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1دورية أكاديمية
المؤلفون: Bradley, Samuel A, Lehka, Beata J, Hansson, Frederik G, Adhikari, Khem B, Rago, Daniela, Rubaszka, Paulina, Haidar, Ahmad K, Chen, Ling, Hansen, Lea G, Gudich, Olga, Giannakou, Konstantina, Lengger, Bettina, Gill, Ryan T, Nakamura, Yoko, de Bernonville, Thomas Dugé, Koudounas, Konstantinos, Romero-Suarez, David, Ding, Ling, Qiao, Yijun, Frimurer, Thomas M, Petersen, Anja A, Besseau, Sébastien, Kumar, Sandeep, Gautron, Nicolas, Melin, Celine, Marc, Jillian, Jeanneau, Remi, O’Connor, Sarah E, Courdavault, Vincent, Keasling, Jay D, Zhang, Jie, Jensen, Michael K
المصدر: Nature Chemical Biology. 19(12)
مصطلحات موضوعية: Biological Sciences, Industrial Biotechnology, Good Health and Well Being, Saccharomyces cerevisiae, Monoterpenes, Indole Alkaloids, Plants, Pharmaceutical Preparations, Catharanthus, Plant Proteins, Medicinal and Biomolecular Chemistry, Biochemistry and Cell Biology, Biochemistry & Molecular Biology, Biochemistry and cell biology, Medicinal and biomolecular chemistry
الوصف: Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.
وصف الملف: application/pdf
الوصول الحر: https://escholarship.org/uc/item/47w2x3txTest
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2دورية أكاديمية
المؤلفون: Bradley, Samuel A., Lehka, Beata J., Hansson, Frederik G., Adhikari, Khem B., Rago, Daniela, Rubaszka, Paulina, Haidar, Ahmad K., Chen, Ling, Hansen, Lea G., Gudich, Olga, Giannakou, Konstantina, Lengger, Bettina, Gill, Ryan T., Nakamura, Yoko, de Bernonville, Thomas Dugé, Koudounas, Konstantinos, Romero-Suarez, David, Ding, Ling, Qiao, Yijun, Frimurer, Thomas M., Petersen, Anja A., Besseau, Sébastien, Kumar, Sandeep, Gautron, Nicolas, Melin, Celine, Marc, Jillian, Jeanneau, Remi, O’Connor, Sarah E., Courdavault, Vincent, Keasling, Jay D., Zhang, Jie, Jensen, Michael K.
المصدر: Bradley , S A , Lehka , B J , Hansson , F G , Adhikari , K B , Rago , D , Rubaszka , P , Haidar , A K , Chen , L , Hansen , L G , Gudich , O , Giannakou , K , Lengger , B , Gill , R T , Nakamura , Y , de Bernonville , T D , Koudounas , K , Romero-Suarez , D , Ding , L , Qiao , Y , Frimurer , T M , Petersen , A A , Besseau , S , ....
مصطلحات موضوعية: /dk/atira/pure/sustainabledevelopmentgoals/good_health_and_well_being, name=SDG 3 - Good Health and Well-being
الوصف: Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker’s yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.
