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Crispr streptomyces

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https://read.qxmd.com/read/30746093/triggering-the-expression-of-a-silent-gene-cluster-from-genetically-intractable-bacteria-results-in-scleric-acid-discovery
#1
Fabrizio Alberti, Daniel J Leng, Ina Wilkening, Lijiang Song, Manuela Tosin, Christophe Corre
In this study, we report the rapid characterisation of a novel microbial natural product resulting from the rational derepression of a silent gene cluster. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable Streptomyces sclerotialus bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-l-proline, named scleric acid...
January 14, 2019: Chemical Science
https://read.qxmd.com/read/30633974/phage-serine-integrase-mediated-genome-engineering-for-efficient-expression-of-chemical-biosynthetic-pathway-in-gas-fermenting-clostridium-ljungdahlii
#2
He Huang, Changsheng Chai, Sheng Yang, Weihong Jiang, Yang Gu
The real value of gas-fermenting clostridia, capable of using CO and CO2 , resides in their potential of being developed into cell factories to produce various bulk chemicals and fuels. This process requires rapid chromosomal integration of heterologous chemical biosynthetic pathways, which is impeded by the absence of genetic tools competent for efficient genome engineering in these anaerobes. Here, we developed a phage serine integrase-mediated site-specific genome engineering technique in Clostridium ljungdahlii, one of the major acetogenic gas-fermenting microbes...
January 8, 2019: Metabolic Engineering
https://read.qxmd.com/read/30548045/crispr-cas-based-genome-engineering-in-natural-product-discovery
#3
REVIEW
Yaojun Tong, Tilmann Weber, Sang Yup Lee
Covering: up to February, 2018This review briefly introduces and summarizes current knowledge about the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) - CRISPR/Cas system and how it was engineered to become one of the most important and versatile genome editing techniques that are currently revolutionizing the whole field of molecular biology. It aims to highlight and discuss the applications and remaining challenges of CRISPR/Cas (mainly focusing on CRISPR/SpCas9)-based genome editing in natural product discovery...
December 14, 2018: Natural Product Reports
https://read.qxmd.com/read/30191290/the-psg5-based-thermosensitive-vector-family-for-genome-editing-and-gene-expression-in-actinomycetes
#4
REVIEW
G√ľnther Muth
Actinomycetes are the most important producers of secondary metabolites for medical, agricultural and industrial applications. Efficient engineering of bacterial genomes to improve their biosynthetic capabilities largely depends on the available arsenal of tools and vectors. One of the most widely used vector systems for actinomycetes is derived from the Streptomyces ghanaensis DSM2932 plasmid pSG5. pSG5 is a broad host range multicopy plasmid replicating via a rolling circle mechanism. The unique feature of pSG5, which distinguishes it from other Streptomyces plasmids, is its naturally thermosensitive mode of replication...
November 2018: Applied Microbiology and Biotechnology
https://read.qxmd.com/read/30087666/crispr-cas9-based-editing-of-streptomyces-for-discovery-characterization-and-production-of-natural-products
#5
REVIEW
Weixin Tao, Anna Yang, Zixin Deng, Yuhui Sun
Microbial natural products (NPs) especially of the Streptomyces genus have been regarded as an unparalleled resource for pharmaceutical drugs discovery. Moreover, recent progress in sequencing technologies and computational resources further reinforces to identify numerous NP biosynthetic gene clusters (BGCs) from the genomes of Streptomyces . However, the majority of these BGCs are silent or poorly expressed in native strains and remain to be activated and investigated, which relies heavily on efficient genome editing approaches...
