The human gut microbiome,a complex ecosystem,significantly influences host health,impacting crucial aspects such as metabolism and immunity.To enhance our comprehension and control of the molecular mechanisms orchestr...The human gut microbiome,a complex ecosystem,significantly influences host health,impacting crucial aspects such as metabolism and immunity.To enhance our comprehension and control of the molecular mechanisms orchestrating the intricate interplay between gut commensal bacteria and human health,the exploration of genome engineering for gut microbes is a promising frontier.Nevertheless,the complexities and diversities inherent in the gut microbiome pose substantial challenges to the development of effective genome engineering tools for human gut microbes.In this comprehensive review,we provide an overview of the current progress and challenges in genome engineering of human gut commensal bacteria,whether executed in vitro or in situ.A specific focus is directed towards the advancements and prospects in cargo DNA delivery and high-throughput techniques.Additionally,we elucidate the immense potential of genome engineering methods to enhance our understanding of the human gut microbiome and engineer the microorganisms to enhance human health.展开更多
Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine...Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep se- quencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phyloge- nomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epi- plasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.展开更多
High-quality genome-scale metabolic models(GEMs)could play critical roles on rational design of microbial cell factories in the classical Design-Build-Test-Learn cycle of synthetic biology studies.Despite of the const...High-quality genome-scale metabolic models(GEMs)could play critical roles on rational design of microbial cell factories in the classical Design-Build-Test-Learn cycle of synthetic biology studies.Despite of the constant establishment and update of GEMs for model microorganisms such as Escherichia coli and Saccharomyces cerevisiae,high-quality GEMs for non-model industrial microorganisms are still scarce.Zymomonas mobilis subsp.mobilis ZM4 is a non-model ethanologenic microorganism with many excellent industrial characteristics that has been developing as microbial cell factories for biochemical production.Although five GEMs of Z.mobilis have been constructed,these models are either generating ATP incorrectly,or lacking information of plasmid genes,or not providing standard format file.In this study,a high-quality GEM iZM516 of Z.mobilis ZM4 was constructed.The information from the improved genome annotation,literature,datasets of Biolog Phenotype Microarray studies,and recently updated Gene-Protein-Reaction information was combined for the curation of iZM516.Finally,516 genes,1389 reactions,1437 metabolites,and 3 cell compartments are included in iZM516,which also had the highest MEMOTE score of 91%among all published GEMs of Z.mobilis.Cell growth was then predicted by iZM516,which had 79.4%agreement with the experimental results of the substrate utilization.In addition,the potential endogenous succinate synthesis pathway of Z.mobilis ZM4 was proposed through simulation and analysis using iZM516.Furthermore,metabolic engineering strategies to produce succinate and 1,4-butanediol(1,4-BDO)were designed and then simulated under anaerobic condition using iZM516.The results indicated that 1.68 mol/mol succinate and 1.07 mol/mol 1,4-BDO can be achieved through combinational metabolic engineering strategies,which was comparable to that of the model species E.coli.Our study thus not only established a high-quality GEM iZM516 to help understand and design microbial cell factories for economic biochemical production using展开更多