وصف الملف: application/pdf
الإتاحة: https://doi.org/10.1038/s41589-023-01430-2Test
https://orbit.dtu.dk/en/publications/abf8d0bd-d55a-44e6-8cb7-c464bd315701Test
https://backend.orbit.dtu.dk/ws/files/346096133/s41589-023-01430-2.pdfTest -
3دورية أكاديمية
المؤلفون: Mohr, Marina, Damas, Nkerorema, Gudmand-Høyer, Johanne, Zeeberg, Katrine, Jedrzejczyk, Dominika, Vlassis, Arsenios, Morera-Gómez, Martí, Pereira-Schoning, Sara, Puš, Urška, Oliver-Almirall, Anna, Lyholm Jensen, Tanja, Baumgartner, Roland, Tate Weinert, Brian, Gill, Ryan T., Warnecke, Tanya
المصدر: Mohr , M , Damas , N , Gudmand-Høyer , J , Zeeberg , K , Jedrzejczyk , D , Vlassis , A , Morera-Gómez , M , Pereira-Schoning , S , Puš , U , Oliver-Almirall , A , Lyholm Jensen , T , Baumgartner , R , Tate Weinert , B , Gill , R T & Warnecke , T 2023 , ' The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells ' , ACS Synthetic Biology , vol. 12 , no. 2 , pp. 375-389 . https://doi.org/10.1021/acssynbio.2c00179Test
مصطلحات موضوعية: CRISPR, MAD7, NHEJ, HDR, Frameshift mutations, CAR T-cells
الوصف: CRISPR-Cas12a nucleases have expanded the toolbox for targeted genome engineering in a broad range of organisms. Here, using a high-throughput engineering approach, we explored the potential of a novel CRISPR-MAD7 system for genome editing in human cells. We evaluated several thousand optimization conditions and demonstrated accurate genome reprogramming with modified MAD7. We identified crRNAs that allow for ≤95% non-homologous end joining (NHEJ) and 66% frameshift mutations in various genes and observed the high-cleavage fidelity of MAD7 resulting in undetectable off-target activity. We explored the dsDNA delivery efficiency of CRISPR-MAD7, and by using our optimized transfection protocol, we obtained ≤85% chimeric antigen receptor (CAR) insertions in primary T cells, thus exceeding the baseline integration efficiencies of therapeutically relevant transgenes using currently available virus-free technologies. Finally, we evaluated multiplex editing efficiency with CRISPR-MAD7 and demonstrated simultaneous ≤35% CAR transgene insertions and ≤80% gene disruption efficiencies. Both the platform and our transfection procedure are easily adaptable for further preclinical studies and could potentially be used for clinical manufacturing of CAR T cells.
وصف الملف: application/pdf
الإتاحة: https://doi.org/10.1021/acssynbio.2c00179Test
https://orbit.dtu.dk/en/publications/4da7de01-4a9e-47c4-9a1b-b91f6592ce6cTest
https://backend.orbit.dtu.dk/ws/files/341113601/mohr-et-al-2023-the-crispr-cas12a-platform-for-accurate-genome-editing-gene-disruption-and-efficient-transgene.pdfTest -
4دورية أكاديمية
المؤلفون: Choudhury, Alaksh, Gachet, Benoit, Dixit, Zoya, Faure, Roland, Gill, Ryan T., Tenaillon, Olivier
المصدر: Choudhury , A , Gachet , B , Dixit , Z , Faure , R , Gill , R T & Tenaillon , O 2023 , ' Deep mutational scanning reveals the molecular determinants of RNA polymerase-mediated adaptation and tradeoffs ' , Nature Communications , vol. 14 , 6319 . https://doi.org/10.1038/s41467-023-41882-7Test
الوصف: RNA polymerase (RNAP) is emblematic of complex biological systems that control multiple traits involving trade-offs such as growth versus maintenance. Laboratory evolution has revealed that mutations in RNAP subunits, including RpoB, are frequently selected. However, we lack a systems view of how mutations alter the RNAP molecular functions to promote adaptation. We, therefore, measured the fitness of thousands of mutations within a region of rpoB under multiple conditions and genetic backgrounds, to find that adaptive mutations cluster in two modules. Mutations in one module favor growth over maintenance through a partial loss of an interaction associated with faster elongation. Mutations in the other favor maintenance over growth through a destabilized RNAP-DNA complex. The two molecular handles capture the versatile RNAP-mediated adaptations. Combining both interaction losses simultaneously improved maintenance and growth, challenging the idea that growth-maintenance tradeoff resorts only from limited resources, and revealing how compensatory evolution operates within RNAP.
وصف الملف: application/pdf
الإتاحة: https://doi.org/10.1038/s41467-023-41882-7Test
https://orbit.dtu.dk/en/publications/0b46a461-efb2-49e9-8b79-03f4b4a655d7Test
https://backend.orbit.dtu.dk/ws/files/341100970/s41467-023-41882-7.pdfTest -
5دورية أكاديمية
المؤلفون: Vlassis, Arsenios, Jensen, Tanja L., Mohr, Marina, Jedrzejczyk, Dominika J., Meng, Xiangyou, Kovacs, Gergo, Barghetti, Andrea, Morera-Gómez, Martí, Muyo Abad, Sergi, Baumgartner, Roland F., Natarajan, Kedar N., Nielsen, Lars K., Warnecke, Tanya, Gill, Ryan T.