2018: Frontiers in Microbiology
https://read.qxmd.com/read/30030786/comparative-analysis-of-crispr-loci-found-in-streptomyces-genome-sequences
#6
Jinqi Zhang, Xiaobin Li, Zixin Deng, Hong-Yu Ou
The interspaced short palindromic repeats (CRISPR) system is an immune system widely distributed in prokaryotes, resisting the invasion of the foreign mobile genetic elements like phages or plasmids. In this study, we present the comparative analysis of 182 CRISPR loci found in 46 publicly available complete genome sequences of Streptomyces. Overall, nine direct repeats (DRs) groups are identified while all the 2104 spacers are divided into three main groups according to the multiple sequence alignment. Only 11 spacers are identical with parts of 10 plasmid sequences, which indicates a possible origin...
July 20, 2018: Interdisciplinary Sciences, Computational Life Sciences
https://read.qxmd.com/read/29980561/crispr-cpf1-assisted-multiplex-genome-editing-and-transcriptional-repression-in-streptomyces
#7
Lei Li, Keke Wei, Guosong Zheng, Xiaocao Liu, Shaoxin Chen, Weihong Jiang, Yinhua Lu
Streptomyces has a strong capability for producing a large number of bioactive natural products and remains invaluable as a source for the discovery of novel drug leads. Although the Streptococcus pyogenes CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in Streptomyces , it has a number of limitations, including the toxicity of Sp Cas9 expression in some important industrial Streptomyces strains and the need for complex expression constructs when targeting multiple genomic loci...
September 15, 2018: Applied and Environmental Microbiology
https://read.qxmd.com/read/29862648/crispr-dcas9-mediated-multiplex-gene-repression-in-streptomyces
#8
Yawei Zhao, Lei Li, Guosong Zheng, Weihong Jiang, Zixin Deng, Zhijun Wang, Yinhua Lu
Streptomycetes are Gram-positive bacteria with the capacity to produce copious bioactive secondary metabolites, which are the main source of medically and industrially relevant drugs. However, genetic manipulation of Streptomyces strains is much more difficult than other model microorganisms like Escherichia coli and Saccharomyces cerevisiae. Recently, CRISPR/Cas9 or dCas9-mediated genetic manipulation tools have been developed and facilitated Streptomyces genome editing. However, till now, CRISPR/dCas9-based interference system (CRISPRi) is only designed to repress single gene expression...
September 2018: Biotechnology Journal
https://read.qxmd.com/read/29799651/auroramycin-a-potent-antibiotic-from-streptomyces-roseosporus-by-crispr-cas9-activation
#9
Yee Hwee Lim, Fong Tian Wong, Wan Lin Yeo, Kuan Chieh Ching, Yi Wee Lim, Elena Heng, Shuwen Chen, De-Juin Tsai, Tsai-Ling Lauderdale, Kak-Shan Shia, Ying Swan Ho, Shawn Hoon, Ee Lui Ang, Mingzi M Zhang, Huimin Zhao
Silent biosynthetic gene clusters represent a potentially rich source of new bioactive compounds. We report the discovery, characterization, and biosynthesis of a novel doubly glycosylated 24-membered polyene macrolactam from a silent biosynthetic gene cluster in Streptomyces roseosporus by using the CRISPR-Cas9 gene cluster activation strategy. Structural characterization of this polyketide, named auroramycin, revealed a rare isobutyrylmalonyl extender unit and a unique pair of amino sugars. Relative and absolute stereochemistry were determined by using a combination of spectroscopic analyses, chemical derivatization, and computational analysis...
May 25, 2018: Chembiochem: a European Journal of Chemical Biology
https://read.qxmd.com/read/29693372/biosynthesis-of-the-15-membered-ring-depsipeptide-neoantimycin
#10
Will Skyrud, Joyce Liu, Divya Thankachan, Maria Cabrera, Ryan F Seipke, Wenjun Zhang
Antimycins are a family of natural products possessing outstanding biological activities and unique structures, which have intrigued chemists for over a half century. Of particular interest are the ring-expanded antimycins that show promising anticancer potential and whose biosynthesis remains uncharacterized. Specifically, neoantimycin and its analogs have been shown to be effective regulators of the oncogenic proteins GRP78/BiP and K-Ras. The neoantimycin structural skeleton is built on a 15-membered tetralactone ring containing one methyl, one hydroxy, one benzyl, and three alkyl moieties, as well as an amide linkage to a conserved 3-formamidosalicylic acid moiety...
