Pub Date : 2022-08-19eCollection Date: 2022-01-01DOI: 10.34133/2022/9802168
Ryan R Cochrane, Arina Shrestha, Mariana M Severo de Almeida, Michelle Agyare-Tabbi, Stephanie L Brumwell, Samir Hamadache, Jordyn S Meaney, Daniel P Nucifora, Henry Heng Say, Jehoshua Sharma, Maximillian P M Soltysiak, Cheryl Tong, Katherine Van Belois, Emma J L Walker, Marc-André Lachance, Gregory B Gloor, David R Edgell, Rebecca S Shapiro, Bogumil J Karas
Fungi are nature's recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals. Still, the great untapped potential exists within the diverse fungal kingdom. However, many yeasts are intractable, preventing their use in biotechnology or in the development of novel treatments for pathogenic fungi. Therefore, as a first step for the domestication of new fungi, an efficient DNA delivery method needs to be developed. Here, we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen Candida auris. To create our superior plasmids, derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions, as well as some unintentional mutations. Specifically, a cluster mutation in the promoter of the conjugative gene traJ had the most significant effect on improving conjugation to yeasts. In addition, we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic cassettes. Finally, we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent, with important applications for the development of next-generation antifungal therapeutics.
{"title":"Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi.","authors":"Ryan R Cochrane, Arina Shrestha, Mariana M Severo de Almeida, Michelle Agyare-Tabbi, Stephanie L Brumwell, Samir Hamadache, Jordyn S Meaney, Daniel P Nucifora, Henry Heng Say, Jehoshua Sharma, Maximillian P M Soltysiak, Cheryl Tong, Katherine Van Belois, Emma J L Walker, Marc-André Lachance, Gregory B Gloor, David R Edgell, Rebecca S Shapiro, Bogumil J Karas","doi":"10.34133/2022/9802168","DOIUrl":"10.34133/2022/9802168","url":null,"abstract":"<p><p>Fungi are nature's recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals. Still, the great untapped potential exists within the diverse fungal kingdom. However, many yeasts are intractable, preventing their use in biotechnology or in the development of novel treatments for pathogenic fungi. Therefore, as a first step for the domestication of new fungi, an efficient DNA delivery method needs to be developed. Here, we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen <i>Candida auris</i>. To create our superior plasmids, derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions, as well as some unintentional mutations. Specifically, a cluster mutation in the promoter of the conjugative gene <i>traJ</i> had the most significant effect on improving conjugation to yeasts. In addition, we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic cassettes. Finally, we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent, with important applications for the development of next-generation antifungal therapeutics.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521675/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241381","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-08-09eCollection Date: 2022-01-01DOI: 10.34133/2022/9819272
Ernesto Segredo-Otero, Rafael Sanjuán
Despite extensive evidence of virus-virus interactions, not much is known about their biological significance. Importantly, virus-virus interactions could have evolved as a form of cooperation or simply be a by-product of other processes. Here, we review and discuss different types of virus-virus interactions from the point of view of social evolution, which provides a well-established framework for interpreting the fitness costs and benefits of such traits. We also classify interactions according to their mechanisms of action and speculate on their evolutionary implications. As in any other biological system, the evolutionary stability of viral cooperation critically requires cheaters to be excluded from cooperative interactions. We discuss how cheater viruses exploit cooperative traits and how viral populations are able to counteract this maladaptive process.
{"title":"Cooperative Virus-Virus Interactions: An Evolutionary Perspective.","authors":"Ernesto Segredo-Otero, Rafael Sanjuán","doi":"10.34133/2022/9819272","DOIUrl":"https://doi.org/10.34133/2022/9819272","url":null,"abstract":"<p><p>Despite extensive evidence of virus-virus interactions, not much is known about their biological significance. Importantly, virus-virus interactions could have evolved as a form of cooperation or simply be a by-product of other processes. Here, we review and discuss different types of virus-virus interactions from the point of view of social evolution, which provides a well-established framework for interpreting the fitness costs and benefits of such traits. We also classify interactions according to their mechanisms of action and speculate on their evolutionary implications. As in any other biological system, the evolutionary stability of viral cooperation critically requires cheaters to be excluded from cooperative interactions. We discuss how cheater viruses exploit cooperative traits and how viral populations are able to counteract this maladaptive process.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521650/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-08-06eCollection Date: 2022-01-01DOI: 10.34133/2022/9853416
Michael Melesse Vergara, Jesse Labbé, Joanna Tannous
Ongoing pest and disease outbreaks pose a serious threat to human, crop, and animal lives, emphasizing the need for constant genetic discoveries that could serve as mitigation strategies. Gene drives are genetic engineering approaches discovered decades ago that may allow quick, super-Mendelian dissemination of genetic modifications in wild populations, offering hopes for medicine, agriculture, and ecology in combating diseases. Following its first discovery, several naturally occurring selfish genetic elements were identified and several gene drive mechanisms that could attain relatively high threshold population replacement have been proposed. This review provides a comprehensive overview of the recent advances in gene drive research with a particular emphasis on CRISPR-Cas gene drives, the technology that has revolutionized the process of genome engineering. Herein, we discuss the benefits and caveats of this technology and place it within the context of natural gene drives discovered to date and various synthetic drives engineered. Later, we elaborate on the strategies for designing synthetic drive systems to address resistance issues and prevent them from altering the entire wild populations. Lastly, we highlight the major applications of synthetic CRISPR-based gene drives in different living organisms, including plants, animals, and microorganisms.