المصدر: Vlassis , A , Jensen , T L , Mohr , M , Jedrzejczyk , D J , Meng , X , Kovacs , G , Barghetti , A , Morera-Gómez , M , Muyo Abad , S , Baumgartner , R F , Natarajan , K N , Nielsen , L K , Warnecke , T & Gill , R T 2023 , ' CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells ' , iScience , vol. 26 , no. 12 , 108287 . https://doi.org/10.1016/j.isci.2023.108287Test
الوصف: Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34+ hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies.
وصف الملف: application/pdf
الإتاحة: https://doi.org/10.1016/j.isci.2023.108287Test
https://orbit.dtu.dk/en/publications/bafbc18a-6fbd-48a9-9936-3daa4e4ee9b0Test
https://backend.orbit.dtu.dk/ws/files/341191156/1-s2.0-S2589004223023647-main.pdfTest -
6دورية أكاديمية
المؤلفون: Gupta, Mamta, Wong, Matthew, Jawed, Kamran, Gedeon, Kamil, Barrett, Hannah, Bassalo, Marcelo, Morrison, Clifford, Eqbal, Danish, Yazdani, Syed Shams, Gill, Ryan T., Huang, Jiaqi, Douaisi, Marc, Dordick, Jonathan, Belfort, Georges, Koffas, Mattheos A.G.
المساهمون: Science and Engineering Research Board, U.S. Department of Energy, University of California, National Institutes of Health, Indo-US Science and Technology Forum
المصدر: Metabolic Engineering Communications ; volume 15, page e00210 ; ISSN 2214-0301
مصطلحات موضوعية: Biomedical Engineering, Endocrinology, Diabetes and Metabolism
الإتاحة: https://doi.org/10.1016/j.mec.2022.e00210Test
https://api.elsevier.com/content/article/PII:S2214030122000190?httpAccept=text/xmlTest
https://api.elsevier.com/content/article/PII:S2214030122000190?httpAccept=text/plainTest -
7دورية أكاديمية
المؤلفون: Weiss, Sophie J, Mansell, Thomas J, Mortazavi, Pooneh, Knight, Rob, Gill, Ryan T
المصدر: PloS one. 11(1)
مصطلحات موضوعية: Escherichia coli, Anti-Bacterial Agents, Chromosome Mapping, Drug Resistance, Microbial, Genomic Library, Alleles, Genome, Bacterial, General Science & Technology
الوصف: Chemical genomics expands our understanding of microbial tolerance to inhibitory chemicals, but its scope is often limited by the throughput of genome-scale library construction and genotype-phenotype mapping. Here we report a method for rapid, parallel, and deep characterization of the response to antibiotics in Escherichia coli using a barcoded genome-scale library, next-generation sequencing, and streamlined bioinformatics software. The method provides quantitative growth data (over 200,000 measurements) and identifies contributing antimicrobial resistance and susceptibility alleles. Using multivariate analysis, we also find that subtle differences in the population responses resonate across multiple levels of functional hierarchy. Finally, we use machine learning to identify a unique allelic and proteomic fingerprint for each antibiotic. The method can be broadly applied to tolerance for any chemical from toxic metabolites to next-generation biofuels and antibiotics.
وصف الملف: application/pdf
الوصول الحر: https://escholarship.org/uc/item/0935w020Test
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8دورية أكاديمية
المؤلفون: Karp, Eric M., Eaton, Todd R., Nogué, Violeta Sànchez i, Vorotnikov, Vassili, Biddy, Mary J., Tan, Eric C. D., Brandner, David G., Cywar, Robin M., Liu, Rongming, Manker, Lorenz P., Michener, William E., Gilhespy, Michelle, Skoufa, Zinovia, Watson, Michael J., Fruchey, O. Stanley, Vardon, Derek R., Gill, Ryan T., Bratis, Adam D., Beckham, Gregg T.