May 18, 2018: ACS Chemical Biology
https://read.qxmd.com/read/29371699/identification-of-a-biosynthetic-gene-cluster-for-the-polyene-macrolactam-sceliphrolactam-in-a-streptomyces-strain-isolated-from-mangrove-sediment
#11
Zhen Jie Low, Li Mei Pang, Yichen Ding, Qing Wei Cheang, Kim Le Mai Hoang, Hoa Thi Tran, Jinming Li, Xue-Wei Liu, Yoganathan Kanagasundaram, Liang Yang, Zhao-Xun Liang
Streptomyces are a genus of Actinobacteria capable of producing structurally diverse natural products. Here we report the isolation and characterization of a biosynthetically talented Streptomyces (Streptomyces sp. SD85) from tropical mangrove sediments. Whole-genome sequencing revealed that Streptomyces sp. SD85 harbors at least 52 biosynthetic gene clusters (BGCs), which constitute 21.2% of the 8.6-Mb genome. When cultivated under lab conditions, Streptomyces sp. SD85 produces sceliphrolactam, a 26-membered polyene macrolactam with unknown biosynthetic origin...
January 25, 2018: Scientific Reports
https://read.qxmd.com/read/29170959/crispr-cas9-toolkit-for-actinomycete-genome-editing
#12
Yaojun Tong, Helene Lunde Robertsen, Kai Blin, Tilmann Weber, Sang Yup Lee
Bacteria of the order Actinomycetales are one of the most important sources of bioactive natural products, which are the source of many drugs. However, many of them still lack efficient genome editing methods, some strains even cannot be manipulated at all. This restricts systematic metabolic engineering approaches for boosting known and discovering novel natural products. In order to facilitate the genome editing for actinomycetes, we developed a CRISPR-Cas9 toolkit with high efficiency for actinomyces genome editing...
2018: Methods in Molecular Biology
https://read.qxmd.com/read/28742008/development-of-a-crispr-cas9-mediated-gene-editing-tool-in-streptomyces-rimosus
#13
Haiyan Jia, Longmei Zhang, Tongtong Wang, Jin Han, Hui Tang, Liping Zhang
Clustered regularly interspaced short palindromic repeats, associated proteins (CRISPR/Cas), has been developed into a powerful, targeted genome-editing tool in a wide variety of species. Here, we report an extensive investigation of the type II CRISPR/Cas9 system for targeted gene editing in Streptomyces rimosus. S. rimosus is used in the production of the antibiotic oxytetracycline, and its genome differs greatly from other species of the genus Streptomyces in the conserved chromosome terminal and core regions, which is of major production and scientific research value...
August 2017: Microbiology
https://read.qxmd.com/read/28507698/formicamycins-antibacterial-polyketides-produced-by-streptomyces-formicae-isolated-from-african-tetraponera-plant-ants
#14
Zhiwei Qin, John T Munnoch, Rebecca Devine, Neil A Holmes, Ryan F Seipke, Karl A Wilkinson, Barrie Wilkinson, Matthew I Hutchings
We report a new Streptomyces species named S. formicae that was isolated from the African fungus-growing plant-ant Tetraponera penzigi and show that it produces novel pentacyclic polyketides that are active against MRSA and VRE. The chemical scaffold of these compounds, which we have called the formicamycins, is similar to the fasamycins identified from the heterologous expression of clones isolated from environmental DNA, but has significant differences that allow the scaffold to be decorated with up to four halogen atoms...
April 1, 2017: Chemical Science
https://read.qxmd.com/read/28398287/crispr-cas9-strategy-for-activation-of-silent-streptomyces-biosynthetic-gene-clusters
#15
Mingzi M Zhang, Fong Tian Wong, Yajie Wang, Shangwen Luo, Yee Hwee Lim, Elena Heng, Wan Lin Yeo, Ryan E Cobb, Behnam Enghiad, Ee Lui Ang, Huimin Zhao
Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities...