Targeted genotyping is an extremely powerful approach for the detection of known genetic variations that are biologically or clinically important.However,for non-model organisms,large-scale target geno-typing in a cos...Targeted genotyping is an extremely powerful approach for the detection of known genetic variations that are biologically or clinically important.However,for non-model organisms,large-scale target geno-typing in a cost-effective manner remains a major challenge.To address this issue,we present an ultrahigh-multiplex,in-solution probe array-based high-throughput diverse marker genotyping(HD-Marker)approach that is capable of targeted genotyping of up to 86000 loci,with coverage of the whole gene repertoire,in what is a 27-fold and six-fold multiplex increase in comparison with the conventional Illumina GoldenGate and original HD-Marker assays,respectively.We perform extensive analyses of var-ious ultrahigh-multiplex levels of HD-Marker(30 k-plex,56 k-plex,and 86 k-plex)and show the power and excellent performance of the proposed method with an extremely high capture rate(about 96%)and genotyping accuracy(about 96%).With great advantages in terms of cost(as low as 0.0006 USD per geno-type)and high technical flexibility,HD-Marker is a highly efficient and powerful tool with broad appli-cation potential for genetic,ecological,and evolutionary studies of non-model organisms.展开更多
Background:Plateau zokor inhabits in sealed burrows from 2,000 to 4,200 meters at Qinghai-Tibet Plateau.This extreme living env ironment makes it a great model to study animal adaptation to hypoxia,low temperature,and...Background:Plateau zokor inhabits in sealed burrows from 2,000 to 4,200 meters at Qinghai-Tibet Plateau.This extreme living env ironment makes it a great model to study animal adaptation to hypoxia,low temperature,and high carbon dioxide concentration.Methods:We provide an integrated resource,ZokorDB,for tissue specific regulatory network annotation for zokor.ZokorDB is based on a high-quality draft genome of a plateau zokor at 3,300 m and its transcriptional profiles in brain,heart,liver,kidney,and lung.The conserved non-coding elements of zokor are annotated by their nearest genes and upstream transcriptional factor motif binding sites.Results:ZokorDB provides a general draft gene regulatory network(GRN),Le?potential transcription factor(TF)binds to non-coding regulatory elements and regulates the expression of target genes(TG).Furthermore,we refined the GRN by incorporating matched RNA-seq and DNase-seq data from mouse ENCODE project and reconstructed five tissue-specific regulatory networks.Conclusions:A web-based,open-access database is developed for easily searching,visualizing,and downloading the annotation and data.The pipeline of non-coding region annotation for zokor will be useful for other non-model species.ZokorDB is free available at the website(bigd.big.ac.cn/zokordb/).展开更多
基金National Key R&D Program of China(2019YFA0906700)Guangdong Basic and Applied Basic Research Foundation(2020A1515110184)+1 种基金Dr.Neher's Biophysics Laboratory for Innovative Drug Discovery(001/2020/ALC),regular grants(0056/2020/AMJ&0063/2022/A2)from Macao Science and Technology Development Fund2020 Young Qihuang Scholar funded by National Administration of Traditional Chinese Medicine and also financially supported by the Start-up Research Grant of University of Macao(SRG2022-00020-FHS)and the Faculty of Health Sciences,University of Macao.
文摘The human gut microbiome,a complex ecosystem,significantly influences host health,impacting crucial aspects such as metabolism and immunity.To enhance our comprehension and control of the molecular mechanisms orchestrating the intricate interplay between gut commensal bacteria and human health,the exploration of genome engineering for gut microbes is a promising frontier.Nevertheless,the complexities and diversities inherent in the gut microbiome pose substantial challenges to the development of effective genome engineering tools for human gut microbes.In this comprehensive review,we provide an overview of the current progress and challenges in genome engineering of human gut commensal bacteria,whether executed in vitro or in situ.A specific focus is directed towards the advancements and prospects in cargo DNA delivery and high-throughput techniques.Additionally,we elucidate the immense potential of genome engineering methods to enhance our understanding of the human gut microbiome and engineer the microorganisms to enhance human health.
基金This work was supported by the Natural Science Foundation of China （Grant Nos. 31430077, 91131013, and 31272285）. Many thanks are given to Prof. Weibo Song （Ocean University of China）, for his indispensable advice during the preparation of the manuscript.
文摘Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep se- quencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phyloge- nomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epi- plasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.
