ACS Synthetic Biology最新文献

{"title":"Construction of Chitinase Complexes Using Self-Assembly Systems for Efficient Hydrolysis of Chitin","authors":"Zhewei Shen,&nbsp;Yuchen Pan,&nbsp;Yuansheng Liu,&nbsp;Houhui Song* and Chenggang Xu*,&nbsp;","doi":"10.1021/acssynbio.4c0061310.1021/acssynbio.4c00613","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00613https://doi.org/10.1021/acssynbio.4c00613","url":null,"abstract":"<p >Chitin biomass is the second most abundant natural polysaccharide after cellulose on the earth, yet its recalcitrance to degrade and utilize severely limits its application. However, many microorganisms, such as <i>Serratia marcescen</i>, can secrete a range of free chitinases to degrade chitin, though their activity is typically insufficient to meet industrial demands. In this study, we employed self-assembly systems, named SpyTag/SpyCatcher and SnoopTag/SnoopCatcher, to modularize the molecular design of CHB, ChiB, ChiC, and CBP21 derived from <i>S. marcescens</i> ATCC14756, and we successfully constructed a variety of chitinase complexes. The assembled complexes showed higher chitinolytic activity and stability, compared to free chitinase mixture. Moreover, the distinct arrangements and combinations of chitinases within these complexes led to varied activities, suggesting that the spatial proximity and substrate channeling effects contribute to the synergy of chitinase complexes. The findings lay a solid technical foundation for the application of chitinosome in the industrial production of <i>N</i>-acetylglucosamine and chitooligosaccharides.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"13 12","pages":"4143–4153 4143–4153"},"PeriodicalIF":3.7,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142858807","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":"Red Light Responsive Cre Recombinase for Bacterial Optogenetics","authors":"Fereshteh Jafarbeglou,&nbsp; and ,&nbsp;Mary J. Dunlop*,&nbsp;","doi":"10.1021/acssynbio.4c0038810.1021/acssynbio.4c00388","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00388https://doi.org/10.1021/acssynbio.4c00388","url":null,"abstract":"<p >Optogenetic tools have been used in a wide range of microbial engineering applications that benefit from the tunable, spatiotemporal control that light affords. However, the majority of current optogenetic constructs for bacteria respond to blue light, limiting the potential for multichromatic control. In addition, other wavelengths offer potential benefits over blue light, including improved penetration of dense cultures and reduced potential for toxicity. In this study, we introduce OptoCre-REDMAP, a red light inducible Cre recombinase system in <i>Escherichia coli</i>. This system harnesses the plant photoreceptors PhyA and FHY1 and a split version of Cre recombinase to achieve precise control over gene expression and DNA excision. We optimized the design by modifying the start codon of Cre and characterized the impact of different levels of induction to find conditions that produced minimal basal expression in the dark and induced full activation within 4 h of red light exposure. We characterized the system’s sensitivity to ambient light, red light intensity, and exposure time, finding OptoCre-REDMAP to be reliable and flexible across a range of conditions. In coculture experiments with OptoCre-REDMAP and the blue light responsive OptoCre-VVD, we found that the systems responded orthogonally to red and blue light inputs. Direct comparisons between red and blue light induction with OptoCre-REDMAP and OptoCre-VVD demonstrated the superior penetration properties of red light. OptoCre-REDMAP’s robust and selective response to red light makes it suitable for advanced synthetic biology applications, particularly those requiring precise multichromatic control.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"13 12","pages":"3991–4001 3991–4001"},"PeriodicalIF":3.7,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142867819","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":"Engineering an αCD206-synNotch Receptor: Insights into the Development of Novel Synthetic Receptors","authors":"Sofija Semeniuk,&nbsp;Bin-Zhi Qian and Elise Cachat*,&nbsp;","doi":"10.1021/acssynbio.4c0014910.1021/acssynbio.4c00149","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00149https://doi.org/10.1021/acssynbio.4c00149","url":null,"abstract":"<p >Immune cells play a pivotal role in the establishment, growth, and progression of tumors at primary and metastatic sites. Macrophages, in particular, play a critical role in suppressing immune responses and promoting an anti-inflammatory environment through both direct and indirect cell–cell interactions. However, our understanding of the mechanisms underlying such interactions is limited due to a lack of reliable tools for studying transient interactions between cancer cells and macrophages within the tumor microenvironment. Recent advances in mammalian synthetic biology have