ACS Synthetic Biology最新文献_第2页

Synthetic Genetic Elements Enable Rapid Characterization of Inorganic Carbon Uptake Systems in Cupriavidus necator H16.

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-03-06 DOI: 10.1021/acssynbio.4c00869

Akira K Nakamura, Emily M Fulk, Christopher W Johnson, Farren J Isaacs

{"title":"Synthetic Genetic Elements Enable Rapid Characterization of Inorganic Carbon Uptake Systems in Cupriavidus necator H16.","authors":"Akira K Nakamura, Emily M Fulk, Christopher W Johnson, Farren J Isaacs","doi":"10.1021/acssynbio.4c00869","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00869","url":null,"abstract":"Cupriavidus necator 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 C. necator, 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 C. necator. 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 C. necator. 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 C. necator.","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":"143565495","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}

引用次数: 0

Developing Biosensors for Specific Assessment of Trans-Translation in Pseudomonas aeruginosa.

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-03-06 DOI: 10.1021/acssynbio.4c00801

Bastien L'Hermitte, Thomas Chauvet, Sylvie Georgeault-Daguenet, Nicolas Le Yondre, Philippe Jehan, Reynald Gillet, Christine Baysse

引用次数: 0

Improving Bacillus subtilis as Biological Chassis Performance by the CRISPR Genetic Toolkit.

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-03-04 DOI: 10.1021/acssynbio.4c00844

Xianhai Cao, Xiaojuan Wang, Ruirui Chen, Lu Chen, Yang Liu, Meng Wang

{"title":"Improving Bacillus subtilis 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":"Bacillus subtilis 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 B. subtilis have lagged behind the most widely used industrial chassis Saccharomyces cerevisiae and Escherichia coli. CRISPR (an acronym for clustered regularly interspaced short palindromic repeats) enables efficient, site-specific, and programmable DNA cleavage, which has revolutionized the manner of genome editing. In 2016, CRISPR technology was first introduced into B. subtilis and has been intensely upgraded since then. In this Review, we discuss recently developed key additions to CRISPR toolkit design in B. subtilis with gene editing, transcriptional regulation, and enzyme modulation. Second, advances in the B. subtilis 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 B. subtilis, wishing that B. subtilis can be comparable to traditional industrial microorganisms such as E. coli and S. cerevisiae someday soon.","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}

引用次数: 0

Programming Nutrient Detection with Modular Regulators for Dynamic Control of Microbial Biosynthesis.

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-03-04 DOI: 10.1021/acssynbio.4c00720

Nhu Nguyen, Vincenzo Kennedy, Jung Yeon Lee, Noel Y Chan, Clement T Y Chan

{"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":"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 Escherichia coli and Pseudomonas putida. 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.","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}

引用次数: 0

Developing Thermosensitive Metabolic Regulation Strategies in the Fermentation Process of Saccharomyces cerevisiae to Enhance α-Bisabolene Production.

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-03-03 DOI: 10.1021/acssynbio.4c00728

Pan Feng, Bowen Sun, Haoran Bi, Yufei Bao, Meng Wang, Huili Zhang, Yunming Fang

{"title":"Developing Thermosensitive Metabolic Regulation Strategies in the Fermentation Process of Saccharomyces cerevisiae 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":"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 Saccharomyces cerevisiae remains an outstanding challenge due to cell growth limitations and insufficient supply of the α-bisabolene precursor farnesyl pyrophosphate. In this work, a new S. cerevisiae 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.","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}

引用次数: 0

The iGEM Grand Jamboree and the Serious Fun of Engineering Biology. iGEM 大露营和工程生物学的严肃乐趣。

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-02-28 DOI: 10.1021/acssynbio.5c00083

Edwin H Wintermute

引用次数: 0

Toward Antibody Production in Genome-Minimized Bacillus subtilis Strains.

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-02-27 DOI: 10.1021/acssynbio.4c00688

Tobias Schilling, Rebekka Biedendieck, Rafael Moran-Torres, Mirva J Saaranen, Lloyd W Ruddock, Rolf Daniel, Jan Maarten van Dijl

{"title":"Toward Antibody Production in Genome-Minimized Bacillus subtilis 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":"Bacillus subtilis 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 B. subtilis, 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 Midi- and MiniBacillus 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 N-ethylmaleimide, and nano-differential scanning fluorimetry, MidiBacillus strains can secrete fully functional, natively folded, disulfide-bonded, and thermostable antibody fragments. We therefore conclude that genome-reduced B. subtilis chassis strains are capable of secreting high-quality antibody fragments.","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}

引用次数: 0

Mechanistic Modeling of In Vivo Translation in Escherichia coli Reliably Identifies Well-Adapted and Optimized RNA Sequences.

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-02-27 DOI: 10.1021/acssynbio.4c00578

Jan Spindler, Christina Giakissiklis, Catharina Stierle, Marc Buschlüter, Klaus Liebeton, Martin Siemann-Herzberg, Ralf Takors

{"title":"Mechanistic Modeling of In Vivo Translation in Escherichia coli 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":"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 Escherichia coli cells. We emphasize that endogenous E. coli sequences typically adapt to these effects, particularly in highly expressed genes, where adaptation ensures efficient translation. Conversely, heterologous gene sequences from Saccharomyces cerevisiae 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 E. coli.","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}

引用次数: 0

Efficient Biosynthesis of Furanocoumarin Intermediate Marmesin by Engineered Escherichia coli.

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-02-27 DOI: 10.1021/acssynbio.4c00892

Baodong Hu, Jingwen Zhou, Jianghua Li, Jian Chen, Guocheng Du, Fang Zhong, Yucheng Zhao, Xinrui Zhao

{"title":"Efficient Biosynthesis of Furanocoumarin Intermediate Marmesin by Engineered Escherichia coli.","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":"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 Escherichia coli by screening the robust 6-prenyltransferase PpPT1 and marmesin synthase PpDCΔ2-29 from Peucedanum praeruptorum. Next, the activities of PpPT1 and PpDCΔ2-29 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.","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}

引用次数: 0

An Arrayed CRISPR Screen Identifies Knockout Combinations Improving Antibody Productivity in HEK293 Cells. 通过 CRISPR 阵列筛选，确定了可提高 HEK293 细胞抗体生产率的基因敲除组合。

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2025-02-27 DOI: 10.1021/acssynbio.4c00772

Eric Edward Bryant, Danyang Gong, Cai Guo, Fernando Garces, René Hubert, Irwin Chen

{"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":"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.","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}

引用次数: 0