Pub Date : 2024-04-28DOI: 10.1021/acssynbio.4c00257
Anže Verbič, Tina Lebar, Arne Praznik and Roman Jerala*,
{"title":"Correction to Subunits of E3 Ligase Complex as Degrons for Efficient Degradation of Cytosolic, Nuclear and Membrane Proteins","authors":"Anže Verbič, Tina Lebar, Arne Praznik and Roman Jerala*, ","doi":"10.1021/acssynbio.4c00257","DOIUrl":"10.1021/acssynbio.4c00257","url":null,"abstract":"","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00257","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140808805","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}
Pub Date : 2024-04-28DOI: 10.1021/acssynbio.4c00193
Zhijuan Hu*, Di Gu, Will Skyrud, Yongle Du, Rui Zhai, Juan Wang and Wenjun Zhang*,
Respirantins are 18-membered antimycin-type depsipeptides produced by Streptomyces sp. and Kitasatospora sp. These compounds have shown extraordinary anticancer activities against a panel of cancer cell lines with nanomolar levels of IC50 values. However, further investigation has been impeded by the low titers of the natural producers and the challenging chemical synthesis due to their structural complexity. The biosynthetic gene cluster (BGC) of respirantin was previously proposed based on a bioinformatic comparison of the four members of antimycin-type depsipeptides. In this study, we report the first successful reconstitution of respirantin in Streptomyces albus using a synthetic BGC. This heterologous system serves as an accessible platform for the production and diversification of respirantins. Through polyketide synthase pathway engineering, biocatalysis, and chemical derivatization, we generated nine respirantin compounds, including six new derivatives. Cytotoxicity screening against human MCF-7 and Hela cancer cell lines revealed a unique biphasic dose–response profile of respirantin. Furthermore, a structure–activity relationship study has elucidated the essential functional groups that contribute to its remarkable cytotoxicity. This work paves the way for respirantin-based anticancer drug discovery and development.
{"title":"Engineered Biosynthesis and Anticancer Studies of Ring-Expanded Antimycin-Type Depsipeptides","authors":"Zhijuan Hu*, Di Gu, Will Skyrud, Yongle Du, Rui Zhai, Juan Wang and Wenjun Zhang*, ","doi":"10.1021/acssynbio.4c00193","DOIUrl":"10.1021/acssynbio.4c00193","url":null,"abstract":"<p >Respirantins are 18-membered antimycin-type depsipeptides produced by <i>Streptomyces</i> sp. and <i>Kitasatospora</i> sp. These compounds have shown extraordinary anticancer activities against a panel of cancer cell lines with nanomolar levels of IC<sub>50</sub> values. However, further investigation has been impeded by the low titers of the natural producers and the challenging chemical synthesis due to their structural complexity. The biosynthetic gene cluster (BGC) of respirantin was previously proposed based on a bioinformatic comparison of the four members of antimycin-type depsipeptides. In this study, we report the first successful reconstitution of respirantin in <i>Streptomyces albus</i> using a synthetic BGC. This heterologous system serves as an accessible platform for the production and diversification of respirantins. Through polyketide synthase pathway engineering, biocatalysis, and chemical derivatization, we generated nine respirantin compounds, including six new derivatives. Cytotoxicity screening against human MCF-7 and Hela cancer cell lines revealed a unique biphasic dose–response profile of respirantin. Furthermore, a structure–activity relationship study has elucidated the essential functional groups that contribute to its remarkable cytotoxicity. This work paves the way for respirantin-based anticancer drug discovery and development.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140808951","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}
Pub Date : 2024-04-27DOI: 10.1021/acssynbio.3c00720
Alister J. Cumming, Diana Khananisho, Mateusz Balka, Nicklas Liljestrand and Daniel O. Daley*,
Escherichia coli is often used as a factory to produce recombinant proteins. In many cases, the recombinant protein needs disulfide bonds to fold and function correctly. These proteins are genetically fused to a signal peptide so that they are secreted to the oxidizing environment of the periplasm (where the enzymes required for disulfide bond formation exist). Currently, it is difficult to determine in vivo whether a recombinant protein is efficiently secreted from the cytoplasm and folded in the periplasm or if there is a bottleneck in one of these steps because cellular capacity has been exceeded. To address this problem, we have developed a biosensor that detects cellular stress caused by (1) inefficient secretion of proteins from the cytoplasm and (2) aggregation of proteins in the periplasm. We demonstrate how the fluorescence fingerprint obtained from the biosensor can be used to identify induction conditions that do not exceed the capacity of the cell and therefore do not cause cellular stress. These induction conditions result in more effective biomass and in some cases higher titers of soluble recombinant proteins.