introduced a wide range of synthetic receptors that have been used in diverse biosensing applications. One such synthetic receptor is the synNotch receptor, which can be tailored to sense specific ligands displayed on the surface of target cells. With this study, we aimed at developing a novel αCD206-synNotch receptor, targeting CD206⁺ macrophages, a population of macrophages that play a crucial role in promoting metastatic seeding and persistent growth. Engineered in cancer cells and used in mouse metastasis models, such a tool could help monitor─and provide an understanding of─the effects that cell–cell interactions between macrophages and cancer cells have on metastasis establishment. Here, we report the development of cancer landing-pad cells for versatile applications and the engineering of αCD206-synNotch cancer cells in particular. We report the measurement of their activity and specificity, and discuss unexpected caveats regarding their <i>in vivo</i> applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"13 12","pages":"3876–3884 3876–3884"},"PeriodicalIF":3.7,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00149","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142858801","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 Exopolysaccharide Biosynthesis of <i>Shewanella oneidensis</i> to Promote Electroactive Biofilm Formation for Liquor Wastewater Treatment.","authors":"Zixuan You, Huan Yu, Baocai Zhang, Qijing Liu, Bo Xiong, Chao Li, Chunxiao Qiao, Longhai Dai, Jianxun Li, Wenwei Li, Guosheng Xin, Zhanying Liu, Feng Li, Hao Song","doi":"10.1021/acssynbio.4c00417","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00417","url":null,"abstract":"<p><p>Microbial electrochemical systems (MESs), as a green and sustainable technology, can decompose organics in wastewater to recover bioelectricity. Electroactive biofilms, a microbial community structure encased in a self-produced matrix, play a decisive role in determining the efficiency of MESs. However, as an essential component of the biofilm matrix, the role of exopolysaccharides in electroactive biofilm formation and their influence on extracellular electron transfer (EET) have been rarely studied. Herein, to explore the effects of exopolysaccharides on biofilm formation and EET rate, we first inhibited the key genes responsible for exopolysaccharide biosynthesis (namely, <i>so_3171</i>, <i>so_3172</i>, <i>so_3177</i>, and <i>so_3178</i>) by using antisense RNA in <i>Shewanella oneidensis</i> MR-1. Then, to explore the underlying mechanisms why inhibition of exopolysaccharide synthesis could enhance biofilm formation and promote the EET rate, we characterized cell physiology and electrophysiology. The results showed inhibition of exopolysaccharide biosynthesis not only altered cell surface hydrophobicity and promoted intercellular adhesion and aggregation, but also increased biosynthesis of <i>c</i>-type cytochromes and decreased interfacial resistance, thus promoting electroactive biofilm formation and improving the EET rate of <i>S. oneidensis</i>. Lastly, to evaluate and intensify the capability of exopolysaccharide-reduced strains in harvesting electrical energy from actual liquor wastewater, engineered strain Δ3171-as3177 was further constructed to treat an actual thin stillage. The results showed that the output power density reached 380.98 mW m^-2, 11.1-fold higher than that of WT strain, which exhibited excellent capability of harvesting electricity from actual liquor wastewater. This study sheds light on the underlying mechanism of how inhibition of exopolysaccharides impacts electroactive biofilm formation and EET rate, which suggested that regulating exopolysaccharide biosynthesis is a promising avenue for increasing the EET rate.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142646143","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 Plug-and-Play T7 Expression System for Heterologous Production of Lanthipeptides in <i>Bacillus subtilis</i>.","authors":"Chengyou Shi, Huimin Zhao","doi":"10.1021/acssynbio.4c00594","DOIUrl":"10.1021/acssynbio.4c00594","url":null,"abstract":"<p><p>Ribosomally synthesized lanthionine-containing peptides (lanthipeptides) have emerged as a promising source of antimicrobials against multidrug resistance pathogens. An effective way to discover and engineer lanthipeptides is through heterologous expression of their biosynthetic gene clusters (BGCs) in a host of choice. Here we report a plug-and-play pathway refactoring strategy for rapid evaluation of lanthipeptide BGCs in <i>Bacillus subtilis</i> based on the T7 expression system. As a proof of concept, we used this strategy to not only observe the successful production of a known lanthipeptide haloduracin β but also discover two new human-microbiota-derived lanthipeptides that previously failed to be produced in <i>Escherichia coli</i>. The resulting <i>B. subtilis</i> plug-and-play T7 expression system should enable the genome mining of new lanthipeptides in a high-throughput manner.