{"title":"Reflection on the Challenges, Accomplishments, and New Frontiers of Gene Drives.","authors":"Michael Melesse Vergara, Jesse Labbé, Joanna Tannous","doi":"10.34133/2022/9853416","DOIUrl":"https://doi.org/10.34133/2022/9853416","url":null,"abstract":"<p><p>Ongoing pest and disease outbreaks pose a serious threat to human, crop, and animal lives, emphasizing the need for constant genetic discoveries that could serve as mitigation strategies. Gene drives are genetic engineering approaches discovered decades ago that may allow quick, super-Mendelian dissemination of genetic modifications in wild populations, offering hopes for medicine, agriculture, and ecology in combating diseases. Following its first discovery, several naturally occurring selfish genetic elements were identified and several gene drive mechanisms that could attain relatively high threshold population replacement have been proposed. This review provides a comprehensive overview of the recent advances in gene drive research with a particular emphasis on CRISPR-Cas gene drives, the technology that has revolutionized the process of genome engineering. Herein, we discuss the benefits and caveats of this technology and place it within the context of natural gene drives discovered to date and various synthetic drives engineered. Later, we elaborate on the strategies for designing synthetic drive systems to address resistance issues and prevent them from altering the entire wild populations. Lastly, we highlight the major applications of synthetic CRISPR-based gene drives in different living organisms, including plants, animals, and microorganisms.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521683/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241378","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-08-02eCollection Date: 2022-01-01DOI: 10.34133/2022/9850305
Tomás Aparicio, Jillian Silbert, Sherezade Cepeda, Víctor de Lorenzo
The promiscuous conjugation machinery of the Gram-negative plasmid RP4 has been reassembled in a minimized, highly transmissible vector for propagating genetically encoded traits through diverse types of naturally occurring microbial communities. To this end, the whole of the RP4-encoded transfer determinants (tra, mob genes, and origin of transfer oriT) was excised from their natural context, minimized, and recreated in the form of a streamlined DNA segment borne by an autoselective replicon. The resulting constructs (the pMATING series) could be self-transferred through a variety of prokaryotic and eukaryotic recipients employing such a rationally designed conjugal delivery device. Insertion of GFP reporter into pMATING exposed the value of this genetic tool for delivering heterologous genes to both specific mating partners and complex consortia (e.g., plant/soil rhizosphere). The results accredited the effective and functional transfer of the recombinant plasmids to a diversity of hosts. Yet the inspection of factors that limit interspecies DNA transfer in such scenarios uncovered type VI secretion systems as one of the factual barriers that check otherwise high conjugal frequencies of tested RP4 derivatives. We argue that the hereby presented programming of hyperpromiscuous gene transfer can become a phenomenal asset for the propagation of beneficial traits through various scales of the environmental microbiome.
