Pub Date : 2025-02-06DOI: 10.1021/acssynbio.4c00810
Yu Fang, Xiangfeng Meng, Lin Liu, Zhongye Li, Kaili Jia, Weifeng Liu
The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers and also has the potential to be developed into a tractable fungal host for biosynthesizing secondary metabolite products. To expedite the genetic engineering of filamentous fungi, efficient DNA assembly processes that can facilitate the transfer of large-sized DNA to fungal hosts, including T. reesei, are still in demand. Here, we developed a method for the simultaneous in vivo assembly and targeted genome integration of multiple DNA fragments (SATIMD) in T. reesei. While efficient orderly DNA end fusions were achieved by homologous recombination (HR) with various lengths of sequence overlaps (100-500 bp), the assembled DNA was also precisely integrated into a specific locus when combined with CRISPR/Cas9-mediated genome cutting. Specifically, we have used this method to achieve the assembly and functional expression of T. reesei key transcriptional activator Xyr1 for cellulase genes. Moreover, fusions and targeted integration of up to 10 different DNA fragments comprising the 32.7 kb sorbicillinoids biosynthetic gene cluster via a single-step transformation was demonstrated. We envision that SATIMD is a powerful tool not only useful for direct large heterologous gene cluster assembly in T. reesei but also can facilitate large-scale fungal strain genetic engineering.
{"title":"Simultaneous <i>In Vivo</i> Assembly and Targeted Genome Integration of Gene Clusters in <i>Trichoderma reesei</i>.","authors":"Yu Fang, Xiangfeng Meng, Lin Liu, Zhongye Li, Kaili Jia, Weifeng Liu","doi":"10.1021/acssynbio.4c00810","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00810","url":null,"abstract":"<p><p>The saprophytic filamentous fungus <i>Trichoderma reesei</i> represents one of the most prolific cellulase producers and also has the potential to be developed into a tractable fungal host for biosynthesizing secondary metabolite products. To expedite the genetic engineering of filamentous fungi, efficient DNA assembly processes that can facilitate the transfer of large-sized DNA to fungal hosts, including <i>T. reesei</i>, are still in demand. Here, we developed a method for the simultaneous <i>in vivo</i> assembly and targeted genome integration of multiple DNA fragments (SATIMD) in <i>T. reesei</i>. While efficient orderly DNA end fusions were achieved by homologous recombination (HR) with various lengths of sequence overlaps (100-500 bp), the assembled DNA was also precisely integrated into a specific locus when combined with CRISPR/Cas9-mediated genome cutting. Specifically, we have used this method to achieve the assembly and functional expression of <i>T. reesei</i> key transcriptional activator Xyr1 for cellulase genes. Moreover, fusions and targeted integration of up to 10 different DNA fragments comprising the 32.7 kb sorbicillinoids biosynthetic gene cluster via a single-step transformation was demonstrated. We envision that SATIMD is a powerful tool not only useful for direct large heterologous gene cluster assembly in <i>T. reesei</i> but also can facilitate large-scale fungal strain genetic engineering.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143363267","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}
Combinatorial Ordered Gene Assembly in Bacillus subtilis (Combi-OGAB) enables construction of combinatorial libraries of various genetic elements, such as promoters in a biosynthetic gene cluster (BGC), and screening of highly productive combinations from the library. The combinations are limited by the library design, and the selectable productivity is defined within the combination. To refine the selected BGC using conventional Combi-OGAB with expanded diversity, we devised a directed evolutionary method called as random mutagenesis with Combi-OGAB (rmCombi-OGAB), which includes random mutagenesis by error-prone PCR and Combi-OGAB. In the present study, Gramicidin S (GS)-producing plasmids were used to examine the utility of rmCombi-OGAB. GS plasmids, originally generated using conventional Combi-OGAB, were successfully evolved using rmCombi-OGAB. B. subtilis carrying the evolved plasmid with unpredictable mutations showed a 1.5-fold improvement in the GS productivity. We thus expect that rmCombi-OGAB can be applied to various BGCs for useful products, such as antibiotics, to improve their productivity.