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"3746-3753"},"PeriodicalIF":3.7,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142542838","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":"Engineering <i>Yarrowia lipolytica</i> to Produce l-Malic Acid from Glycerol.","authors":"Yaping Wang, Yuqing Han, Chang Liu, Liyan Cao, Qingqing Ye, Chen Ding, Yuyang Wang, Qingeng Huang, Jiwei Mao, Cui-Ying Zhang, Aiqun Yu","doi":"10.1021/acssynbio.4c00445","DOIUrl":"10.1021/acssynbio.4c00445","url":null,"abstract":"<p><p>The declining availability of cheap fossil-based resources has sparked growing interest in the sustainable biosynthesis of organic acids. l-Malic acid, a crucial four-carbon dicarboxylic acid, finds extensive applications in the food, chemical, and pharmaceutical industries. Synthetic biology and metabolic engineering have enabled the efficient microbial production of l-malic acid, albeit not in <i>Yarrowia lipolytica</i>, an important industrial microorganism. The present study aimed to explore the potential of this fungal species for the production of l-malic acid. First, endogenous biosynthetic genes and heterologous transporter genes were overexpressed in <i>Y. lipolytica</i> to identify bottlenecks in the l-malic acid biosynthesis pathway grown on glycerol. Second, overexpression of isocitrate lyase, malate synthase, and malate dehydrogenase in the glyoxylate cycle pathway and introduction of a malate transporter from <i>Schizosaccharomyces pombe</i> significantly boosted l-malic acid production, which reached 27.0 g/L. A subsequent increase to 37.0 g/L was attained through shake flask medium optimization. Third, adaptive laboratory evolution allowed the engineered strain <i>Po1g-CEE2+Sp</i> to tolerate a lower pH and to accumulate a higher amount of l-malic acid (56.0 g/L). Finally, when scaling up to a 5 L bioreactor, a titer of 112.5 g/L was attained. In conclusion, this study demonstrates for the first time the successful production of l-malic acid in <i>Y. lipolytica</i> by combining metabolic engineering and laboratory evolution, paving the way for large-scale sustainable biosynthesis of this and other organic acids.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"3635-3645"},"PeriodicalIF":3.7,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142491076","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":"Recent Advances in Genetic Engineering Strategies of <i>Sinorhizobium meliloti</i>.","authors":"Xuan Liu, Huina Dong, Huiying Wang, Xinyi Ren, Xia Yang, Tingting Li, Gang Fu, Miaomiao Xia, Huan Fang, Guangqing Du, Zhaoxia Jin, Dawei Zhang","doi":"10.1021/acssynbio.4c00348","DOIUrl":"10.1021/acssynbio.4c00348","url":null,"abstract":"<p><p><i>Sinorhizobium meliloti</i> is a free-living soil Gram-negative bacterium that participates in nitrogen-fixation symbiosis with several legumes. <i>S. meliloti</i> has the potential to be utilized for the production of high-value nutritional compounds, such as vitamin B₁₂. Advances in gene editing tools play a vital role in the development of <i>S. meliloti</i> strains with enhanced characteristics for biotechnological applications. Several novel genetic engineering strategies have emerged in recent years to investigate genetic modifications in <i>S. meliloti</i>. This review provides a comprehensive overview of the mechanism and application of the extensively used Tn5-mediated genetic engineering strategies. Strategies based on homologous recombination and site-specific recombination were also discussed. Subsequently, the development and application of the genetic engineering strategies utilizing various CRISPR/Cas systems in <i>S. meliloti</i> are summarized. This review may stimulate research interest among scientists, foster studies in the application areas of <i>S. meliloti</i>, and serve as a reference for the utilization of genome editing tools for other Rhizobium species.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"3497-3506"},"PeriodicalIF":3.7,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11574922/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142556578","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":"Cohesive Living Bacterial Films with Tunable Mechanical Properties from Cell Surface Protein Display.","authors":"Hanwei Liu, Priya K Chittur, Julia A Kornfield, David A Tirrell","doi":"10.1021/acssynbio.4c00528","DOIUrl":"10.1021/acssynbio.4c00528","url":null,"abstract":"<p><p>Engineered living materials (ELMs) constitute a novel class of functional materials that contain living organisms. The mechanical properties of many such systems are dominated by the polymeric matrices used to encapsulate the cellular components of the material, making it hard to tune the mechanical behavior through genetic manipulation. To address this issue, we have developed living materials in which mechanical properties are controlled by the cell-surface display of engineered proteins. Here, we