ACS Synthetic Biology最新文献

{"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}

{"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":"https://doi.org/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":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","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":"https://doi.org/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":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-Free Multistep Gene Regulatory Cascades Using Eukaryotic ON-Riboswitches Responsive to <i>in Situ</i> Expressed Protein Ligands.","authors":"Atsushi Ogawa, Masahiro Fujikawa, Riku Tanimoto, Kiho Matsuno, Riko Uehara, Honami Inoue, Hajime Takahashi","doi":"10.1021/acssynbio.4c00840","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00840","url":null,"abstract":"<p><p>One of the most pressing challenges in cell-free synthetic biology is to assemble well-controlled genetic circuits. However, no complex circuits have been reported in eukaryotic cell-free systems, unlike the case in bacterial ones, despite several unique advantages of the former. We here developed protein-responsive upregulating riboswitches (ON-riboswitches) that function in wheat germ extract to create multistep gene regulatory cascades. Although the initial two types of ON-riboswitches we first designed were less efficient than desired, we improved one of them by incorporating hybridization switches to successfully construct a pair of highly efficient, protein-responsive ON-riboswitches. Both upregulated expression up to 20-fold through self-cleavage by a hammerhead ribozyme (HHR) in response to the corresponding protein ligands expressed <i>in situ</i>. We then combined them with similar types of HHR-based, small-molecule-responsive ON-riboswitches regulating protein ligand expression, to create four kinds of two-step regulatory cascades. Due to the high orthogonality of all the riboswitches used, we also succeeded in regulating two-step cascades concurrently and even in creating three-step cascades. Interestingly, the switching efficiency of each multistep cascade constructed was equivalent to that of the worst step within it. Therefore, more complex cascades with additional steps could be constructed using other efficient and orthogonal, protein-responsive ON-riboswitches with minimal loss of total switching efficiency, although the reaction conditions must be optimized to prevent a reduction of expression efficiencies. Riboswitch-based cascades fashioned through our proposed strategy would aid in the construction of eukaryotic genetic circuits for programmed cell-free systems or artificial cells with functionalities surpassing those of natural cells.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143481899","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":"CnRed: Efficient, Marker-free Genome Engineering of <i>Cupriavidus necator</i> H16 by Adapted Lambda Red Recombineering.","authors":"Simon Arhar, Johanna Pirchner, Holly Stolterfoht-Stock, Karin Reicher, Robert Kourist, Anita Emmerstorfer-Augustin","doi":"10.1021/acssynbio.4c00757","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00757","url":null,"abstract":"<p><p>Due to its ability to utilize carbon dioxide, native intracellular accumulation of bioplastic precursors, and a high protein content, the bacterium <i>Cupriavidus necator</i> offers potential solutions for social problems tackled by modern biotechnology. Yet, engineering of high-performing chemolithotrophic production strains has so far been hindered by the lack of adequate genome editing methods. In this work we present the establishment of a lambda Red recombineering system for use in <i>Cupriavidus necator</i> H16. In combination with electroporation as DNA delivery system, it enables an efficient and fast gene deletion methodology utilizing either suicide plasmids or, for the first time, linear PCR product. The novel lambda Red system was validated for the modification of three different genomic loci and, as a proof-of-concept, ultimately utilized for stable genomic integration of <i>Escherichia coli</i> phytase gene <i>appA</i> into the <i>phaC1</i> locus. A Cre/<i>loxP</i> system further enabled efficient marker recycling. The combination of a minimal transformation protocol with lambda Red recombineering and a Cre/<i>loxP</i> system offers a robust, freedom-to-operate synthetic biology tool in an increasingly important bacterial production host. This approach simplifies and accelerates genome engineering in <i>C. necator</i> and is expected to significantly enhance future strain development efforts.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143481902","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":"Novel Reprogramming of Polyketide Synthase for Valerolactam Production.","authors":"Jikai Zong, Kaixing Xiao, Dan Wang, Yaqi Kang, Zhiyao Peng, Bo Yu","doi":"10.1021/acssynbio.4c00758","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00758","url":null,"abstract":"<p><p>δ-Valerolactam (VL), as an organic compound, is an important precursor chemical for nylon and has a wide range of applications in organic synthesis, pharmaceutical synthesis, polymer materials, and other fields. This study introduces a novel biosynthetic method for producing VL in the engineered strain <i>Escherichia coli</i> BL21 through the reprogramming of polyketide synthases (PKS). Initially, an in vitro multienzyme system was constructed to verify the reliability of the VL synthesis pathway. Subsequently, an optimized biosynthetic pathway was established in <i>E. coli</i>, converting l-aspartate to VL with a yield of 3.66 mg/L in a 250 mL shake flask. Various engineering strategies were then implemented to enhance VL production, including substrate-enzyme affinity modification and multidimensional substrate optimization. These methods resulted in a 3.7-fold increase in VL yield, reaching 13.5 mg/L in shake flask cultures. Further scale-up in a 5 L fed-batch fermenter achieved a VL concentration of 76.2 mg/L. This research provides innovative insights into the optimization of VL production pathways and industrial-scale production.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143475876","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}