{"title":"<i>rm</i>Combi-OGAB for the Directed Evolution of a Biosynthetic Gene Cluster toward Productivity Improvement.","authors":"Naoki Miyamoto, Kentaro Hayashi, Naohisa Ogata, Naoyuki Yamada, Kenji Tsuge","doi":"10.1021/acssynbio.4c00734","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00734","url":null,"abstract":"<p><p><u><b>Combi</b></u>natorial <u><b>O</b></u>rdered <u><b>G</b></u>ene <u><b>A</b></u>ssembly in <u><b><i>B</i></b></u><i>acillus subtilis</i> (Combi-OGAB) enables construction of combinatorial libraries of various genetic elements, such as promoters in a biosynthetic gene cluster (BGC), and screening of highly productive combinations from the library. The combinations are limited by the library design, and the selectable productivity is defined within the combination. To refine the selected BGC using conventional Combi-OGAB with expanded diversity, we devised a directed evolutionary method called as <u><b>r</b></u>andom <u><b>m</b></u>utagenesis with <u><b>Combi-OGAB</b></u> (<i>rm</i>Combi-OGAB), which includes random mutagenesis by error-prone PCR and Combi-OGAB. In the present study, Gramicidin S (GS)-producing plasmids were used to examine the utility of <i>rm</i>Combi-OGAB. GS plasmids, originally generated using conventional Combi-OGAB, were successfully evolved using <i>rm</i>Combi-OGAB. <i>B. subtilis</i> carrying the evolved plasmid with unpredictable mutations showed a 1.5-fold improvement in the GS productivity. We thus expect that <i>rm</i>Combi-OGAB can be applied to various BGCs for useful products, such as antibiotics, to improve their productivity.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254160","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}
To overcome the difficulty of building large nonribosomal peptide synthetase (NRPS) gene cluster libraries, an efficient one-pot method using Bacillus subtilis was developed. This new method, named Seamed Express Assembly Method (SEAM)-combi-Ordered Gene Assembly in Bacillus subtilis (OGAB), combines the SEAM-OGAB approach for NRPS gene cluster construction with the combi-OGAB method for combinatorial DNA library construction to randomly swap DNA fragments for NRPS modules. In this study, NRPS gene clusters of plipastatin and gramicidin S were used as the starting material. The full length of each gene cluster was prepared as plasmid DNA by introducing restriction enzyme SfiI sites into the module border according to SEAM-OGAB. These two plasmids were mixed, digested with SfiI, ligated in a tandem repeat form, and used to transform B. subtilis according to the combi-OGAB method. While 64 of all the possible combinations were used in the calculation, 32 types of plasmid DNA were obtained from 50 randomly selected transformants. These transformants produced at least 30 types of peptides, including cyclic and linear variations with lengths ranging from 5 to 10 amino acids. Thus, this method enabled an efficient construction of NRPS gene cluster libraries with more than five module members, making it advantageous for applications in peptide libraries.
{"title":"Combinatorial Nonribosomal Peptide Synthetase Libraries Using the SEAM-Combi-OGAB Method.","authors":"Varada Jagadeesh, Nobuyuki Okahashi, Fumio Matsuda, Kenji Tsuge, Akihiko Kondo","doi":"10.1021/acssynbio.4c00671","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00671","url":null,"abstract":"<p><p>To overcome the difficulty of building large nonribosomal peptide synthetase (NRPS) gene cluster libraries, an efficient one-pot method using <i>Bacillus subtilis</i> was developed. This new method, named <u>S</u>eamed <u>E</u>xpress <u>A</u>ssembly <u>M</u>ethod (SEAM)-combi-<u>O</u>rdered <u>G</u>ene <u>A</u>ssembly in <i>Bacillus subtilis</i> (OGAB), combines the SEAM-OGAB approach for NRPS gene cluster construction with the combi-OGAB method for combinatorial DNA library construction to randomly swap DNA fragments for NRPS modules. In this study, NRPS gene clusters of plipastatin and gramicidin S were used as the starting material. The full length of each gene cluster was prepared as plasmid DNA by introducing restriction enzyme SfiI sites into the module border according to SEAM-OGAB. These two plasmids were mixed, digested with SfiI, ligated in a tandem repeat form, and used to transform <i>B. subtilis</i> according to the combi-OGAB method. While 64 of all the possible combinations were used in the calculation, 32 types of plasmid DNA were obtained from 50 randomly selected transformants. These transformants produced at least 30 types of peptides, including cyclic and linear variations with lengths ranging from 5 to 10 amino acids. Thus, this method enabled an efficient construction of NRPS gene cluster libraries with more than five module members, making it advantageous for applications in peptide libraries.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143187543","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}
Synthetic riboswitches, genetic regulatory elements composed entirely of RNA, have been engineered to control a variety of mechanisms at the level of both transcription and translation in all domains of life. The efficiency of riboswitch regulation can be increased by inserting two of them into an mRNA sequence in close proximity, resulting in a tandem riboswitch. The tandem state results in improved regulation beyond that of a single riboswitch by allowing both binding pockets to contribute to a higher dynamic range. The focus of this study was to create a novel tandem riboswitch design by integrating the binding pockets of two different riboswitches into one continuous structure, thereby creating a dual-input hybrid riboswitch. These hybrids remain compact in size with a shorter sequence length compared to a tandem riboswitch, while taking advantage of the binding pockets and scaffold sequences provided by both parental riboswitches. Through rational design, hybrid constructs derived from the combination of tetracycline-, tobramycin-, neomycin-, and paromomycin-binding riboswitches were engineered that significantly increase the dynamic range (e.g., from 14- to 36-fold for tobramycin) while increasing their expression levels in the absence of ligand (e.g., 28% to 68% expression for tetracycline). This study expands the toolbox of synthetic riboswitches and establishes general design guidelines applicable to similar riboswitches. Additionally, the dual-input state makes hybrid riboswitches an interesting target for the design of genetic regulators following Boolean logic.