April 10, 2017: Nature Chemical Biology
https://read.qxmd.com/read/27417933/high-efficiency-genome-editing-of-streptomyces-species-by-an-engineered-crispr-cas-system
#16
Y Wang, R E Cobb, H Zhao
Next-generation sequencing technologies have rapidly expanded the genomic information of numerous organisms and revealed a rich reservoir of natural product gene clusters from microbial genomes, especially from Streptomyces, the largest genus of known actinobacteria at present. However, genetic engineering of these bacteria is often time consuming and labor intensive, if even possible. In this chapter, we describe the design and construction of pCRISPomyces, an engineered Type II CRISPR/Cas system, for targeted multiplex gene deletions in Streptomyces lividans, Streptomyces albus, and Streptomyces viridochromogenes with editing efficiency ranging from 70% to 100%...
2016: Methods in Enzymology
https://read.qxmd.com/read/26901661/an-active-type-i-e-crispr-cas-system-identified-in-streptomyces-avermitilis
#17
Yi Qiu, Shiwei Wang, Zhi Chen, Yajie Guo, Yuan Song
CRISPR-Cas systems, the small RNA-dependent immune systems, are widely distributed in prokaryotes. However, only a small proportion of CRISPR-Cas systems have been identified to be active in bacteria. In this work, a naturally active type I-E CRISPR-Cas system was found in Streptomyces avermitilis. The system shares many common genetic features with the type I-E system of Escherichia coli, and meanwhile shows unique characteristics. It not only degrades plasmid DNA with target protospacers, but also acquires new spacers from the target plasmid DNA...
2016: PloS One
https://read.qxmd.com/read/26556277/in-vitro-crispr-cas9-system-for-efficient-targeted-dna-editing
#18
Yunkun Liu, Weixin Tao, Shishi Wen, Zhengyuan Li, Anna Yang, Zixin Deng, Yuhui Sun
UNLABELLED: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments...
2015: MBio
https://read.qxmd.com/read/26318449/highly-efficient-editing-of-the-actinorhodin-polyketide-chain-length-factor-gene-in-streptomyces-coelicolor-m145-using-crispr-cas9-coda-sm-combined-system
#19
Hu Zeng, Shishi Wen, Wei Xu, Zhaoren He, Guifa Zhai, Yunkun Liu, Zixin Deng, Yuhui Sun
The current diminishing returns in finding useful antibiotics and the occurrence of drug-resistant bacteria call for the need to find new antibiotics. Moreover, the whole genome sequencing revealed that the biosynthetic potential of Streptomyces, which has produced the highest numbers of approved and clinical-trial drugs, has been greatly underestimated. Considering the known gene editing toolkits were arduous and inefficient, novel and efficient gene editing system are desirable. Here, we developed an engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) combined with the counterselection system CodA(sm), the D314A mutant of cytosine deaminase, to rapidly and effectively edit Streptomyces genomes...
December 2015: Applied Microbiology and Biotechnology
https://read.qxmd.com/read/25806970/crispr-cas9-based-engineering-of-actinomycetal-genomes
#20
Yaojun Tong, Pep Charusanti, Lixin Zhang, Tilmann Weber, Sang Yup Lee
Bacteria of the order Actinomycetales are one of the most important sources of pharmacologically active and industrially relevant secondary metabolites. Unfortunately, many of them are still recalcitrant to genetic manipulation, which is a bottleneck for systematic metabolic engineering. To facilitate the genetic manipulation of actinomycetes, we developed a highly efficient CRISPR-Cas9 system to delete gene(s) or gene cluster(s), implement precise gene replacements, and reversibly control gene expression in actinomycetes...
September 18, 2015: ACS Synthetic Biology
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