{"title":"Propagation of Recombinant Genes through Complex Microbiomes with Synthetic Mini-RP4 Plasmid Vectors.","authors":"Tomás Aparicio, Jillian Silbert, Sherezade Cepeda, Víctor de Lorenzo","doi":"10.34133/2022/9850305","DOIUrl":"10.34133/2022/9850305","url":null,"abstract":"<p><p>The promiscuous conjugation machinery of the Gram-negative plasmid RP4 has been reassembled in a minimized, highly transmissible vector for propagating genetically encoded traits through diverse types of naturally occurring microbial communities. To this end, the whole of the RP4-encoded transfer determinants (<i>tra</i>, <i>mob</i> genes, and origin of transfer <i>oriT</i>) was excised from their natural context, minimized, and recreated in the form of a streamlined DNA segment borne by an autoselective replicon. The resulting constructs (the pMATING series) could be self-transferred through a variety of prokaryotic and eukaryotic recipients employing such a rationally designed conjugal delivery device. Insertion of GFP reporter into pMATING exposed the value of this genetic tool for delivering heterologous genes to both specific mating partners and complex consortia (e.g., plant/soil rhizosphere). The results accredited the effective and functional transfer of the recombinant plasmids to a diversity of hosts. Yet the inspection of factors that limit interspecies DNA transfer in such scenarios uncovered type VI secretion systems as one of the factual barriers that check otherwise high conjugal frequencies of tested RP4 derivatives. We argue that the hereby presented programming of hyperpromiscuous gene transfer can become a phenomenal asset for the propagation of beneficial traits through various scales of the environmental microbiome.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521647/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Camelid single-domain antibody fragments (nanobodies) are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent years. Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting the large-scale application of nanobodies. In this study, we developed three kinds of gene designated-region pan-editing (GDP) technologies to introduce multiple mutations in complementarity-determining regions (CDRs) of nanobodies in vitro. Including the integration of G-quadruplex fragments in CDRs, which induces the spontaneous multiple mutations in CDRs; however, these mutant sequences are highly similar, resulting in a lack of sequences diversity in the CDRs. We also used CDR-targeting traditional gRNA-guided base-editors, which effectively diversify the CDRs. And most importantly, we developed the self-assembling gRNAs, which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as crRNAs. Using base-editors guided by self-assembling gRNAs, we can realize the iteratively diversify the CDRs. And we believe the last GDP technology is highly promising in immunization-free nanobody library construction, and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies in vitro.
{"title":"<i>In Vitro</i> Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology.","authors":"Zhiyuan Niu, Zhixia Luo, Pengyang Sun, Linwei Ning, Xinru Jin, Guanxu Chen, Changjiang Guo, Lingtong Zhi, Wei Chang, Wuling Zhu","doi":"10.34133/2022/9823578","DOIUrl":"10.34133/2022/9823578","url":null,"abstract":"<p><p>Camelid single-domain antibody fragments (nanobodies) are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent years. Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting the large-scale application of nanobodies. In this study, we developed three kinds of gene designated-region pan-editing (GDP) technologies to introduce multiple mutations in complementarity-determining regions (CDRs) of nanobodies <i>in vitro</i>. Including the integration of G-quadruplex fragments in CDRs, which induces the spontaneous multiple mutations in CDRs; however, these mutant sequences are highly similar, resulting in a lack of sequences diversity in the CDRs. We also used CDR-targeting traditional gRNA-guided base-editors, which effectively diversify the CDRs. And most importantly, we developed the self-assembling gRNAs, which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as crRNAs. Using base-editors guided by self-assembling gRNAs, we can realize the iteratively diversify the CDRs. And we believe the last GDP technology is highly promising in immunization-free nanobody library construction, and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies <i>in vitro</i>.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521727/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-07-01eCollection Date: 2022-01-01DOI: 10.34133/2022/9806749
Guowei Li, Xinlei Wei, Ranran Wu, Wei Zhou, Yunjie Li, Zhiguang Zhu, Chun You
Maltose is a natural α-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used for in vitro biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP) yields β-glucose 1-phosphate (β-G1P) that cannot be utilized by α-phosphoglucomutase (α-PGM) commonly found in in vitro synthetic enzymatic biosystems previously constructed by our group. Herein, we designed an in vitro synthetic enzymatic reaction module comprised of MP, β-phosphoglucomutase (β-PGM), and polyphosphate glucokinase (PPGK) for the stoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this synthetic module, we further constructed two in vitro synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP), respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6 mW/cm2, whereas the 5-enzyme in vitro FDP-producing biosystem yielded 187.0 mM FDP from 50 g/L (139 mM) maltose by adopting a fed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novel strategies for the high-efficient production of bioelectricity and value-added biochemicals.