{"title":"Synthetic Dual-Input Hybrid Riboswitches─Optimized Genetic Regulators in Yeast.","authors":"Daniel Kelvin, Janette Arias Rodriguez, Ann-Christin Groher, Kiara Petras, Beatrix Suess","doi":"10.1021/acssynbio.4c00660","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00660","url":null,"abstract":"<p><p>Synthetic riboswitches, genetic regulatory elements composed entirely of RNA, have been engineered to control a variety of mechanisms at the level of both transcription and translation in all domains of life. The efficiency of riboswitch regulation can be increased by inserting two of them into an mRNA sequence in close proximity, resulting in a tandem riboswitch. The tandem state results in improved regulation beyond that of a single riboswitch by allowing both binding pockets to contribute to a higher dynamic range. The focus of this study was to create a novel tandem riboswitch design by integrating the binding pockets of two different riboswitches into one continuous structure, thereby creating a dual-input hybrid riboswitch. These hybrids remain compact in size with a shorter sequence length compared to a tandem riboswitch, while taking advantage of the binding pockets and scaffold sequences provided by both parental riboswitches. Through rational design, hybrid constructs derived from the combination of tetracycline-, tobramycin-, neomycin-, and paromomycin-binding riboswitches were engineered that significantly increase the dynamic range (e.g., from 14- to 36-fold for tobramycin) while increasing their expression levels in the absence of ligand (e.g., 28% to 68% expression for tetracycline). This study expands the toolbox of synthetic riboswitches and establishes general design guidelines applicable to similar riboswitches. Additionally, the dual-input state makes hybrid riboswitches an interesting target for the design of genetic regulators following Boolean logic.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143187547","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 : 2025-02-04DOI: 10.1021/acssynbio.4c00649
Leidy D Caraballo G, Inci Cevher Zeytin, Purva Rathi, Che-Hsing Li, Ai-Ni Tsao, Yaery J Salvador L, Manish Ranjan, Brendan Magee Traynor, Andras A Heczey
DNA modification and synthesis are fundamental to genetic engineering, and systems that enable time- and cost-effective execution of these processes are crucial. Iteration of genetic construct variants takes significant time, cost and effort to develop new therapeutic strategies to treat diseases including cancer. Thus, decreasing cost and enhancing simplicity while accelerating the speed of advancement is critical. We have developed a PCR-based platform that allows for deletion, replacement, insertion, mutagenesis, and synthesis of DNA (DRIMS). These modifications rely on the recA-independent recombination pathway and are carried out in a single amplification step followed by DpnI digestion and transformation into competent cells. DNA synthesis is accomplished through sequential PCR amplification reactions without the need for a DNA template. Here, we provide proof-of-concept for the DRIMS platform by performing four deletions within DNA fragments of various sizes, sixty-four replacements of DNA binding sequences that incorporate repeat sequences, four replacements of chimeric antigen receptor components, fifty-one insertions of artificial microRNAs that form complex tertiary structures, five varieties of point mutations, and synthesis of eight DNA sequences including two with high GC content. Compared to other advanced cloning methods including Gibson and "in vivo assembly", we demonstrate the significant advantages of the DRIMS platform. In summary, DRIMS allows for efficient modification and synthesis of DNA in a simple, rapid and cost-effective manner to accelerate the synthetic biology field and development of therapeutics.
