ACS Synthetic Biology最新文献

{"title":"High Ectoine Production from Lignocellulosic Hydrolysate by <i>Escherichia coli</i> through Metabolic and Fermentation Engineering.","authors":"Yifan Feng, Wenlong Xiao, Xinyi Li, Weiyu Cao, Yujia Jiang, Wenming Zhang, Wankui Jiang, Fengxue Xin, Min Jiang","doi":"10.1021/acssynbio.4c00899","DOIUrl":"10.1021/acssynbio.4c00899","url":null,"abstract":"<p><p>Ectoine, a major compatible solute in halophilic micro-organisms, shows great potential in cosmetics and pharmaceuticals areas owing to its water-binding properties and capability to prevent oxidative damage. In this study, the <i>ectABC</i> gene cluster responsible for the ectoine synthesis originated from halophilic bacterium <i>Halomonas venusta</i> was first assembled into <i>Escherichia coli</i>. Subsequently, the <i>crr</i> gene in PTS was knocked out to further drive the metabolic flux from phosphoenolpyruvate to oxaloacetate, resulting in 1.27 g/L of ectoine. Then, the rate-limiting enzyme LysC in the ectoine synthesis pathway was identified and modified. The recombinant <i>E. coli</i> with the further overexpression of feedback-insensitive mutant <i>EclysC</i>* increased the ectoine titer to 2.51 g/L with a yield of 0.37 g/g in shake flasks. After the medium optimization including the carbon and nitrogen source, sodium chloride, and magnesium sulfate concentration, the ectoine titer was improved to 4.55 g/L. 115.15 g/L of ectoine with a yield of 0.23 g/g was obtained in the 5.0 L bioreactor through the optimization of substrate feeding and IPTG supplementation in the fed-batch fermentation. To achieve the cost-effective production of ectoine, lignocellulosic hydrolysate from wheat straw was adopted. 134.08 g/L of ectoine with a yield of 0.33 g/g sugar and a productivity of 3.7 g/L/h was finally produced, representing a relatively high level of ectoine production from renewable resources compared to other studies. This study provides valuable insights into a cost-effective and efficient method for industrial-scale ectoine production.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"609-620"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143397494","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":"Metabolic Engineering of <i>Escherichia coli</i> BL21(DE3) for 2'-Fucosyllactose Synthesis in a Higher Productivity.","authors":"Na Li, Saifeng Yan, Hongzhi Xia, Yin Fang, Kun Niu, Guyue Li, Zheng Xu, Yang Sun, Hong Xu, Xiaoqi Xu","doi":"10.1021/acssynbio.4c00598","DOIUrl":"10.1021/acssynbio.4c00598","url":null,"abstract":"<p><p>2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharides (HMOs). 2'-FL exhibits great benefits for infant health, such as preventing infantile diarrhea and promoting the growth of intestinal probiotics. The microbial cell factory technique has shown promise for the massive production of 2'-FL. Here, we aimed to construct a recombinant <i>E. coli</i> BL21(DE3) strain for the hyperproduction of 2'-FL. Initially, multicopy genomic integration and expression of the lactose permease gene <i>lacY</i> reduced the formation of byproducts. Furthermore, a more efficient Shine-Dalgarno sequence was used to replace the wild-type sequence in the <i>manC</i>-<i>manB</i> and <i>gmd</i>-<i>wcaG</i> gene clusters, which significantly increased the 2'-FL titer. Based on these results, we overexpressed the sugar efflux transporter SetA and knocked out the <i>pgi</i> gene. This further improved 2'-FL synthesis when glycerol was used as the sole carbon source. Finally, a new α-1,2-fucosyltransferase was identified in <i>Neisseria</i> sp., which exhibited a higher capacity for 2'-FL production. Fed-batch fermentation produced 141.27 g/L 2'-FL in 45 h with a productivity of 3.14 g/L × h. This productivity rate achieved the highest recorded 2'-FL levels, indicating the potential of engineered <i>E. coli</i> BL21 (DE3) strains for use in the industrial production of 2'-FL.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"441-452"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142996173","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":"CRISPR-Cas9 Cytidine-Base-Editor Mediated Continuous <i>In Vivo</i> Evolution in <i>Aspergillus nidulans</i>.","authors":"Yuan Tian, Qing Xu, Meng Pang, Youchu Ma, Zhiruo Zhang, Dongfang Zhang, Donghui Guo, Lupeng Wang, Qingbin Li, Yanling Li, Fanglong Zhao","doi":"10.1021/acssynbio.4c00716","DOIUrl":"10.1021/acssynbio.4c00716","url":null,"abstract":"<p><p>Filamentous fungi are important cell factories for producing chemicals, organic acids, and enzymes. Although several genome editing tools are available for filamentous fungi, few effectively enable continuous evolution for rational engineering of complex phenotype. Here, we present CRISPR-Cas9 cytidine-base-editor (CBE) assisted <i>in vivo</i> evolution by continuously delivering a combinatorial sgRNA library to filamentous fungi. The method was validated by targeting core genes of 46 natural product biosynthetic gene clusters in <i>Aspergillus nidulans</i> NRRL 8112 to eliminate fungal toxins via six rounds of evolution. NGS analysis revealed the average C-to-T conversion rates in the first, third, and sixth rounds were 2.02%, 5.25%, and 9.34%, respectively. Metabolic profiles of the evolved mutants exhibited significant changes, allowing for the isolation of clean-background strains with enhanced production of an antifungal compound Echinocandin B. This study demonstrates that CBE-mediated <i>in vivo</i> evolution greatly facilitates the iterative refinement of complex morphogenetic traits in filamentous fungi.