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A flexible,modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

Synthetic and Systems Biotechnology 2024年第1期9卷 69-77页

作者： Xuejin Zhao Yeqing Zong Qiuli Lou Chenrui Qin Chunbo Lou CAS Key Laboratory of Pathogenic Microbiology and Immunology Institute of MicrobiologyChinese Academy of SciencesBeijing100101China CAS Key Laboratory of Microbial Physiological and Metabolic Engineering Institute of MicrobiologyChinese Academy of SciencesBeijing100101China Center for Cell and gene Circuit Design Key Laboratory of Quantitative Synthetic BiologyShenzhen Institute of Synthetic BiologyShenzhen Institutes of Advanced TechnologyChinese Academy of Sciences1068 Xueyuan AvenueUniversity TownNanshanShenzhen518055China Peking-Tsinghua Joint Center for Life Sciences Peking UniversityBeijing100871China Center for Quantitative Biology Academy for Advanced Interdisciplinary StudiesSchool of PhysicsPeking UniversityBeijing100871China College of Life Sciences University of Chinese Academy of SciencesBeijing100149China

Streptomyces has enormous potential to produce novel natural products(NPs)as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters(BGCs).However,the lack of efficient gene cluster engineering strategies has hampered the pace of new drug ***,we developed an easy-to-use,highly flexible DNA assembly toolkit for gene cluster *** DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick,Golden Gate,CATCH,yeast homologous recombination-based DNA assembly and homing endonuclease-mediated *** compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene *** demonstrate the utility of this toolkit,we quantified a library of modular regulatory parts,and engineered a gene cluster(act)using characterized promoters that led to increased ***,this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.

关键词： Streptomyces Standard toolkit DNA assembly gene cluster Synthetic biology

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Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments

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Science China(Life Sciences) 2017年第9期60卷 968-979页

作者： Jiming Zhuo Binbin Ma Jingjing Xu Weihong Hu Jihui Zhang Huarong Tan Yuqing Tian State Key Laboratory of Microbial Resources Institute of Microbiology Chinese Academy of Sciences Beijing 100101 China University of Chinese Academy of Sciences Beijing 100049 China School of Life Sciences Yunnan University Kunming 650091 China

genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and *** this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson *** this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting *** reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and *** study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.

关键词： large DNA fragment PCR targeting Gibson assembly gene cluster hybrid antibiotic

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Characterization of inthomycin biosynthetic gene cluster revealing new insights into carboxamide formation

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Chinese Journal of Natural Medicines 2020年第9期18卷 677-683页

作者： WANG Yong-Jiang WANG Li HE Xin XU Dong-Dong TANG Jun MA Ya-Tuan YAN Yi-Jun GU Yu-Cheng YANG Jing HUANG Sheng-Xiong State Key Laboratory of Phytochemistry and P/ant Resources in West China and CAS Center for Excellence in Molecular Plant SciencesKunming Institute of BotanyChinese Academy of SciencesKunming 650201.China College of Chemistry and Pharmacy Norhwest Agriculture and Forestry UniversityYangling 712100.China Savaid Medical School University of Chinese Academy of ScienrcesBejing 100049.China Syngenta Jelott's Hill International Research Centre BracknellBerkshire RG426EYU.K.

Inthomycins are polyketide antibiotics which contain a terminal carboxamide group and a triene chain. Inthomycin B(1) and its two new analogues 2 and 3 were isolated from the crude extract of Streptomyces pactum L8. Identification of the gene cluster for inthomycin biosynthesis as well as the ^(15)N-labeled glycine incorporation into inthomycins are described. Combined with the gene deletion of the rare P450 domain in the NRPS module, a formation mechanism of carboxamide moiety in inthomycins was proposed via an oxidative release of the assembly chain assisted by the P450 domain.