{"title":"Biosensor that Detects Stress Caused by Periplasmic Proteins","authors":"Alister J. Cumming, Diana Khananisho, Mateusz Balka, Nicklas Liljestrand and Daniel O. Daley*, ","doi":"10.1021/acssynbio.3c00720","DOIUrl":"10.1021/acssynbio.3c00720","url":null,"abstract":"<p ><i>Escherichia coli</i> is often used as a factory to produce recombinant proteins. In many cases, the recombinant protein needs disulfide bonds to fold and function correctly. These proteins are genetically fused to a signal peptide so that they are secreted to the oxidizing environment of the periplasm (where the enzymes required for disulfide bond formation exist). Currently, it is difficult to determine <i>in vivo</i> whether a recombinant protein is efficiently secreted from the cytoplasm and folded in the periplasm or if there is a bottleneck in one of these steps because cellular capacity has been exceeded. To address this problem, we have developed a biosensor that detects cellular stress caused by (1) inefficient secretion of proteins from the cytoplasm and (2) aggregation of proteins in the periplasm. We demonstrate how the fluorescence fingerprint obtained from the biosensor can be used to identify induction conditions that do not exceed the capacity of the cell and therefore do not cause cellular stress. These induction conditions result in more effective biomass and in some cases higher titers of soluble recombinant proteins.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.3c00720","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140799399","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}
Pub Date : 2024-04-26DOI: 10.1021/acssynbio.4c00048
Arik Shams, Alexandria Fischer, Anastasia Bodnar and Melinda Kliegman*,
Genetically engineered microorganisms (GEMs) represent a new paradigm in our ability to address the needs of a growing, changing world. GEMs are being used in agriculture, food production and additives, manufacturing, commodity and noncommodity products, environmental remediation, etc., with even more applications in the pipeline. Along with modern advances in genome-manipulating technologies, new manufacturing processes, markets, and attitudes are driving a boom in more products that contain or are derived from GEMs. Consequentially, researchers and developers are poised to interact with biotechnology regulatory policies that have been in effect for decades, but which are out of pace with rapidly changing scientific advances and knowledge. In the United States, biotechnology is regulated by multiple agencies with overlapping responsibilities. This poses a challenge for both developers and regulators to simultaneously allow new innovation and products into the market while also ensuring their safety and efficacy for the public and environment. This article attempts to highlight the various factors that interact between regulatory policy and development of GEMs in the United States, with perspectives from both regulators and developers. We present insights from a 2022 workshop hosted at the University of California, Berkeley that convened regulators from U.S. regulatory agencies and industry developers of various GEMs and GEM-derived products. We highlight several new biotechnologies and applications that are driving innovation in this space, and how regulatory agencies evaluate and assess these products according to current policies. Additionally, we describe recent updates to regulations that incorporate new technology and knowledge and how they can adapt further to effectively continue regulating for the future.
