ACS Synthetic Biology最新文献

{"title":"Developing Biosensors for Specific Assessment of <i>Trans</i>-Translation in <i>Pseudomonas aeruginosa</i>.","authors":"Bastien L'Hermitte, Thomas Chauvet, Sylvie Georgeault-Daguenet, Nicolas Le Yondre, Philippe Jehan, Reynald Gillet, Christine Baysse","doi":"10.1021/acssynbio.4c00801","DOIUrl":"10.1021/acssynbio.4c00801","url":null,"abstract":"<p><p>We developed two <i>Pseudomonas aeruginosa</i> biosensors to detect <i>trans</i>-translation inhibitors in this medically relevant pathogen. These biosensors leverage the red fluorescence produced by the accumulation of protoporphyrin IX, the substrate of ferrochelatase. The first biosensor monitors tmRNA-SmpB-mediated tagging and degradation of ferrochelatase, while the second serves as a control by tracking ClpP1-mediated proteolysis and porphyrin biosynthesis. Both biosensors were tested in wild-type and mutant backgrounds, and red fluorescence was measured relative to absorbance at 600 nm in microtiter plates. The results confirmed a link between fluorescence and <i>trans</i>-translation or proteolysis activity. These biosensors offer a promising tool for high-throughput screening of <i>trans</i>-translation inhibitors in <i>P. aeruginosa</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565594","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":"https://doi.org/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":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-04","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":"Programming Nutrient Detection with Modular Regulators for Dynamic Control of Microbial Biosynthesis.","authors":"Nhu Nguyen, Vincenzo Kennedy, Jung Yeon Lee, Noel Y Chan, Clement T Y Chan","doi":"10.1021/acssynbio.4c00720","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00720","url":null,"abstract":"<p><p>Dynamic control of biosynthetic pathways improves the bioproduction efficiency. One common approach is to use genetic sensors that control pathway expression in response to a nutrient molecule in the target feedstock. However, programming the cellular response requires the engineering of numerous genetic parts, which poses a significant barrier to explore the use of different nutrients as cellular signals. Here we created a dynamic control platform based on a set of modular transcriptional regulators; these regulators control the same promoter for driving gene expression, but each of them responds to a unique signal. We demonstrated that by replacing only the regulator, a different nutrient molecule can then be used for induction of the same genetic circuit. To show host versatility, we implemented this platform in both <i>Escherichia coli</i> and <i>Pseudomonas putida</i>. This platform was then used to program the induction of ethanol production by three nutrients, fructose, cellobiose, and galactose, of which each molecule can be present in a different set of crops. These results suggest that our platform facilitates the use of different agricultural products for the dynamic control of biosynthesis.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539427","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":"Developing Thermosensitive Metabolic Regulation Strategies in the Fermentation Process of <i>Saccharomyces cerevisiae</i> to Enhance α-Bisabolene Production.","authors":"Pan Feng, Bowen Sun, Haoran Bi, Yufei Bao, Meng Wang, Huili Zhang, Yunming Fang","doi":"10.1021/acssynbio.4c00728","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00728","url":null,"abstract":"<p><p>α-Bisabolene's distinctive aroma is highly prized in fragrances and cosmetics, while its antioxidant properties hold significant pharmaceutical potential. However, the production of α-bisabolene in <i>Saccharomyces cerevisiae</i> remains an outstanding challenge due to cell growth limitations and insufficient supply of the α-bisabolene precursor farnesyl pyrophosphate. In this work, a new <i>S. cerevisiae</i> platform strain capable of producing high levels of α-bisabolene was presented. Carbon flux in the α-bisabolene synthesis pathway was maximized by iterative enhancement of the mevalonate metabolic pathway. The effects of MVA pathway intermediates on cell growth were addressed through a two-stage fermentation controlled based on a temperature-sensitive regulation strategy. The fermentation medium was optimized based on metabolomics and response surface model analysis. Under the optimal fermentation process, the titer of α-bisabolene reached 18.6 g/L during fed-batch fermentation, representing the highest titer reported to date. These strategies open up new avenues for industrial-scale terpene biosynthesis.