关键词： Inthomycins Biosynthesis gene cluster Carboxamide P450

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Heat-inducible SlWRKY3 confers thermotolerance by activating the SlGRXS1 gene cluster in tomato

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Horticultural Plant Journal 2024年第2期10卷 515-531页

作者： Ying Wang Wenxian Gai Liangdan Yuan Lele Shang Fangman Li Zhao Gong Pingfei Ge Yaru Wang Jinbao Tao Xingyu Zhang Haiqiang Dong Yuyang Zhang National Key Laboratory for Germplasm Innovation and Utilization for Fruit and Vegetable Horticultural Crops Huazhong Agricultural UniversityWuhanHubei 430070China Hubei Hongshan Laboratory WuhanHubei 430070China

High temperature stress is one of the major environmental factors that affect the growth and development of plants. Although WRKY transcription factors play a critical role in stress responses, there are few studies on the regulation of heat stress by WRKY transcription factors,especially in tomato. Here, we identified a group I WRKY transcription factor, SlWRKY3, involved in thermotolerance in tomato. First, SlWRKY3 was induced and upregulated under heat stress. Accordingly, overexpression of SlWRKY3 led to an increase, whereas knock-out of SlWRKY3 resulted in decreased tolerance to heat stress. Overexpression of SlWRKY3 accumulated less reactive oxygen species(ROS), whereas knock-out of SlWRKY3 accumulated more ROS under heat stress. This indicated that SlWRKY3 positively regulates heat stress in tomato. In addition,SlWRKY3 activated the expression of a range of abiotic stress-responsive genes involved in ROS scavenging, such as a SlGRXS1 gene *** analysis showed that SlWRKY3 can bind to the promoters of the SlGRXS1 gene cluster and activate their expression. Collectively, these results imply that SlWRKY3 is a positive regulator of thermotolerance through direct binding to the promoters of the SlGRXS1 gene cluster and activating their expression and ROS scavenging.

关键词： Tomato WRKY transcription factor SlWRKY3 Thermotolerance SlGRXS1 gene cluster Abiotic stress

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An Oryza-specific hydroxycinnamoyl tyramine gene cluster contributes to enhanced disease resistance

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Science Bulletin 2021年第23期66卷 2369-2380页

作者： Shuangqian Shen Meng Peng Hong Fang Zixuan Wang Shen Zhou Xinyu Jing Meng Zhang Chenkun Yang Hao Guo Yufei Li Long Lei Yuheng Shi Yangyang Sun Xianqing Liu Congping Xu Takayuki Tohge Meng Yuan Alisdair R.Fernie Yuese Ning Guo-Liang Wang Jie Luo National Key Laboratory of Crop genetic Improvement and National Center of Plant gene Research(Wuhan) Huazhong Agricultural UniversityWuhan 430070China College of Tropical Crops Hainan UniversityHaikou 570228China State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China Department of Plant Pathology Ohio State UniversityColumbus OH 43210USA VIB-UGent Center for Plant Systems Biology Ghent UniversityGhent 9052Belgium Max Planck Institute of Molecular Plant Physiology Potsdam-Golm 14476Germany Center of Plant Systems Biology and Biotechnology Plovdiv 4000Bulgaria Graduate School of Biological Sciences Nara Institute of Science and TechnologyIkoma Nara 630-0192Japan

Genomic clustering of non-homologous genes for the biosynthesis of plant defensive compounds is an emerging theme, but insights into their formation and physiological function remain limited. Here we report the identification of a newly discovered hydroxycinnamoyl tyramine(HT) gene cluster in *** cluster contains a pyridoxamine 50-phosphate oxidase(Os PDX3) producing the cofactor pyridoxal50-phosphate(PLP), a PLP-dependent tyrosine decarboxylase(Os Ty DC1), and two duplicated hydroxycinnamoyl transferases(Os THT1 and Os THT2). These members were combined to represent an enzymological innovation gene cluster. Natural variation analysis showed that the abundance of the toxic tyramine intermediate of the gene cluster among different rice accessions is mainly determined by the coordinated transcription of Os Ty DC1 and Os THT1. Further pathogen incubation assays demonstrated that the end products of the HT gene cluster displayed enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. Oryzae(Xoo) and fungal pathogen Magnaporthe oryzae(M. oryzae), and the enhanced resistance is associated with the boost of phytoalexins and the activation of defense response. The unique presence of the HT gene cluster in Oryza AA genome, together with the enrichment of transposon elements within this gene cluster region, provides an evolutionary background to accelerate cluster member combinations. Our study not only discovered a gene cluster involved in the phenylpropanoid metabolism but also addressed the key aspects of gene cluster formation. In addition, our results provide a new metabolic pool for plant defense against pathogens.

关键词： Rice gene cluster Hydroxycinnamic acid amide Phytoalexin Xanthomonas oryzae pv.oryzae Magnaporthe oryzae Transposon element

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Genomics-driven discovery of pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis

Genomics-driven discovery of pneumocandin biosynthetic gene ...