{"title":"Perspectives on Genetically Engineered Microorganisms and Their Regulation in the United States","authors":"Arik Shams, Alexandria Fischer, Anastasia Bodnar and Melinda Kliegman*, ","doi":"10.1021/acssynbio.4c00048","DOIUrl":"10.1021/acssynbio.4c00048","url":null,"abstract":"<p >Genetically engineered microorganisms (GEMs) represent a new paradigm in our ability to address the needs of a growing, changing world. GEMs are being used in agriculture, food production and additives, manufacturing, commodity and noncommodity products, environmental remediation, etc., with even more applications in the pipeline. Along with modern advances in genome-manipulating technologies, new manufacturing processes, markets, and attitudes are driving a boom in more products that contain or are derived from GEMs. Consequentially, researchers and developers are poised to interact with biotechnology regulatory policies that have been in effect for decades, but which are out of pace with rapidly changing scientific advances and knowledge. In the United States, biotechnology is regulated by multiple agencies with overlapping responsibilities. This poses a challenge for both developers and regulators to simultaneously allow new innovation and products into the market while also ensuring their safety and efficacy for the public and environment. This article attempts to highlight the various factors that interact between regulatory policy and development of GEMs in the United States, with perspectives from both regulators and developers. We present insights from a 2022 workshop hosted at the University of California, Berkeley that convened regulators from U.S. regulatory agencies and industry developers of various GEMs and GEM-derived products. We highlight several new biotechnologies and applications that are driving innovation in this space, and how regulatory agencies evaluate and assess these products according to current policies. Additionally, we describe recent updates to regulations that incorporate new technology and knowledge and how they can adapt further to effectively continue regulating for the future.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00048","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140808790","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}
Pub Date : 2024-04-25DOI: 10.1021/acssynbio.4c00043
Rongbin Wang, Benjamin Nji Wandi, Nora Schwartz, Jacob Hecht, Larissa Ponomareva, Kendall Paige, Alexis West, Kathryn Desanti, Jennifer Nguyen, Jarmo Niemi, Jon S. Thorson, Khaled A. Shaaban*, Mikko Metsä-Ketelä* and S. Eric Nybo*,
Streptomyces spp. are “nature’s antibiotic factories” that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.
{"title":"Diverse Combinatorial Biosynthesis Strategies for C–H Functionalization of Anthracyclinones","authors":"Rongbin Wang, Benjamin Nji Wandi, Nora Schwartz, Jacob Hecht, Larissa Ponomareva, Kendall Paige, Alexis West, Kathryn Desanti, Jennifer Nguyen, Jarmo Niemi, Jon S. Thorson, Khaled A. Shaaban*, Mikko Metsä-Ketelä* and S. Eric Nybo*, ","doi":"10.1021/acssynbio.4c00043","DOIUrl":"10.1021/acssynbio.4c00043","url":null,"abstract":"<p ><i>Streptomyces</i> spp. are “nature’s antibiotic factories” that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in <i>Streptomyces coelicolor</i> M1152Δ<i>matAB</i> that could produce aklavinone, 9-<i>epi</i>-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues <i>via</i> two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and <i>S. coelicolor</i> M1152Δ<i>matAB</i> expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant <i>Streptomyces spp.</i> hosts.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00043","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140658696","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}
Pub Date : 2024-04-25DOI: 10.1021/acssynbio.3c00570
Yingjia Tong, Ning Li, Shenghu Zhou, Liang Zhang, Sha Xu and Jingwen Zhou*,
Chalcone synthase (CHS) catalyzes the rate-limiting step of (2S)-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2S)-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2S)-naringenin biosensor based on the TtgR operon was constructed in Escherichia coli and its detection range was expanded by promoter optimization to 0–300 mg/L, the widest range for (2S)-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, SjCHS1S208N with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for de novo biosynthesis of (2S)-naringenin by SjCHS1S208N was constructed in Saccharomyces cerevisiae, combined with CHS precursor pathway optimization, increasing the (2S)-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2S)-naringenin to 2513 ± 105 mg/L, the highest reported so far. These findings will facilitate efficient flavonoid biosynthesis and further modification of the CHS in the future.
{"title":"Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution","authors":"Yingjia Tong, Ning Li, Shenghu Zhou, Liang Zhang, Sha Xu and Jingwen Zhou*, ","doi":"10.1021/acssynbio.3c00570","DOIUrl":"10.1021/acssynbio.3c00570","url":null,"abstract":"<p >Chalcone synthase (CHS) catalyzes the rate-limiting step of (2<i>S</i>)-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2<i>S</i>)-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2<i>S</i>)-naringenin biosensor based on the TtgR operon was constructed in <i>Escherichia coli</i> and its detection range was expanded by promoter optimization to 0–300 mg/L, the widest range for (2<i>S</i>)-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, <i>Sj</i>CHS1<sup>S208N</sup> with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for <i>de novo</i> biosynthesis of (2<i>S</i>)-naringenin by <i>Sj</i>CHS1<sup>S208N</sup> was constructed in <i>Saccharomyces cerevisiae</i>, combined with CHS precursor pathway optimization, increasing the (2<i>S</i>)-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2<i>S</i>)-naringenin to 2513 ± 105 mg/L, the highest reported so far. These findings will facilitate efficient flavonoid biosynthesis and further modification of the CHS in the future.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140656277","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}
Pub Date : 2024-04-24DOI: 10.1021/acssynbio.3c00542
Viviana Nguyen, Yifei Li and Ting Lu*,
Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of Saccharomyces cerevisiae. Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. This study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.