show that engineered <i>Esherichia coli</i> cells outfitted with surface-displayed elastin-like proteins (ELPs, designated E6) grow into soft, cohesive bacterial films with biaxial moduli around 14 kPa. When subjected to bulge-testing, such films yielded at strains of approximately 10%. Introduction of a single cysteine residue near the exposed N-terminus of the ELP (to afford a protein designated CE6) increases the film modulus 3-fold to 44 kPa and eliminates the yielding behavior. When subjected to oscillatory stress, films prepared from <i>E. coli</i> strains bearing CE6 exhibit modest hysteresis and full strain recovery; in E6 films much more significant hysteresis and substantial plastic deformation are observed. CE6 films heal autonomously after damage, with the biaxial modulus fully restored after a few hours. This work establishes an approach to living materials with genetically programmable mechanical properties and a capacity for self-healing. Such materials may find application in biomanufacturing, biosensing, and bioremediation.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"3686-3697"},"PeriodicalIF":3.7,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11574920/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142562380","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 Cyborg Pathogens through Intracellular Hydrogelation.","authors":"Shahid Khan, Pin-Ru Lin, Cheemeng Tan","doi":"10.1021/acssynbio.4c00420","DOIUrl":"10.1021/acssynbio.4c00420","url":null,"abstract":"<p><p>Synthetic biology primarily focuses on two kinds of cell chassis: living cells and nonliving systems. Living cells are autoreplicating systems that have active metabolism. Nonliving systems, including artificial cells and nanoparticles, are nonreplicating systems typically lacking active metabolism. In recent work, Cyborg bacteria that are nonreplicating-but-metabolically active have been engineered through intracellular hydrogelation. Intracellular hydrogelation is conducted by infusing gel monomers and photoactivators into cells, followed by the activation of polymerization of the gel monomers inside the cells. However, the previous work investigated only <i>Escherichia coli</i> cells. Extending the Cyborg-Cell method to pathogenic bacteria could enable the exploitation of their pathogenic properties in biomedical applications. Here, we focus on different strains of <i>Pseudomonas aeruginosa</i>, <i>Staphylococcus aureus</i>, and <i>Klebsiella pneumoniae</i>. To synthesize the Cyborg pathogens, we first reveal the impact of different hydrogel concentrations on the metabolism, replication, and intracellular gelation of Cyborg pathogens. Next, we demonstrate that the Cyborg pathogens are taken up by macrophages in a similar magnitude as wild-type pathogens through confocal microscopy and real-time PCR. Finally, we show that the macrophage that takes up the Cyborg pathogen exhibits a similar phenotypic response to the wild-type pathogen. Our work generalizes the intracellular hydrogelation approach from lab strains of <i>E. coli</i> to bacterial pathogens. The new Cyborg pathogens could be applied in biomedical applications ranging from drug delivery to immunotherapy.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"3609-3620"},"PeriodicalIF":3.7,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453377","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":"Application of a Cell-Free Synthetic Biology Platform for the Reconstitution of Teleocidin B and UK-2A Precursor Biosynthetic Pathways.","authors":"Krishna Madduri, Deepa Acharya, Adam Lescallette, Jeremy McFadden, Paul Ketterer, Jade Bing, Babu Raman","doi":"10.1021/acssynbio.4c00560","DOIUrl":"10.1021/acssynbio.4c00560","url":null,"abstract":"<p><p>We report the successful cell-free reconstitution of two natural product biosynthetic pathways of divergent complexity and structural classes. We first constructed the teleocidin biosynthetic pathway using our BY-2 (tobacco) cell-free protein synthesis (CFPS) system. We discovered a direct interaction between TleA and MbtH, and showed that the BY-2 system is capable of producing more than 80 mg/L teleocidin B-3 with cofactor supplementation and ∼20 mg/L with no cofactors supplemented, demonstrating the high metabolic activity of the system. We then extended our methodology and report the first successful cell-free biosynthesis of UK-2 diol (precursor to the commercially valuable secondary metabolite UK-2A) from simple building blocks by refactoring a complex pathway of 10 proteins in the wheat germ CFPS system. We show that plant CFPS systems are suitable for reconstructing pathways and identifying the functions of uncharacterized genes linked to biosynthetic gene clusters and rate-limiting biosynthetic steps.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"3711-3723"},"PeriodicalIF":3.7,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142520316","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}