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2012年中国菌物学会学术年会

作者： Javier Ortiz-López Gerald F.Bills Zhiqaing An Fundación MEDINA Centro de Excelencia en Investigación de Medicamentos Innovadores en AndaluciaGranadaSpain Brown Foundation Institute of Molecular Medicine University of Texas Health Science Center at HoustonHoustonTX 77030USA

The antifungal caspofungin is a semi-synthetic derivative of the lipohexapetide pneumocandin B produced by the fungus Glarea lozoyensis.A whole genome sequencing approach has identified the pneumocandin biosynthetic gene cluster which contains a nonribosomal peptide synthetase(NRPS,GLNRPS4),a polyketide synthase(PKS,GLPKS4),and at least nine other modifying *** mutants of GLNRPS4 or GLPKS4 no longer produce pneumocandin and lose their antibiotic activity against Candida *** additional to pneumocandins,the *** genome reveals a rich repertoire of natural product-encoding gene clusters which include 24 PKSs,6 NRPSs,5 PKS-NRPS hybrids,2 dimethylallyltryptophan synthases,and 3 terpene *** of the gene cluster provides a blueprint for bioengineering novel pneumocandin derivatives with more desirable pharmacological properties such as oral *** genome estimation of secondary metabolite-encoding genes from *** illustrates the huge potential for natural products drug discovery from the vast fungal kingdom.

关键词： Caspofungin pneumocandin Glarea lozoyensis genome gene cluster

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Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9

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Journal of Molecular Cell Biology 2015年第4期7卷 284-298页

作者： Jinhuan Li Jia Shou YaGuo Yuanxiao Tang YonghuWu Zhilian Jia Yanan Zhai Zhifeng Chen Quan Xu Qiang Wu Key Laboratory of Systems Biomedicine(Ministry of Education) Center for Comparative BiomedicineInstitute of Systems BiomedicineShanghai Jiao Tong UniversityShanghai 200240China State Key Laboratory of Oncogenes and Related genes Shanghai Cancer InstituteRenji HospitalSchool of MedicineShanghai Jiao Tong UniversityShanghai 200240China Key Laboratory for the genetics of Developmental and Neuropsychiatric Disorders Ministry of EducationBio-X CenterSchool of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghai 200240China Collaborative Innovation Center of Systems Biomedicine Shanghai Jiao Tong University School of MedicineShanghai 200240China

The human genome contains millions of DNA regulatory elements and a large number of gene clusters,most of which have not been tested *** clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)programed with a synthetic single-guide RNA(sgRNA)emerges as a method for genome editing in virtually any *** we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two ***,we found that,in cultured human cells and mice,efficient precise inversions of DNA fragments ranging in size froma few tens of bp to hundreds of kb could be *** addition,DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks(DSBs)on two homologous chromosomes(chromatids).Moreover,junctions of combinatorial inversions and duplications of the protocadherin(Pcdh)gene clusters induced by Cas9 with four sgRNAs could be *** mice,we obtained founders with alleles of precise inversions,duplications,and deletions of DNA fragments of variable sizes by ***,we found that very efficient inversions were mediated by microhomology-mediated end joining(MMEJ)through short inverted *** showed for the first time that DNA fragment inversions could be transmitted through germlines in ***,we applied this CRISPR method to a regulatory element of the Pcdha cluster and found a new role in the regulation of members of the Pcdhg *** simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.

关键词： DNA regulatory element inversion duplication deletion CRISPR/Cas9 enhancer genome manipulation gene cluster

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Two gene clusters and their positive regulator SlMYB13 that have undergone domestication-associated negative selection control phenolamide accumulation and drought tolerance in tomato

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Molecular Plant 2024年第4期17卷 579-597页

作者： Peng Cao Jun Yang Linghao Xia Zhonghui Zhang Zeyong Wu Yingchen Hao Penghui Liu Chao Wang Chun Li Jie Yang Jun Lai Xianggui Li Meng Deng Shouchuang Wang School of Breeding and Multiplication(Sanya Institute of Breeding and Multiplication) Hainan UniversitySanya 572025China College of Tropical Agriculture and Forestry Hainan UniversityHaikou 572208China Collaborative Innovation Center of Nanfan and High-Efficiency Tropical Agriculture Hainan UniversityHaikou 572208China