{"title":"Emergence of Orchestrated and Dynamic Metabolism of Saccharomyces cerevisiae","authors":"Viviana Nguyen, Yifei Li and Ting Lu*, ","doi":"10.1021/acssynbio.3c00542","DOIUrl":"10.1021/acssynbio.3c00542","url":null,"abstract":"<p >Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of <i>Saccharomyces cerevisiae</i>. Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. This study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140664770","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}
Pub Date : 2024-04-18DOI: 10.1021/acssynbio.4c00039
Samuel A. Bradley, Frederik G. Hansson, Beata J. Lehka, Daniela Rago, Pedro Pinho, Huadong Peng, Khem B. Adhikari, Ahmad K. Haidar, Lea G. Hansen, Daria Volkova, Maxence Holtz, Sergi Muyo Abad, Xin Ma, Konstantinos Koudounas, Sébastien Besseau, Nicolas Gautron, Céline Mélin, Jillian Marc, Caroline Birer Williams, Vincent Courdavault, Emil D. Jensen, Jay D. Keasling, Jie Zhang* and Michael K. Jensen*,
Monoterpene indole alkaloids (MIAs) make up a highly bioactive class of metabolites produced by a range of tropical and subtropical plants. The corynanthe-type MIAs are a stereochemically complex subclass with therapeutic potential against a large number of indications including cancer, psychotic disorders, and erectile dysfunction. Here, we report yeast-based cell factories capable of de novo production of corynanthe-type MIAs rauwolscine, yohimbine, tetrahydroalstonine, and corynanthine. From this, we demonstrate regioselective biosynthesis of 4 fluorinated derivatives of these compounds and de novo biosynthesis of 7-chlororauwolscine by coexpression of a halogenase with the biosynthetic pathway. Finally, we capitalize on the ability of these cell factories to produce derivatives of these bioactive scaffolds to establish a proof-of-principle drug discovery pipeline in which the corynanthe-type MIAs are screened for bioactivity on human drug targets, expressed in yeast. In doing so, we identify antagonistic and agonistic behavior against the human adrenergic G protein-coupled receptors ADRA2A and ADRA2B, and the serotonergic receptor 5HT4b, respectively. This study thus demonstrates a proto-drug discovery pipeline for bioactive plant-inspired small molecules based on one-pot biocatalysis of natural and new-to-nature corynanthe-type MIAs in yeast.
{"title":"Yeast Platforms for Production and Screening of Bioactive Derivatives of Rauwolscine","authors":"Samuel A. Bradley, Frederik G. Hansson, Beata J. Lehka, Daniela Rago, Pedro Pinho, Huadong Peng, Khem B. Adhikari, Ahmad K. Haidar, Lea G. Hansen, Daria Volkova, Maxence Holtz, Sergi Muyo Abad, Xin Ma, Konstantinos Koudounas, Sébastien Besseau, Nicolas Gautron, Céline Mélin, Jillian Marc, Caroline Birer Williams, Vincent Courdavault, Emil D. Jensen, Jay D. Keasling, Jie Zhang* and Michael K. Jensen*, ","doi":"10.1021/acssynbio.4c00039","DOIUrl":"10.1021/acssynbio.4c00039","url":null,"abstract":"<p >Monoterpene indole alkaloids (MIAs) make up a highly bioactive class of metabolites produced by a range of tropical and subtropical plants. The corynanthe-type MIAs are a stereochemically complex subclass with therapeutic potential against a large number of indications including cancer, psychotic disorders, and erectile dysfunction. Here, we report yeast-based cell factories capable of de novo production of corynanthe-type MIAs rauwolscine, yohimbine, tetrahydroalstonine, and corynanthine. From this, we demonstrate regioselective biosynthesis of 4 fluorinated derivatives of these compounds and de novo biosynthesis of 7-chlororauwolscine by coexpression of a halogenase with the biosynthetic pathway. Finally, we capitalize on the ability of these cell factories to produce derivatives of these bioactive scaffolds to establish a proof-of-principle drug discovery pipeline in which the corynanthe-type MIAs are screened for bioactivity on human drug targets, expressed in yeast. In doing so, we identify antagonistic and agonistic behavior against the human adrenergic G protein-coupled receptors ADRA2A and ADRA2B, and the serotonergic receptor 5HT4b, respectively. This study thus demonstrates a proto-drug discovery pipeline for bioactive plant-inspired small molecules based on one-pot biocatalysis of natural and new-to-nature corynanthe-type MIAs in yeast.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140608934","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}
Pub Date : 2024-04-17DOI: 10.1021/acssynbio.4c00073
Eleonora Bailoni, Miyer F. Patiño-Ruiz, Andreea R. Stan, Gea K. Schuurman-Wolters, Marten Exterkate, Arnold J. M. Driessen and Bert Poolman*,
ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder–utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.