DNA 修饰和合成是基因工程的基础,而能够以经济高效的方式及时执行这些过程的系统则至关重要。基因构建变体的迭代需要花费大量的时间、成本和精力,才能开发出治疗癌症等疾病的新疗法。因此,在加快进度的同时降低成本和提高简便性至关重要。我们开发了一种基于 PCR 的平台,可以进行 DNA 的删除、替换、插入、诱变和合成(DRIMS)。这些修饰依赖于不依赖 recA 的重组途径,在一个扩增步骤中完成,然后进行 DpnI 消化并转化为合格细胞。DNA 合成是通过连续的 PCR 扩增反应完成的,无需 DNA 模板。在这里,我们对 DRIMS 平台进行了概念验证,对不同大小的 DNA 片段进行了四次缺失,对包含重复序列的 DNA 结合序列进行了六十四次替换,对嵌合抗原受体成分进行了四次替换,对形成复杂三级结构的人工 microRNA 进行了五十一次插入,进行了五种点突变,并合成了八种 DNA 序列,其中包括两种高 GC 含量的 DNA 序列。与其他先进的克隆方法(包括 Gibson 和 "体内组装")相比,我们展示了 DRIMS 平台的显著优势。总之,DRIMS 允许以简单、快速和经济高效的方式高效修饰和合成 DNA,从而加速合成生物学领域和治疗药物的开发。
{"title":"DRIMS: A Synthetic Biology Platform that Enables Deletion, Replacement, Insertion, Mutagenesis, and Synthesis of DNA.","authors":"Leidy D Caraballo G, Inci Cevher Zeytin, Purva Rathi, Che-Hsing Li, Ai-Ni Tsao, Yaery J Salvador L, Manish Ranjan, Brendan Magee Traynor, Andras A Heczey","doi":"10.1021/acssynbio.4c00649","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00649","url":null,"abstract":"<p><p>DNA modification and synthesis are fundamental to genetic engineering, and systems that enable time- and cost-effective execution of these processes are crucial. Iteration of genetic construct variants takes significant time, cost and effort to develop new therapeutic strategies to treat diseases including cancer. Thus, decreasing cost and enhancing simplicity while accelerating the speed of advancement is critical. We have developed a PCR-based platform that allows for <b>d</b>eletion, <b>r</b>eplacement, <b>i</b>nsertion, <b>m</b>utagenesis, and <b>s</b>ynthesis of DNA (DRIMS). These modifications rely on the recA-independent recombination pathway and are carried out in a single amplification step followed by DpnI digestion and transformation into competent cells. DNA synthesis is accomplished through sequential PCR amplification reactions without the need for a DNA template. Here, we provide proof-of-concept for the DRIMS platform by performing four deletions within DNA fragments of various sizes, sixty-four replacements of DNA binding sequences that incorporate repeat sequences, four replacements of chimeric antigen receptor components, fifty-one insertions of artificial microRNAs that form complex tertiary structures, five varieties of point mutations, and synthesis of eight DNA sequences including two with high GC content. Compared to other advanced cloning methods including Gibson and \"in vivo assembly\", we demonstrate the significant advantages of the DRIMS platform. In summary, DRIMS allows for efficient modification and synthesis of DNA in a simple, rapid and cost-effective manner to accelerate the synthetic biology field and development of therapeutics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143121669","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 : 2025-02-03DOI: 10.1021/acssynbio.4c00569
Rosa Selenia Guerra-Resendez, Samantha LeGoff Lydon, Alex J Ma, Guy C Bedford, Daniel R Reed, Sunghwan Kim, Erik R Terán, Tomoki Nishiguchi, Mario Escobar, Andrew R DiNardo, Isaac B Hilton
Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.
{"title":"Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.","authors":"Rosa Selenia Guerra-Resendez, Samantha LeGoff Lydon, Alex J Ma, Guy C Bedford, Daniel R Reed, Sunghwan Kim, Erik R Terán, Tomoki Nishiguchi, Mario Escobar, Andrew R DiNardo, Isaac B Hilton","doi":"10.1021/acssynbio.4c00569","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00569","url":null,"abstract":"<p><p>Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143078034","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 : 2025-02-03DOI: 10.1021/acssynbio.4c00619
Hinako Kawabe, Luran Manfio, Sebastian Magana Pena, Nicolette A Zhou, Kevin M Bradley, Cen Chen, Chris McLendon, Steven A Benner, Karen Levy, Zunyi Yang, Jorge A Marchand, Erica R Fuhrmeister
Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.