Among plant metabolites,phenolamides,which are conjugates of hydroxycinnamic acid derivatives and polyamines,play important roles in plant adaptation to abiotic and biotic ***,the molecular mechanisms underlying phenolamide metabolism and regulation as well as the effects of domestication and breeding on phenolamide diversity in tomato remain largely *** this study,we performed a metabolite-based genome-wide association study and identified two biosynthetic gene clusters(BGC7 and BGC11)containing 12 genes involved in phenolamide metabolism,including four biosynthesis genes(two 4CL genes,one C3H gene,and one CPA gene),seven decoration genes(five AT genes and two UGT genes),and one transport protein gene(DTX29).Using gene co-expression network analysis we further discovered that SlMYB13 positively regulates the expression of two gene clusters,thereby promoting phenolamide *** and physiological analyses showed that BGC7,BGC11 and SlMYB13 enhance drought tolerance by enhancing scavenging of reactive oxygen species and increasing abscisic acid content in *** variation analysis suggested that BGC7,BGC11 and SlMYB13 were negatively selected during tomato domestication and improvement,leading to reduced phenolamide content and drought tolerance of cultivated ***,our study discovers a key mechanism of phenolamide biosynthesis and regulation in tomato and reveals that crop domestication and improvement shapes metabolic diversity to affect plant environmental adaptation.

关键词： tomato phenolamides gene cluster drought tolerance natural variation evolution

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Assembly and features of secondary metabolite biosynthetic gene clusters in Streptomyces ansochromogenes

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Science China(Life Sciences) 2013年第7期56卷 609-618页

作者： ZHONG XingYu TIAN YuQing NIU GuoQing TAN HuaRong State Key Laboratory of Microbial Resources Institute of Microbiology Chinese Academy of Sciences University of Chinese Academy of Sciences

A draft genome sequence of Streptomyces ansochromogenes 7100 was generated using 454 sequencing technology. In combination with local BLAST searches and gap filling techniques, a comprehensive antiSMASH-based method was adopted to assemble the secondary metabolite biosynthetic gene clusters in the draft genome of S. ansochromogenes. A total of at least 35 putative gene clusters were identified and assembled. Transcriptional analysis showed that 20 of the 35 gene clusters were expressed in either or all of the three different media tested, whereas the other 15 gene clusters were silent in all three different media. This study provides a comprehensive method to identify and assemble secondary metabolite biosynthetic gene clusters in draft genomes of Streptomyces, and will significantly promote functional studies of these secondary metabolite biosynthetic gene clusters.

关键词： secondary metabolite gene cluster assemble features draft genome

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Involvement of clustered oyster Wnt genes in gut formation

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Journal of Oceanology and Limnology 2018年第5期36卷 1746-1752页

作者： LIU Jun XU Fei JI Peng LI Li ZHANG Guofan Key Laboratory of Experimental Marine Biology Institute of OceanologyChinese Academy of SciencesQingdao 266071China University of Chinese Academy of Sciences Beijing 100049China Laboratory for Marine Biology and Biotechnology Qingdao National Laboratory for Marine Science and TechnologyQingdao 266000China National ＆ Local Joint Engineering Laboratory of Ecological Mariculture Institute of OceanologyChinese Academy of SciencesQingdao 266071China Laboratory for Marine Fisheries and Aquaculture Qingdao National Laboratory for Marine Science and TechnologyQingdao 266000China

genes encoding Wnt ligands play important roles in organ development. The Wnt10-Wnt6-Wnt1-Wnt9 cluster widely presents in many metazoan genomes, indicating the importance of gene arrangement. Hypothesis has been proposed that they may be coordinately regulated. However, few expression correlations were identified in model animals. We analyzed the tissue expression pattern of clustered oyster Wnt10, Wnt6, Wnt1, and Wnt9 a genes in this study. The results indicated the highest expression level in adult gut system of these clustered W nt genes, except for Wnt6, which had highest expression in mantle. Further whole-mount immunofluorescence assay indicated that Wnt6 protein was restricted to gut region in oyster larvae. These results suggest the possible important role of the W nt10-Wnt6-Wnt1-Wnt9 cluster in oyster gut formation.

关键词： gene cluster tissue distribution digestive gland immunofluorescence Crassostrea gigas

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