通过脱氨基作用降解精氨酸是在合成细胞中生成 ATP 的一种有效方法,但目前这种方法受限于缓慢的精氨酸/鸟氨酸交换。目前,我们正在采用一种动力学性能更好的新型反转运体,以获得更快的 ATP 循环。我们利用依赖于精氨酸的 ATP 形成,通过甘油激酶和甘油-3-磷酸/Pi 反转运体,持续合成和输出甘油-3-磷酸。输出的甘油-3-磷酸酯是第二组囊泡中磷脂生物合成的前体,是细胞膜扩张的基础。因此,我们开发了一个用于 ATP 循环的失衡代谢网络,并将其与脂质合成结合起来。这种馈源-利用系统可作为系统建立合成细胞的原理验证,但小泡也可用于研究封闭的单个反应网络。
{"title":"Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis†","authors":"Eleonora Bailoni, Miyer F. Patiño-Ruiz, Andreea R. Stan, Gea K. Schuurman-Wolters, Marten Exterkate, Arnold J. M. Driessen and Bert Poolman*, ","doi":"10.1021/acssynbio.4c00073","DOIUrl":"10.1021/acssynbio.4c00073","url":null,"abstract":"<p >ATP is a universal energy currency that is essential for life. <span>l</span>-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow <span>l</span>-arginine/<span>l</span>-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use <span>l</span>-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder–utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00073","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140608939","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}
Computer-aided promoter design is a major development trend in synthetic promoter engineering. Various deep learning models have been used to evaluate or screen synthetic promoters, but there have been few works on de novo promoter design. To explore the potential ability of generative models in promoter design, we established a diffusion-based generative model for promoter design in Escherichia coli. The model was completely driven by sequence data and could study the essential characteristics of natural promoters, thus generating synthetic promoters similar to natural promoters in structure and component. We also improved the calculation method of FID indicator, using a convolution layer to extract the feature matrix of the promoter sequence instead. As a result, we got an FID equal to 1.37, which meant synthetic promoters have a distribution similar to that of natural ones. Our work provides a fresh approach to de novo promoter design, indicating that a completely data-driven generative model is feasible for promoter design.
{"title":"Diffusion-Based Generative Network for de Novo Synthetic Promoter Design","authors":"Jianfeng Lin, Xin Wang, Tuoyu Liu, Yue Teng* and Wei Cui*, ","doi":"10.1021/acssynbio.4c00041","DOIUrl":"10.1021/acssynbio.4c00041","url":null,"abstract":"<p >Computer-aided promoter design is a major development trend in synthetic promoter engineering. Various deep learning models have been used to evaluate or screen synthetic promoters, but there have been few works on de novo promoter design. To explore the potential ability of generative models in promoter design, we established a diffusion-based generative model for promoter design in <i>Escherichia coli</i>. The model was completely driven by sequence data and could study the essential characteristics of natural promoters, thus generating synthetic promoters similar to natural promoters in structure and component. We also improved the calculation method of FID indicator, using a convolution layer to extract the feature matrix of the promoter sequence instead. As a result, we got an FID equal to 1.37, which meant synthetic promoters have a distribution similar to that of natural ones. Our work provides a fresh approach to de novo promoter design, indicating that a completely data-driven generative model is feasible for promoter design.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140571771","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}