{"title":"Harnessing Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance.","authors":"Hinako Kawabe, Luran Manfio, Sebastian Magana Pena, Nicolette A Zhou, Kevin M Bradley, Cen Chen, Chris McLendon, Steven A Benner, Karen Levy, Zunyi Yang, Jorge A Marchand, Erica R Fuhrmeister","doi":"10.1021/acssynbio.4c00619","DOIUrl":"10.1021/acssynbio.4c00619","url":null,"abstract":"<p><p>Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143078035","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}
Yarrowia lipolytica has been widely engineered as a eukaryotic cell factory to produce various important compounds. However, the difficulty of gene editing and the lack of efficient neutral sites make rewiring of Y. lipolytica metabolism challenging. Herein, a Cas9 system was established to redesign the Y. lipolytica homologous recombination system, which caused a more than 56-fold increase in the HR efficiency. The fusion expression of the hBrex27 sequence in the C-terminus of Cas9 recruited more Rad51 protein, and the engineered Cas9 decreased NHEJ, achieving 85% single-gene positive efficiency and 25% multigene editing efficiency. With this system, neutral sites on different chromosomes were characterized, and a deep learning model was developed for gRNA activity prediction, thus providing the corresponding integration efficiency and expression intensity. Subsequently, the tool and platform strains were validated by applying them for the de novo synthesis of (S)-reticuline and (2S)-taxifolin. The developed platform strains and tools helped transform Y. lipolytica into an easy-to-operate model cell factory, similar to Saccharomyces cerevisiae.
{"title":"<i>De Novo</i> Synthesis of Reticuline and Taxifolin Using Re-engineered Homologous Recombination in <i>Yarrowia lipolytica</i>.","authors":"Changtai Zhang, Mengsu Liu, Xinglong Wang, Junyi Cheng, Jinbo Xiang, Mingyu Yue, Yang Ning, Zhengxuan Shao, Chalak Najat Abdullah, Jingwen Zhou","doi":"10.1021/acssynbio.4c00853","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00853","url":null,"abstract":"<p><p><i>Yarrowia lipolytica</i> has been widely engineered as a eukaryotic cell factory to produce various important compounds. However, the difficulty of gene editing and the lack of efficient neutral sites make rewiring of <i>Y. lipolytica</i> metabolism challenging. Herein, a Cas9 system was established to redesign the <i>Y. lipolytica</i> homologous recombination system, which caused a more than 56-fold increase in the HR efficiency. The fusion expression of the hBrex27 sequence in the C-terminus of Cas9 recruited more Rad51 protein, and the engineered Cas9 decreased NHEJ, achieving 85% single-gene positive efficiency and 25% multigene editing efficiency. With this system, neutral sites on different chromosomes were characterized, and a deep learning model was developed for gRNA activity prediction, thus providing the corresponding integration efficiency and expression intensity. Subsequently, the tool and platform strains were validated by applying them for the <i>de novo</i> synthesis of (<i>S</i>)-reticuline and (2<i>S</i>)-taxifolin. The developed platform strains and tools helped transform <i>Y. lipolytica</i> into an easy-to-operate model cell factory, similar to <i>Saccharomyces cerevisiae</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143121666","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 : 2025-01-31DOI: 10.1021/acssynbio.4c00864
Xingyu Liao, Yanyan Li, Shuangyi Li, Long Wen, Xingyi Li, Bin Yu
The continuous advancement of single-cell multimodal omics (scMulti-omics) technologies offers unprecedented opportunities to measure various modalities, including RNA expression, protein abundance, gene perturbation, DNA methylation, and chromatin accessibility at single-cell resolution. These advances hold significant potential for breakthroughs by integrating diverse omics modalities. However, the data generated from different omics layers often face challenges due to high dimensionality, heterogeneity, and sparsity, which can adversely impact the accuracy and efficiency of data integration analyses. To address these challenges, we propose a high-precision analysis method called scMGAT (single-cell multiomics data analysis based on multihead graph attention networks). This method effectively coordinates reliable information across multiomics data sets using a multihead attention mechanism, allowing for better management of the heterogeneous characteristics inherent in scMulti-omics data. We evaluated scMGAT's performance on eight sets of real scMulti-omics data, including samples from both human and mouse. The experimental results demonstrate that scMGAT significantly enhances the quality of multiomics data and improves the accuracy of cell-type annotation compared to state-of-the-art methods. scMGAT is now freely accessible at https://github.com/Xingyu-Liao/scMGAT.