麦芽糖是一种天然的α-(1,4)连接二糖,在食品工业和微生物发酵中有着广泛的应用。然而,麦芽糖几乎没有被用于体外生物合成,可能是因为它被麦芽糖磷酸化酶(MP)磷酸化产生的β-葡萄糖1-磷酸(β-G1P)不能被我们小组先前构建的体外合成酶生物系统中常见的α-磷酸葡糖变位酶(α-PGM)利用。在此,我们设计了一个由MP、β-磷酸葡萄糖变位酶(β-PGM)和多磷酸葡萄糖激酶(PPGK)组成的体外合成酶促反应模块,用于将每个麦芽糖分子化学计量转化为两个葡萄糖-6-磷酸(G6P)分子。在这个合成模块的基础上,我们进一步构建了两个体外合成生物系统,分别产生生物电和1,6-二磷酸果糖(FDP)。14酶生物电池实现了96.4%的法拉第效率和0.6的最大功率密度 mW/cm2,而5-酶体外产生FDP的生物系统产生187.0 mM FDP,50 g/L(139 mM)麦芽糖。我们的研究不仅提出了麦芽糖的新应用场景,还为高效生产生物电和增值生物化学品提供了新的策略。
{"title":"Stoichiometric Conversion of Maltose for Biomanufacturing by <i>In Vitro</i> Synthetic Enzymatic Biosystems.","authors":"Guowei Li, Xinlei Wei, Ranran Wu, Wei Zhou, Yunjie Li, Zhiguang Zhu, Chun You","doi":"10.34133/2022/9806749","DOIUrl":"10.34133/2022/9806749","url":null,"abstract":"<p><p>Maltose is a natural <i>α</i>-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used for <i>in vitro</i> biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP) yields <i>β</i>-glucose 1-phosphate (<i>β</i>-G1P) that cannot be utilized by <i>α</i>-phosphoglucomutase (<i>α</i>-PGM) commonly found in <i>in vitro</i> synthetic enzymatic biosystems previously constructed by our group. Herein, we designed an <i>in vitro</i> synthetic enzymatic reaction module comprised of MP, <i>β</i>-phosphoglucomutase (<i>β</i>-PGM), and polyphosphate glucokinase (PPGK) for the stoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this synthetic module, we further constructed two <i>in vitro</i> synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP), respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6 mW/cm<sup>2</sup>, whereas the 5-enzyme <i>in vitro</i> FDP-producing biosystem yielded 187.0 mM FDP from 50 g/L (139 mM) maltose by adopting a fed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novel strategies for the high-efficient production of bioelectricity and value-added biochemicals.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521662/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-06-15eCollection Date: 2022-01-01DOI: 10.34133/2022/9898461
Zhenkun Shi, Pi Liu, Xiaoping Liao, Zhitao Mao, Jianqi Zhang, Qinhong Wang, Jibin Sun, Hongwu Ma, Yanhe Ma
Revolutionary breakthroughs in artificial intelligence (AI) and machine learning (ML) have had a profound impact on a wide range of scientific disciplines, including the development of artificial cell factories for biomanufacturing. In this paper, we review the latest studies on the application of data-driven methods for the design of new proteins, pathways, and strains. We first briefly introduce the various types of data and databases relevant to industrial biomanufacturing, which are the basis for data-driven research. Different types of algorithms, including traditional ML and more recent deep learning methods, are also presented. We then demonstrate how these data-based approaches can be applied to address various issues in cell factory development using examples from recent studies, including the prediction of protein function, improvement of metabolic models, and estimation of missing kinetic parameters, design of non-natural biosynthesis pathways, and pathway optimization. In the last section, we discuss the current limitations of these data-driven approaches and propose that data-driven methods should be integrated with mechanistic models to complement each other and facilitate the development of synthetic strains for industrial biomanufacturing.
{"title":"Data-Driven Synthetic Cell Factories Development for Industrial Biomanufacturing.","authors":"Zhenkun Shi, Pi Liu, Xiaoping Liao, Zhitao Mao, Jianqi Zhang, Qinhong Wang, Jibin Sun, Hongwu Ma, Yanhe Ma","doi":"10.34133/2022/9898461","DOIUrl":"https://doi.org/10.34133/2022/9898461","url":null,"abstract":"<p><p>Revolutionary breakthroughs in artificial intelligence (AI) and machine learning (ML) have had a profound impact on a wide range of scientific disciplines, including the development of artificial cell factories for biomanufacturing. In this paper, we review the latest studies on the application of data-driven methods for the design of new proteins, pathways, and strains. We first briefly introduce the various types of data and databases relevant to industrial biomanufacturing, which are the basis for data-driven research. Different types of algorithms, including traditional ML and more recent deep learning methods, are also presented. We then demonstrate how these data-based approaches can be applied to address various issues in cell factory development using examples from recent studies, including the prediction of protein function, improvement of metabolic models, and estimation of missing kinetic parameters, design of non-natural biosynthesis pathways, and pathway optimization. In the last section, we discuss the current limitations of these data-driven approaches and propose that data-driven methods should be integrated with mechanistic models to complement each other and facilitate the development of synthetic strains for industrial biomanufacturing.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521697/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-06-15eCollection Date: 2022-01-01DOI: 10.34133/2022/9871087
Xiaodong Lv, Haijie Xue, Lei Qin, Chun Li
Microbial cell factories (MCFs) are typical and widely used platforms in biomanufacturing for designing and constructing synthesis pathways of target compounds in microorganisms. In MCFs, transporter engineering is especially significant for improving the biomanufacturing efficiency and capacity through enhancing substrate absorption, promoting intracellular mass transfer of intermediate metabolites, and improving transmembrane export of target products. This review discusses the current methods and strategies of mining and characterizing suitable transporters and presents the cases of transporter engineering in the production of various chemicals in MCFs.