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539424","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 iGEM Grand Jamboree and the Serious Fun of Engineering Biology.","authors":"Edwin H Wintermute","doi":"10.1021/acssynbio.5c00083","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00083","url":null,"abstract":"<p><p>The iGEM Grand Jamboree, hosted last year in Paris, France, represents a new way of sharing and communicating science. The global event for young synthetic biologists blends the serious biotech of a traditional conference with the spontaneous energy of a music festival, fostering a kind of World's Fair for the built-with-biology future. The broader biotech community should take inspiration from iGEM and seek to enrich more of our public communications and professional events with iGEM-like humanity, authenticity, and fun.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143522196","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":"Toward Antibody Production in Genome-Minimized <i>Bacillus subtilis</i> Strains.","authors":"Tobias Schilling, Rebekka Biedendieck, Rafael Moran-Torres, Mirva J Saaranen, Lloyd W Ruddock, Rolf Daniel, Jan Maarten van Dijl","doi":"10.1021/acssynbio.4c00688","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00688","url":null,"abstract":"<p><p><i>Bacillus subtilis</i> is a bacterial cell factory with outstanding protein secretion capabilities that has been deployed as a workhorse for the production of industrial enzymes for more than a century. Nevertheless, the production of other proteins with <i>B. subtilis</i>, such as antibody formats, has thus far been challenging due to specific requirements that relate to correct protein folding and disulfide bond formation upon export from the cytoplasm. In the present study, we explored the possibility of producing functional antibody formats, such as scFvs and scFabs, using the genome-reduced <i>Midi</i>- and <i>MiniBacillus</i> strain lineage. The applied workflow included selection of optimal chassis strains, appropriate expression vectors, signal peptides, growth media, and analytical methods to verify the functionality of the secreted antibody fragments. The production of scFv fragments was upscaled to the 1 L bioreactor level. As demonstrated for a human C-reactive protein-binding scFv antibody by mass spectrometry, biolayer interferometry, circular dichroism, free thiol cross-linking with <i>N</i>-ethylmaleimide, and nano-differential scanning fluorimetry, <i>MidiBacillus</i> strains can secrete fully functional, natively folded, disulfide-bonded, and thermostable antibody fragments. We therefore conclude that genome-reduced <i>B. subtilis</i> chassis strains are capable of secreting high-quality antibody fragments.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143513998","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":"Mechanistic Modeling of In Vivo Translation in <i>Escherichia coli</i> Reliably Identifies Well-Adapted and Optimized RNA Sequences.","authors":"Jan Spindler, Christina Giakissiklis, Catharina Stierle, Marc Buschlüter, Klaus Liebeton, Martin Siemann-Herzberg, Ralf Takors","doi":"10.1021/acssynbio.4c00578","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00578","url":null,"abstract":"<p><p>Translation elongation is a multifaceted process that intricately links translational resource availability to the biophysical effects arising from the interaction of mRNA sequences, ribosomes, and nascent polypeptide chains. Optimizing (heterologous) gene expression via codon usage or tRNA preference alone may yield suboptimal outcomes. In this study, we present a comprehensive mechanistic model that accounts for the competition of tRNAs at the ribosomal A-site, internal Shine-Dalgarno sequence interactions, and the decelerating effects of positively charged peptide patches. Our model offers a holistic perspective on the effects of translational elongation, including growth rate-dependent variation in translational rates by 22 to 25% between slow- and fast-growing <i>Escherichia coli</i> cells. We emphasize that endogenous <i>E. coli</i> sequences typically adapt to these effects, particularly in highly expressed genes, where adaptation ensures efficient translation. Conversely, heterologous gene sequences from <i>Saccharomyces cerevisiae</i> are predicted to exhibit lower translational elongation rates by 14 to 70% compared to the homologous isoform. Simulated elongation profiles not only underscore potential sites for translation engineering but also suggest feasible synonymous codon exchanges. The implications of our model extend beyond mere codon usage adaptation and shed light on the key factors influencing translation efficiency (e.g., codons for positively charged amino acids reduced elongation rates by ∼6%). This study provides a nuanced understanding