ACS Synthetic Biology最新文献

{"title":"Promiscuity of an Alcohol-Dependent Hemiterpene Pathway for the In Vivo Production of a Non-Natural Alkylated Tryptophan Derivative.","authors":"Anuran K Gayen, Rachael S Pitts Hall, Sean Lund, Gavin J Williams","doi":"10.1021/acssynbio.4c00865","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00865","url":null,"abstract":"<p><p>The prenyl motif determines the biological activity of many natural products. Yet, structural diversification of the prenyl site has been restricted due to the limitations of native biosynthetic pathways to hemiterpenes, the universal isoprenoid building blocks. Previously, we developed the artificial alcohol dependent hemiterpene (ADH) pathway comprising the acid phosphatase PhoN and the isopentenyl kinase IPK to provide natural isoprenoids assembled from hemiterpenes in vivo. Here, we revealed the broad specificity of the first enzyme of the ADH module, PhoN, and a downstream aromatic prenyltransferase. We then showed that the combined promiscuity of the ADH module and prenyltransferase were sufficient to produce a non-natural-alkylated tryptophan derivative in vivo when coupled with the previously described promiscuity of IPK. The short and modular ADH pathway provides a convenient and scalable approach to alkyl-pyrophosphates and facilitates probing the promiscuity of other downstream enzymes involved in isoprenoid biosynthesis without the tedious in vitro preparation of alkyl-pyrophosphates. This sets the stage to leverage the ADH pathway to forward engineer isoprenoid biosynthesis and expand its chemical space accessible to synthetic biology.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707783","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 Reaction System for ATP Regeneration from d-Fructose","authors":"Franziska Kraußer,&nbsp;Kenny Rabe,&nbsp;Christopher M. Topham,&nbsp;Julian Voland,&nbsp;Laura Lilienthal,&nbsp;Jan-Ole Kundoch,&nbsp;Daniel Ohde,&nbsp;Andreas Liese and Thomas Walther*,&nbsp;","doi":"10.1021/acssynbio.4c0087710.1021/acssynbio.4c00877","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00877https://doi.org/10.1021/acssynbio.4c00877","url":null,"abstract":"<p >Adenosine triphosphate (ATP)-dependent <i>in vitro</i> bioprocesses, such as cell-free protein synthesis and the production of phosphorylated fine chemicals, are of considerable industrial significance. However, their implementation is mainly hindered by the high cost of ATP. We propose and demonstrate the feasibility of a cell-free ATP regeneration system based on the <i>in situ</i> generation of the high-energy compound acetyl phosphate from low-cost <span>d</span>-fructose and inorganic phosphate substrates. The enzyme cascade chains <span>d</span>-fructose phosphoketolase, <span>d</span>-erythrose isomerase, <span>d</span>-erythrulose phosphoketolase, and glycolaldehyde phosphoketolase activities theoretically enabling production of 3 mol ATP per mol of <span>d</span>-fructose. Through a semirational engineering approach and the screening of nine single-mutation libraries, we optimized the phosphoketolase (PKT) from <i>Bifidobacterium adolescentis</i>, identifying the improved variant Bad.F6Pkt H548N. This mutant exhibited a 5.6-fold increase in <span>d</span>-fructose activity, a 2.2-fold increase in <span>d</span>-erythrulose activity, and a 1.3-fold increase in glycolaldehyde activity compared to the wild-type enzyme. The Bad.F6Pkt H548N mutant was initially implemented in a cell-free reaction system together with an acetate kinase from <i>Geobacillus stearothermophilus</i> and a glycerol kinase from <i>Cellulomonas</i> sp. for the production of glycerol-3 phosphate from ADP and glycerol. We demonstrated the feasibility of ATP regeneration from 25 mM <span>d</span>-fructose with a stoichiometry of 1 mol of ATP per mol of C₆ ketose. Subsequently, the reaction system was enhanced by incorporating <span>d</span>-erythrose isomerase activity provided by a <span>l</span>-rhamnose isomerase from <i>Pseudomonas stutzeri</i>. In the complete system, the ATP yield increased to 2.53 mol mol_fructose^–1 with a maximum productivity of 7.2 mM h^–1.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 4","pages":"1250–1263 1250–1263"},"PeriodicalIF":3.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00877","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842512","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}

{"title":"Cell-Free Reaction System for ATP Regeneration from d-Fructose.","authors":"Franziska Kraußer, Kenny Rabe, Christopher M Topham, Julian Voland, Laura Lilienthal, Jan-Ole Kundoch, Daniel Ohde, Andreas Liese, Thomas Walther","doi":"10.1021/acssynbio.4c00877","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00877","url":null,"abstract":"<p><p>Adenosine triphosphate (ATP)-dependent <i>in vitro</i> bioprocesses, such as cell-free protein synthesis and the production of phosphorylated fine chemicals, are of considerable industrial significance. However, their implementation is mainly hindered by the high cost of ATP. We propose and demonstrate the feasibility of a cell-free ATP regeneration system based on the <i>in situ</i> generation of the high-energy compound acetyl phosphate from low-cost d-fructose and inorganic phosphate substrates. The enzyme cascade chains d-fructose phosphoketolase, d-erythrose isomerase, d-erythrulose phosphoketolase, and glycolaldehyde