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"621-628"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143044907","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":"Cell-Free Systems to Mimic and Expand Metabolism.","authors":"Blake J Rasor, Tobias J Erb","doi":"10.1021/acssynbio.4c00729","DOIUrl":"10.1021/acssynbio.4c00729","url":null,"abstract":"<p><p>Cell-free synthetic biology incorporates purified components and/or crude cell extracts to carry out metabolic and genetic programs. While protein synthesis has historically been the primary focus, more metabolism researchers are now turning toward cell-free systems either to prototype pathways for cellular implementation or to design new-to-nature reaction networks that incorporate environmentally relevant substrates or new energy sources. The ability to design, build, and test enzyme combinations <i>in vitro</i> has accelerated efforts to understand metabolic bottlenecks and engineer high-yielding pathways. However, only a small fraction of metabolic possibilities has been explored in cell-free systems, and extracts from model organisms remain the most common starting points. Expanding the scope of cell-free metabolism to include extracts from new organisms, alternative metabolic pathways, and non-natural chemistries will enhance our ability to understand and engineer bio-based chemical conversions.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"316-322"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11852204/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057511","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":"Rapid Enzymatic Assay for Antiretroviral Drug Monitoring Using CRISPR-Cas12a-Enabled Readout.","authors":"Maya A Singh, Megan M Chang, Qin Wang, Catherine Rodgers, Barry R Lutz, Ayokunle O Olanrewaju","doi":"10.1021/acssynbio.4c00674","DOIUrl":"10.1021/acssynbio.4c00674","url":null,"abstract":"<p><p>Maintaining the efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse transcriptase ACTivity crispR (REACTR) for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to the synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR's sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"510-519"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11852202/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143397496","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":"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":"https://doi.org/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":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","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":"https://doi.org/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":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-20","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}

{"title":"Metabolic Engineering of <i>Corynebacterium glutamicum</i> for Producing Different Types of Triterpenoids.","authors":"Jingzhi Li, Xinxin Wang, Xahnaz Xokat, Ya Wan, Xiaopeng Gao, Ying Wang, Chun Li","doi":"10.1021/acssynbio.4c00737","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00737","url":null,"abstract":"<p><p>Triterpenoids widely exist in nature with diverse structures and possess various functional properties and biological effects. However, research on triterpenoids biosynthesis in <i>Corynebacterium glutamicum</i> is still limited to squalene, which restricts the development of <i>C. glutamicum</i> to produce high-value triterpenoids. In this study, <i>C. glutamicum</i> was developed as an efficient and flexible platform for the biosynthesis of different types of triterpenoids. Squalene was synthesized and the titer was improved to 400.1 mg/L in flask combining strategies of metabolic engineering and fermentation optimization. Particularly, intracellular squalene accounted for more than 97%, addressing the problem of leaking squalene in <i>C. glutamicum</i>, which may restrict the subsequent synthesis of other triterpenoids derived from squalene. Furthermore, 201.9 mg/L (3S)-2,3-oxidosqualene (SQO) and 264.9 mg/L (3S,22S)-2,3,22,23-dioxidosqualene (SDO) were successfully synthesized in strains harboring heterogeneous squalene epoxidase from <i>Arabidopsis thaliana</i> with different expression