{"title":"Enhanced Integration of Single-Cell Multi-Omics Data Using Graph Attention Networks.","authors":"Xingyu Liao, Yanyan Li, Shuangyi Li, Long Wen, Xingyi Li, Bin Yu","doi":"10.1021/acssynbio.4c00864","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00864","url":null,"abstract":"<p><p>The continuous advancement of single-cell multimodal omics (scMulti-omics) technologies offers unprecedented opportunities to measure various modalities, including RNA expression, protein abundance, gene perturbation, DNA methylation, and chromatin accessibility at single-cell resolution. These advances hold significant potential for breakthroughs by integrating diverse omics modalities. However, the data generated from different omics layers often face challenges due to high dimensionality, heterogeneity, and sparsity, which can adversely impact the accuracy and efficiency of data integration analyses. To address these challenges, we propose a high-precision analysis method called scMGAT (single-cell multiomics data analysis based on multihead graph attention networks). This method effectively coordinates reliable information across multiomics data sets using a multihead attention mechanism, allowing for better management of the heterogeneous characteristics inherent in scMulti-omics data. We evaluated scMGAT's performance on eight sets of real scMulti-omics data, including samples from both human and mouse. The experimental results demonstrate that scMGAT significantly enhances the quality of multiomics data and improves the accuracy of cell-type annotation compared to state-of-the-art methods. scMGAT is now freely accessible at https://github.com/Xingyu-Liao/scMGAT.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143070726","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 : 2025-01-30DOI: 10.1021/acssynbio.4c00672
Randi M Pullen, Stephen R Decker, Venkataramanan Subramanian, Meaghan J Adler, Alexander V Tobias, Matthew Perisin, Christian J Sund, Matthew D Servinsky, Mark T Kozlowski
Fungi, especially filamentous fungi, are a relatively understudied, biotechnologically useful resource with incredible potential for commercial applications. These multicellular eukaryotic organisms have long been exploited for their natural production of useful commodity chemicals and proteins such as enzymes used in starch processing, detergents, food and feed production, pulping and paper making and biofuels production. The ability of filamentous fungi to use a wide range of feedstocks is another key advantage. As chassis organisms, filamentous fungi can express cellular machinery, and metabolic and signal transduction pathways from both prokaryotic and eukaryotic origins. Their genomes abound with novel genetic elements and metabolic processes that can be harnessed for biotechnology applications. Synthetic biology tools are becoming inexpensive, modular, and expansive while systems biology is beginning to provide the level of understanding required to design increasingly complex synthetic systems. This review covers the challenges of working in filamentous fungi and offers a perspective on the approaches needed to exploit fungi as microbial cell factories.
{"title":"Considerations for Domestication of Novel Strains of Filamentous Fungi.","authors":"Randi M Pullen, Stephen R Decker, Venkataramanan Subramanian, Meaghan J Adler, Alexander V Tobias, Matthew Perisin, Christian J Sund, Matthew D Servinsky, Mark T Kozlowski","doi":"10.1021/acssynbio.4c00672","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00672","url":null,"abstract":"<p><p>Fungi, especially filamentous fungi, are a relatively understudied, biotechnologically useful resource with incredible potential for commercial applications. These multicellular eukaryotic organisms have long been exploited for their natural production of useful commodity chemicals and proteins such as enzymes used in starch processing, detergents, food and feed production, pulping and paper making and biofuels production. The ability of filamentous fungi to use a wide range of feedstocks is another key advantage. As chassis organisms, filamentous fungi can express cellular machinery, and metabolic and signal transduction pathways from both prokaryotic and eukaryotic origins. Their genomes abound with novel genetic elements and metabolic processes that can be harnessed for biotechnology applications. Synthetic biology tools are becoming inexpensive, modular, and expansive while systems biology is beginning to provide the level of understanding required to design increasingly complex synthetic systems. This review covers the challenges of working in filamentous fungi and offers a perspective on the approaches needed to exploit fungi as microbial cell factories.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062250","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}