phosphoketolase activities theoretically enabling production of 3 mol ATP per mol of d-fructose. Through a semirational engineering approach and the screening of nine single-mutation libraries, we optimized the phosphoketolase (PKT) from <i>Bifidobacterium adolescentis</i>, identifying the improved variant Bad.F6Pkt H548N. This mutant exhibited a 5.6-fold increase in d-fructose activity, a 2.2-fold increase in d-erythrulose activity, and a 1.3-fold increase in glycolaldehyde activity compared to the wild-type enzyme. The Bad.F6Pkt H548N mutant was initially implemented in a cell-free reaction system together with an acetate kinase from <i>Geobacillus stearothermophilus</i> and a glycerol kinase from <i>Cellulomonas</i> sp. for the production of glycerol-3 phosphate from ADP and glycerol. We demonstrated the feasibility of ATP regeneration from 25 mM d-fructose with a stoichiometry of 1 mol of ATP per mol of C₆ ketose. Subsequently, the reaction system was enhanced by incorporating d-erythrose isomerase activity provided by a l-rhamnose isomerase from <i>Pseudomonas stutzeri</i>. In the complete system, the ATP yield increased to 2.53 mol mol_fructose^-1 with a maximum productivity of 7.2 mM h^-1.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717574","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":"Expression of Fluorescence Reporters and Natural Products in Native Gut <i>Escherichia coli</i>.","authors":"Dake Liu, Phuong M Ton, David Zong, Amir Zarrinpar, Yousong Ding","doi":"10.1021/acssynbio.4c00835","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00835","url":null,"abstract":"<p><p><i>Escherichia coli</i> is a widely studied model organism and an integral component of the human gut microbiome, offering significant potential for bacteria-based therapeutic applications. Despite this promise, engineering native <i>E. coli</i> strains remains challenging. In this study, we employed the chassis-independent recombinase-assisted genome engineering (CRAGE) technique to genetically engineer the native gut strain <i>E. coli</i> EcAZ-1 and the probiotic strain <i>E. coli</i> Nissle 1917 (EcN). We successfully expressed a suite of heterologous genes, including the bioluminescent <i>lux</i> operon, green fluorescent protein (GFP), and oxygen-independent fluorescent protein IFP2.0, in both strains. Optimization of IFP2.0 fluorescence was achieved under both aerobic and anaerobic conditions by coexpressing a heme oxygenase gene and/or supplementing the chromophore biliverdin or hemin. Additionally, we engineered these strains to biosynthesize the bioactive compounds naringenin and mycosporine-like amino acids. This work highlights the potential of native <i>E. coli</i> strains as versatile platforms for synthetic biology, paving the way for innovative applications in biomedical research and therapeutic development.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143727002","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":"Promiscuity of an Alcohol-Dependent Hemiterpene Pathway for the In Vivo Production of a Non-Natural Alkylated Tryptophan Derivative","authors":"Anuran K. Gayen,&nbsp;Rachael S. Pitts Hall,&nbsp;Sean Lund and Gavin J. Williams*,&nbsp;","doi":"10.1021/acssynbio.4c0086510.1021/acssynbio.4c00865","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00865https://doi.org/10.1021/acssynbio.4c00865","url":null,"abstract":"<p >The prenyl motif determines the biological activity of many natural products. Yet, structural diversification of the prenyl site has been restricted due to the limitations of native biosynthetic pathways to hemiterpenes, the universal isoprenoid building blocks. Previously, we developed the artificial alcohol dependent hemiterpene (ADH) pathway comprising the acid phosphatase PhoN and the isopentenyl kinase IPK to provide natural isoprenoids assembled from hemiterpenes in vivo. Here, we revealed the broad specificity of the first enzyme of the ADH module, PhoN, and a downstream aromatic prenyltransferase. We then showed that the combined promiscuity of the ADH module and prenyltransferase were sufficient to produce a non-natural-alkylated tryptophan derivative in vivo when coupled with the previously described promiscuity of IPK. The short and modular ADH pathway provides a convenient and scalable approach to alkyl-pyrophosphates and facilitates probing the promiscuity of other downstream enzymes involved in isoprenoid biosynthesis without the tedious in vitro preparation of alkyl-pyrophosphates. This sets the stage to leverage the ADH pathway to forward engineer isoprenoid biosynthesis and expand its chemical space accessible to synthetic biology.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 4","pages":"1220–1229 1220–1229"},"PeriodicalIF":3.