strengths. Therefore, a platform for de novo triterpenoids synthesis based on SQO or SDO was constructed in <i>C. glutamicum</i>. For instance, biosynthesis of α-amyrin and α-onocerin was achieved for the first time by introducing oxidosqualene cyclases in SQO- and SDO-producing <i>C. glutamicum</i> strains, respectively. After optimization, the titer of α-amyrin and α-onocerin was improved to 65.3 and 136.85 mg/L, respectively. Furthermore, ursolic acid, derived from α-amyrin, was synthesized after expressing cytochrome P450 enzyme and its compatible cytochrome P450 reductases with a titer of 486 μg/L. For the first time, reactions of epoxidation, cyclization, and oxidation from squalene were achieved in <i>C. glutamicum</i>, leading to the production of different types of triterpenoids. Our study provides a new platform for the production of triterpenoids, which will be helpful for the large-scale production of triterpenoids employing <i>C. glutamicum</i> as a chassis strain.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143447433","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":"LAMP Assay Coupled with a <i>Pyrococcus furiosus</i> Argonaute System for the Rapid Detection of Porcine Epidemic Diarrhea Virus.","authors":"Zhaorong Yu, Ying Shao, Yu Zhang, Fanyu Cheng, Peng Fang, Jian Tu, Xiangjun Song, Kezong Qi, Zhenyu Wang","doi":"10.1021/acssynbio.4c00446","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00446","url":null,"abstract":"<p><p>Porcine epidemic diarrhea virus (PEDV) infection can lead to serious acute intestinal infectious disease, bringing huge economic losses to the pig industry. In addition to triggering an extremely high mortality rate for lactating piglets, there is currently a lack of effective treatments and vaccines. Therefore, rapid, accurate, sensitive, and specific detection of PEDV is critical for timely control. In this study, a nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification (RT-LAMP) and <i>Pyrococcus furiosus</i> Argonaute (<i>Pf</i>Ago) was established for the detection of PEDV and performed after optimizing the system (mainly for the design and screening of the LAMP primers and <i>Pf</i>Ago gDNA). The optimized system had a detection limit as low as 2.4 copies/μL. To reach more timely on-site detection of PEDV and overcome the reliance on bulky and complex equipment, a lateral flow strip was introduced into the system, which could detect the target as low as 24 copies/μL. This RT-LAMP-<i>Pf</i>Ago system took about 35 min to react, and the results could be observed and clarified with the naked eyes. Moreover, the method was highly specific and had no cross-reactivity with other swine pathogens. The detection results for the clinical samples were consistent with those obtained by the gold standard method, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), proving its applicability. In conclusion, the established RT-LAMP-<i>Pf</i>Ago system can provide a new solution for the development of a portable, visual PEDV testing platform.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439317","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":"Metabolite-Responsive Control of Transcription by Phase Separation-Based Synthetic Organelles.","authors":"Carolina Jerez-Longres, Wilfried Weber","doi":"10.1021/acssynbio.4c00633","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00633","url":null,"abstract":"<p><p>Living natural materials have remarkable sensing abilities that translate external cues into functional changes of the material. The reconstruction of such sensing materials in bottom-up synthetic biology provides the opportunity to develop synthetic materials with life-like sensing and adaptation ability. Key to such functions are material modules that translate specific input signals into a biomolecular response. Here, we engineer a synthetic organelle based on liquid-liquid phase separation that translates a metabolic signal into the regulation of gene transcription. To this aim, we engineer the pyruvate-dependent repressor PdhR to undergo liquid-liquid phase separation <i>in vitro</i> by fusion to intrinsically disordered regions. We demonstrate that the resulting coacervates bind DNA harboring PdhR-responsive operator sites in a pyruvate dose-dependent and reversible manner. We observed that the activity of transcription units on the DNA was strongly attenuated following recruitment to the coacervates. However, the addition of pyruvate resulted in a reversible and dose-dependent reconstitution of transcriptional activity. The coacervate-based synthetic organelles linking metabolic cues to transcriptional signals represent a materials approach to confer stimulus responsiveness to minimal bottom-up synthetic biological systems and open opportunities in materials for sensor applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143424618","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}