المصدر: Science, 2017 Dec 01. 358(6368), 1307-1310.
الوصول الحر: https://www.jstor.org/stable/26401044Test
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9دورية أكاديميةTranscriptional Regulatory Networks Involved in C3-C4 Alcohol Stress Response and Tolerance in Yeast
المؤلفون: Liang, Liya, Liu, Rongming, Freed, Emily F., Eckert, Carrie A., Gill, Ryan T.
المصدر: Liang , L , Liu , R , Freed , E F , Eckert , C A & Gill , R T 2021 , ' Transcriptional Regulatory Networks Involved in C3-C4 Alcohol Stress Response and Tolerance in Yeast ' , ACS Synthetic Biology , vol. 10 , no. 1 , pp. 19-28 . https://doi.org/10.1021/acssynbio.0c00253Test
مصطلحات موضوعية: Alcohol tolerance, Isobutanol, Isopropanol, Transcriptional regulatory network, Yeast
الوصف: Alcohol toxicity significantly impacts the titer and productivity of industrially produced biofuels. To overcome this limitation, we must find and use strategies to improve stress tolerance in production strains. Previously, we developed a multiplex navigation of a global regulatory network (MINR) library that targeted 25 regulatory genes that are predicted to modify global regulation in yeast under different stress conditions. In this study, we expanded this concept to target the active sites of 47 transcriptional regulators using a saturation mutagenesis library. The 47 targeted regulators interact with more than half of all yeast genes. We then screened and selected for C3-C4 alcohol tolerance. We identified specific mutants that have resistance to isopropanol and isobutanol. Notably, the WAR1_K110N variant improved tolerance to both isopropanol and isobutanol. In addition, we investigated the mechanisms for improvement of isopropanol and isobutanol stress tolerance and found that genes related to glycolysis play a role in tolerance to isobutanol, while changes in ATP synthesis and mitochondrial respiration play a role in tolerance to both isobutanol and isopropanol. Overall, this work sheds light on basic mechanisms for isopropanol and isobutanol toxicity and demonstrates a promising strategy to improve tolerance to C3-C4 alcohols by perturbing the transcriptional regulatory network.
وصف الملف: application/pdf
الإتاحة: https://doi.org/10.1021/acssynbio.0c00253Test
https://orbit.dtu.dk/en/publications/a4474491-8eac-4d27-b1a1-5e49413da753Test
https://backend.orbit.dtu.dk/ws/files/258641754/Liang2021_Transcriptional_Regulatory_Networks_Involved_in_C3_C4_Alcohol_Stress_Response_and_Tolerance_in_Yeast.pdfTest -
10دورية أكاديمية
المؤلفون: Liu, Rongming, Liang, Liya, Freed, Emily F., Gill, Ryan T.
المصدر: Liu , R , Liang , L , Freed , E F & Gill , R T 2021 , ' Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing ' , Trends in Biotechnology , vol. 39 , no. 3 , pp. 262-273 . https://doi.org/10.1016/j.tibtech.2020.07.005Test
الوصف: CRISPR technology is a universal tool for genome engineering that has revolutionized biotechnology. Recently identified unique CRISPR/Cas systems, as well as re-engineered Cas proteins, have rapidly expanded the functions and applications of CRISPR/Cas systems. The structures of Cas proteins are complex, containing multiple functional domains. These protein domains are evolutionarily conserved polypeptide units that generally show independent structural or functional properties. In this review, we propose using protein domains as a new way to classify protein engineering strategies for these proteins and discuss common ways to engineer key domains to modify the functions of CRISPR/Cas systems.
وصف الملف: application/pdf
الإتاحة: https://doi.org/10.1016/j.tibtech.2020.07.005Test
https://orbit.dtu.dk/en/publications/368229dd-ec7a-47d9-ae9d-85f5b12466f8Test
https://backend.orbit.dtu.dk/ws/files/258598178/Liu2021_Directed_Evolution_of_CRISPRCas_Systems_for_Precise_Gene_Editing.pdfTest
