome-editing tools, which could eventually be translated into the clinic.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"636-647"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934138/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143497448","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":"An Arrayed CRISPR Screen Identifies Knockout Combinations Improving Antibody Productivity in HEK293 Cells.","authors":"Eric Edward Bryant, Danyang Gong, Cai Guo, Fernando Garces, René Hubert, Irwin Chen","doi":"10.1021/acssynbio.4c00772","DOIUrl":"10.1021/acssynbio.4c00772","url":null,"abstract":"<p><p>Mammalian cells are used to express complex biologics, such as multispecific antibodies. While multispecifics enable promising new strategies for treating human disease, their production at high expression titer and purity can be challenging. To understand how cells respond to antibody and multispecific expression, five molecules were selected for bulk RNA sequencing (RNA-seq) early after the transfection of a human embryonic kidney 293 (HEK293) host. All five molecules shared a differential expression signature of secretory and protein folding stresses, but this signature was stronger for molecules with low titer. We then designed an arrayed CRISPR knockout screen of 206 differentially expressed target genes and 223 literature-motivated targets to identify knockouts that affect antibody productivity. Eight novel knockout targets were identified that increased expression titers by 20-80%. Notably, seven of these top eight hits were from the differentially expressed set of candidate-gene knockouts. The top knockout target, HIST2H3C, showed evidence for additivity with five other hits, including a knockout combination that increased the titer of a difficult-to-express antibody by up to 100%. Findings for both HIST2H3C and INHBE knockout targets generalized to an alternate HEK293 host expressing an additional antibody and a multispecific host with no meaningful impact on product purity. Thus, we propose HIST2H3C and INHBE disruption as a promising and novel strategy for host-cell engineering to improve antibody and multispecific productivity.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"855-866"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143522193","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":"MultiCRISPR-EGA: Optimizing Guide RNA Array Design for Multiplexed CRISPR Using the Elitist Genetic Algorithm.","authors":"Yangyu Zhang, Guanlin Chen, Ce Liang, Bin Yang, Xin Lei, Tao Chen, Huaiguang Jiang, Wei Xiong","doi":"10.1021/acssynbio.4c00860","DOIUrl":"10.1021/acssynbio.4c00860","url":null,"abstract":"<p><p>Multiplexed CRISPR design, which allows for the concurrent and efficient editing of multiple genomic sites, is a powerful tool for complex genetic modifications. However, designing effective multiplexed guide RNA (gRNA) arrays remains challenging due to the exponential increase in potential gRNA array candidates and the significant impact of different target site selections on efficiency and specificity. Recognizing that more stable gRNAs, characterized by lower minimum free energy (MFE), have prolonged activity and thus higher efficacy, we developed MultiCRISPR-EGA, a graphical user interface (GUI)-based tool that employs the Elitist Genetic Algorithm (EGA) to design optimized single-promoter-driven multiplexed gRNA arrays. Computational experiments on <i>Escherichia coli</i> gene targets demonstrate that the EGA can rapidly optimize multiplexed gRNA arrays, outperforming other intelligent optimization algorithms in CRISPR interference (CRISPRi) applications, while the GUI provides real-time design progress control and compatibility with various CRISPR-Cas systems. This tool aims to advance the multiplexed gRNA array design process, enabling more efficient and cost-effective genome editing for synthetic biology.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"919-930"},"PeriodicalIF":3.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143456261","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}