of the intricate dynamics governing translation in <i>E. coli</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143522195","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":"Efficient Biosynthesis of Furanocoumarin Intermediate Marmesin by Engineered <i>Escherichia coli</i>.","authors":"Baodong Hu, Jingwen Zhou, Jianghua Li, Jian Chen, Guocheng Du, Fang Zhong, Yucheng Zhao, Xinrui Zhao","doi":"10.1021/acssynbio.4c00892","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00892","url":null,"abstract":"<p><p>Marmesin, a plant dihydrofuranocoumarin, is an important intermediate in the synthesis of linear furanocoumarins and exhibits a variety of pharmacological activities. However, due to the lack of efficient prenyltransferases, the incompatibility of redox partners for P450 enzymes, and the insufficient supply of precursor (DMAPP), the microbial synthesis of marmesin remained at an extremely low level. Here, we report the efficient biosynthesis of marmesin in <i>Escherichia coli</i> by screening the robust 6-prenyltransferase PpPT1 and marmesin synthase PpDCΔ_2-29 from <i>Peucedanum praeruptorum</i>. Next, the activities of PpPT1 and PpDCΔ_2-29 were enhanced using fusion protein tags and redox partner engineering, respectively. In addition, the synthesis of marmesin was further improved by strengthening the methylerythritol phosphate (MEP) pathway to increase the availability of DMAPP and by optimizing the modular pathway in the engineered strain. Finally, the titer of marmesin reached 203.69 mg L^-1 in the fed-batch fermentation with a molar conversion rate of umbelliferone of 81.4%, which is the highest titer for marmesin production using engineered microorganisms. The applied strategy and marmesin-producing strain constructed in this study lay the foundation for the green production of valuable complex furanocoumarins.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143522194","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 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":"https://doi.org/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":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-27","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":"Potential and Optimization of Mammalian Splice Riboswitches for the Regulation of Exon Skipping-Dependent Gene Expression and Isoform Switching within the <i>ALOX5</i> Gene.","authors":"Robin W Bruckhoff, Olga Becker, Dieter Steinhilber, Beatrix Suess","doi":"10.1021/acssynbio.4c00731","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00731","url":null,"abstract":"<p><p>Synthetic riboswitches are attracting increasing interest for a diverse range of applications, including synthetic biology, functional genomics, and prospective therapeutic strategies. This study demonstrates that controlling alternative splicing with synthetic riboswitches represents a promising approach to effectively regulating transgene expression in mammalian cells. However, the function of synthetic riboswitches in the eukaryotic system in controlling gene expression is often limited to certain genes or cell types. So far, strategies to increase the dynamic range of regulation have been focused on adapting and modifying the riboswitch sequence itself without taking into account the context in which the riboswitch was inserted. In the present study, the tetracycline riboswitch was chosen to investigate the effects of the context and insertion site of a cassette exon within the gene to control the expression of an artificial arachidonate 5-lipoxygenase gene (<i>ALOX5</i>) in HEK293 cells. We demonstrate here that the use of riboswitch-controlled cassette exons for the control of gene expression via alternative splicing can be easily transferred to another gene through the process of contextual sequence adaptation. This was achieved through the introduction of gene-specific intronic and exonic sequences with different intron lengths and positions being tested. In contrast, the introduction of nonadapted constructs resulted in an unanticipated functionality outcome of the gene switch. Furthermore, we demonstrate that the combination of two cassette exons into a single gene resulted in a notable enhancement in the dynamic range. Finally, we generated a novel riboswitch-controlled splicing concept that enabled us to switch 5-LO wild-type to expression of an <i>ALOX5</i> isoform that lacks exon 13 (5-LOΔ13). Taken together, this study demonstrates that synthetic riboswitches that control alternative splicing are a powerful tool to regulate gene expression when applied in combination with gene-specific intronic and exonic sequences.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143513995","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}