基金the National Key Technology Research and Development Program of China(2018YFA0900300 and 2022YFA0911800)the National Natural Science Foundation of China(21978071 and U1932141)+3 种基金the Key Science and Technology Innovation Project of Hubei Province(2021BAD001)2022 Joint Projects between Chinese and CEEC’s Universities(202004)the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang Province(2018R01014)the Innovation Base for Introducing Talents of Discipline of Hubei Province(2019BJH021)。
文摘High-quality genome-scale metabolic models(GEMs)could play critical roles on rational design of microbial cell factories in the classical Design-Build-Test-Learn cycle of synthetic biology studies.Despite of the constant establishment and update of GEMs for model microorganisms such as Escherichia coli and Saccharomyces cerevisiae,high-quality GEMs for non-model industrial microorganisms are still scarce.Zymomonas mobilis subsp.mobilis ZM4 is a non-model ethanologenic microorganism with many excellent industrial characteristics that has been developing as microbial cell factories for biochemical production.Although five GEMs of Z.mobilis have been constructed,these models are either generating ATP incorrectly,or lacking information of plasmid genes,or not providing standard format file.In this study,a high-quality GEM iZM516 of Z.mobilis ZM4 was constructed.The information from the improved genome annotation,literature,datasets of Biolog Phenotype Microarray studies,and recently updated Gene-Protein-Reaction information was combined for the curation of iZM516.Finally,516 genes,1389 reactions,1437 metabolites,and 3 cell compartments are included in iZM516,which also had the highest MEMOTE score of 91%among all published GEMs of Z.mobilis.Cell growth was then predicted by iZM516,which had 79.4%agreement with the experimental results of the substrate utilization.In addition,the potential endogenous succinate synthesis pathway of Z.mobilis ZM4 was proposed through simulation and analysis using iZM516.Furthermore,metabolic engineering strategies to produce succinate and 1,4-butanediol(1,4-BDO)were designed and then simulated under anaerobic condition using iZM516.The results indicated that 1.68 mol/mol succinate and 1.07 mol/mol 1,4-BDO can be achieved through combinational metabolic engineering strategies,which was comparable to that of the model species E.coli.Our study thus not only established a high-quality GEM iZM516 to help understand and design microbial cell factories for economic biochemical production using
基金the grant support from National Natural Science Foundation of China (32130107, 32002446 and 32102778)Project of Sanya Yazhouwan Science and Technology City Management Foundation (SKJC-KJ-2019KY01)+1 种基金China Agriculture Research System of MOF and MARATaishan Scholar Project Fund of Shandong Province of China
文摘Targeted genotyping is an extremely powerful approach for the detection of known genetic variations that are biologically or clinically important.However,for non-model organisms,large-scale target geno-typing in a cost-effective manner remains a major challenge.To address this issue,we present an ultrahigh-multiplex,in-solution probe array-based high-throughput diverse marker genotyping(HD-Marker)approach that is capable of targeted genotyping of up to 86000 loci,with coverage of the whole gene repertoire,in what is a 27-fold and six-fold multiplex increase in comparison with the conventional Illumina GoldenGate and original HD-Marker assays,respectively.We perform extensive analyses of var-ious ultrahigh-multiplex levels of HD-Marker(30 k-plex,56 k-plex,and 86 k-plex)and show the power and excellent performance of the proposed method with an extremely high capture rate(about 96%)and genotyping accuracy(about 96%).With great advantages in terms of cost(as low as 0.0006 USD per geno-type)and high technical flexibility,HD-Marker is a highly efficient and powerful tool with broad appli-cation potential for genetic,ecological,and evolutionary studies of non-model organisms.
基金ZokorDB is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDB13000000)The authors are also supported by the National Natural Science Foundation of China(NSFC)(Nos.11871463,11871462,61671444 and 61621003)+1 种基金We thank all the lab members for discussions on data collection,genome alignment,annotation,GRN reconstructionWe thank Dr.Yilei Wu and his group for help on database design and management.
文摘Background:Plateau zokor inhabits in sealed burrows from 2,000 to 4,200 meters at Qinghai-Tibet Plateau.This extreme living env ironment makes it a great model to study animal adaptation to hypoxia,low temperature,and high carbon dioxide concentration.Methods:We provide an integrated resource,ZokorDB,for tissue specific regulatory network annotation for zokor.ZokorDB is based on a high-quality draft genome of a plateau zokor at 3,300 m and its transcriptional profiles in brain,heart,liver,kidney,and lung.The conserved non-coding elements of zokor are annotated by their nearest genes and upstream transcriptional factor motif binding sites.Results:ZokorDB provides a general draft gene regulatory network(GRN),Le?potential transcription factor(TF)binds to non-coding regulatory elements and regulates the expression of target genes(TG).Furthermore,we refined the GRN by incorporating matched RNA-seq and DNase-seq data from mouse ENCODE project and reconstructed five tissue-specific regulatory networks.Conclusions:A web-based,open-access database is developed for easily searching,visualizing,and downloading the annotation and data.The pipeline of non-coding region annotation for zokor will be useful for other non-model species.ZokorDB is free available at the website(bigd.big.ac.cn/zokordb/).