{"title":"Transporter Engineering in Microbial Cell Factory Boosts Biomanufacturing Capacity.","authors":"Xiaodong Lv, Haijie Xue, Lei Qin, Chun Li","doi":"10.34133/2022/9871087","DOIUrl":"https://doi.org/10.34133/2022/9871087","url":null,"abstract":"<p><p>Microbial cell factories (MCFs) are typical and widely used platforms in biomanufacturing for designing and constructing synthesis pathways of target compounds in microorganisms. In MCFs, transporter engineering is especially significant for improving the biomanufacturing efficiency and capacity through enhancing substrate absorption, promoting intracellular mass transfer of intermediate metabolites, and improving transmembrane export of target products. This review discusses the current methods and strategies of mining and characterizing suitable transporters and presents the cases of transporter engineering in the production of various chemicals in MCFs.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521751/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-06-06eCollection Date: 2022-01-01DOI: 10.34133/2022/9896125
Xian Fu, Yijian Huang, Yue Shen
The site-specific incorporation of the noncanonical amino acid (ncAA) into proteins via genetic code expansion (GCE) has enabled the development of new and powerful ways to learn, regulate, and evolve biological functions in vivo. However, cellular biosynthesis of ncAA-containing proteins with high efficiency and fidelity is a formidable challenge. In this review, we summarize up-to-date progress towards improving the efficiency and orthogonality of GCE and enhancing intracellular compatibility of introduced translation machinery in the living cells by creation and optimization of orthogonal translation components, constructing genomically recoded organism (GRO), utilization of unnatural base pairs (UBP) and quadruplet codons (four-base codons), and spatial separation of orthogonal translation.
{"title":"Improving the Efficiency and Orthogonality of Genetic Code Expansion.","authors":"Xian Fu, Yijian Huang, Yue Shen","doi":"10.34133/2022/9896125","DOIUrl":"10.34133/2022/9896125","url":null,"abstract":"<p><p>The site-specific incorporation of the noncanonical amino acid (ncAA) into proteins via genetic code expansion (GCE) has enabled the development of new and powerful ways to learn, regulate, and evolve biological functions <i>in vivo</i>. However, cellular biosynthesis of ncAA-containing proteins with high efficiency and fidelity is a formidable challenge. In this review, we summarize up-to-date progress towards improving the efficiency and orthogonality of GCE and enhancing intracellular compatibility of introduced translation machinery in the living cells by creation and optimization of orthogonal translation components, constructing genomically recoded organism (GRO), utilization of unnatural base pairs (UBP) and quadruplet codons (four-base codons), and spatial separation of orthogonal translation.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521639/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241373","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-05-26eCollection Date: 2022-01-01DOI: 10.34133/2022/9834989
Chenfei Tian, Yixin Zhang, Jianhua Li, Yong Wang
The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolites or therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitative characterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used N. benthamiana leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expression of firefly/Renilla luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporter protein and mRNA levels. In addition, we investigated the effects of terminator-promoter combinations on gene expression and found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest and weakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterized promoters to engineer the betalain pathway in N. benthamiana. Seven selected plant promoters with different expression strengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0-362.4 μg/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequences in N. benthamiana and BY2 cells but also adds to the toolbox of plant promoters for plant engineering.
{"title":"Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research.","authors":"Chenfei Tian, Yixin Zhang, Jianhua Li, Yong Wang","doi":"10.34133/2022/9834989","DOIUrl":"https://doi.org/10.34133/2022/9834989","url":null,"abstract":"<p><p>The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolites or therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitative characterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used <i>N. benthamiana</i> leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expression of firefly/<i>Renilla</i> luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporter protein and mRNA levels. In addition, we investigated the effects of terminator-promoter combinations on gene expression and found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest and weakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterized promoters to engineer the betalain pathway in <i>N. benthamiana</i>. Seven selected plant promoters with different expression strengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0-362.4 <i>μ</i>g/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequences in <i>N. benthamiana</i> and BY2 cells but also adds to the toolbox of plant promoters for plant engineering.</p>","PeriodicalId":56832,"journal":{"name":"生物设计研究(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10521690/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41241351","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}