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842513","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":"Correction to \"From Natural Microbe Screening to Sustained Chitinase Activity in Exogenous Hosts\".","authors":"Diptee Chaulagain, Narges S Shamabadi, Skylar A Leslie, David K Karig","doi":"10.1021/acssynbio.5c00187","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00187","url":null,"abstract":"","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707777","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":"Multistep Metabolic Engineering of Escherichia coli for High-Level Ectoine Production","authors":"Zheng Lei,&nbsp;Jinyong Wu,&nbsp;Caiwen Lao,&nbsp;Jin Wang,&nbsp;Yanyi Xu,&nbsp;He Li,&nbsp;Lixia Yuan,&nbsp;Xiangsong Chen* and Jianming Yao*,&nbsp;","doi":"10.1021/acssynbio.4c0087610.1021/acssynbio.4c00876","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00876https://doi.org/10.1021/acssynbio.4c00876","url":null,"abstract":"<p >Ectoine is an important natural macromolecule protector that helps extremophiles maintain cellular stability and function under high-salinity conditions. Recently, the development of microbial strains for high-level ectoine production has become an attractive research direction. In this study, we constructed an efficient plasmid-free ectoine-producing strain. We modified the 5′-untranslated region of the <i>ectABC</i> gene cluster from <i>Halomonas elongate</i> to fine-tune the expression of genes <i>ectA</i>, <i>ectB</i>, and <i>ectC</i>. Furthermore, we optimized the carbon flow across the MEP pathway, the TCA cycle, and the aspartic acid metabolic pathway. Subsequently, we blocked the production of byproducts from the aspartic acid metabolic pathway and dynamically regulated the TCA cycle to coordinate the balance between strain growth and production. The final strain was tested in a 5-L fermenter, which reached 118.5 g/L at 114 h of fermentation. The metabolic engineering strategies employed in this study can be used for the biosynthesis of other aspartate derivatives.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 4","pages":"1230–1239 1230–1239"},"PeriodicalIF":3.7,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842507","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":"Multistep Metabolic Engineering of <i>Escherichia coli</i> for High-Level Ectoine Production.","authors":"Zheng Lei, Jinyong Wu, Caiwen Lao, Jin Wang, Yanyi Xu, He Li, Lixia Yuan, Xiangsong Chen, Jianming Yao","doi":"10.1021/acssynbio.4c00876","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00876","url":null,"abstract":"<p><p>Ectoine is an important natural macromolecule protector that helps extremophiles maintain cellular stability and function under high-salinity conditions. Recently, the development of microbial strains for high-level ectoine production has become an attractive research direction. In this study, we constructed an efficient plasmid-free ectoine-producing strain. We modified the 5'-untranslated region of the <i>ectABC</i> gene cluster from <i>Halomonas elongate</i> to fine-tune the expression of genes <i>ectA</i>, <i>ectB</i>, and <i>ectC</i>. Furthermore, we optimized the carbon flow across the MEP pathway, the TCA cycle, and the aspartic acid metabolic pathway. Subsequently, we blocked the production of byproducts from the aspartic acid metabolic pathway and dynamically regulated the TCA cycle to coordinate the balance between strain growth and production. The final strain was tested in a 5-L fermenter, which reached 118.5 g/L at 114 h of fermentation. The metabolic engineering strategies employed in this study can be used for the biosynthesis of other aspartate derivatives.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699067","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":"Correction to “From Natural Microbe Screening to Sustained Chitinase Activity in Exogenous Hosts”","authors":"Diptee Chaulagain,&nbsp;Narges S. Shamabadi,&nbsp;Skylar A. Leslie and David K. Karig*,&nbsp;","doi":"10.1021/acssynbio.5c0018710.1021/acssynbio.5c00187","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00187https://doi.org/10.1021/acssynbio.5c00187","url":null,"abstract":"","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 4","pages":"1323 1323"},"PeriodicalIF":3.7,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842209","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":"Ultra-efficient Integration of Gene Libraries onto Yeast Cytosolic Plasmids.","authors":"Alexander Olek Pisera, Yutong Yu, Rory L Williams, Chang C Liu","doi":"10.1021/acssynbio.4c00786","DOIUrl":"10.1021/acssynbio.4c00786","url":null,"abstract":"<p><p>Efficient methods for diversifying genes of interest (GOIs) are essential in protein engineering. For example, OrthoRep, a yeast-based orthogonal DNA replication system that achieves the rapid <i>in vivo</i> diversification of GOIs encoded on a cytosolic plasmid (p1), has been successfully used to drive numerous protein engineering campaigns. However, OrthoRep-based GOI evolution has almost always started from single GOI sequences, limiting the number of locations on a fitness landscape from where evolutionary search begins. Here, we present a simple approach for the high-efficiency integration of GOI libraries onto OrthoRep. By leveraging integrases, we demonstrate recombination of donor DNA onto the cytosolic p1 plasmid at exceptionally high transformation efficiencies, even surpassing the transformation efficiency of standard circular plasmids and linearized plasmid fragments into yeast. We demonstrate our method's utility through the straightforward construction of mock nanobody libraries encoded on OrthoRep, from which rare binders were reliably enriched. Overall, integrase-assisted manipulation of yeast cytosolic plasmids should enhance the versatility of OrthoRep in continuous evolution experiments and support the routine construction of large GOI libraries in yeast, in general.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699068","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}