iCatch: a new strategy for capturing large DNA fragments using homing endonucleases

Wang, Jingman; Lu, Anrui; Liu, Jiakun; Huang, Weiren; Wang, Jin; Cai, Zhiming; Zhao, Guoping

doi:10.1093/abbs/gmy139

Cited by 7 publications

(9 citation statements)

References 33 publications

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“…The iCatch [ 89 ] is an upgrade of the 2014 proposed iBrick principle [ 90 ] based on the BioBricks concept [ 91 ]. This tool was developed to facilitate “catching” of large regions (e.g., BGCs from actinomycetes).…”

Section: Genetic Engineering Of Actinomycetesmentioning

confidence: 99%

“…The iBrick strategy is suitable for assembling whole gene clusters in vectors using the BioBricks concept. As iBrick still has some limitations, such as low efficiencies and difficulties in capturing large BGCs from Actinomyces , iCatch was developed [ 89 ]. The target regions are released by genomic DNA (gDNA) digestions with HEs (as the target is flanked by I-SceI and PI-PspI sites) which are in vivo introduced via homologous recombination.…”

Section: Genetic Engineering Of Actinomycetesmentioning

confidence: 99%

“…strain. Furthermore, the obtained ACT cluster was further assembled into other iBrick components, which enabled their subsequent use for other studies [ 89 ].…”

Section: Genetic Engineering Of Actinomycetesmentioning

confidence: 99%

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An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

2020

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The first antibiotic-producing actinomycete (Streptomyces antibioticus) was described by Waksman and Woodruff in 1940. This discovery initiated the “actinomycetes era”, in which several species were identified and demonstrated to be a great source of bioactive compounds. However, the remarkable group of microorganisms and their potential for the production of bioactive agents were only partially exploited. This is caused by the fact that the growth of many actinomycetes cannot be reproduced on artificial media at laboratory conditions. In addition, sequencing, genome mining and bioactivity screening disclosed that numerous biosynthetic gene clusters (BGCs), encoded in actinomycetes genomes are not expressed and thus, the respective potential products remain uncharacterized. Therefore, a lot of effort was put into the development of technologies that facilitate the access to actinomycetes genomes and activation of their biosynthetic pathways. In this review, we mainly focus on molecular tools and methods for genetic engineering of actinomycetes that have emerged in the field in the past five years (2015–2020). In addition, we highlight examples of successful application of the recently developed technologies in genetic engineering of actinomycetes for activation and/or improvement of the biosynthesis of secondary metabolites.

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Section: Genetic Engineering Of Actinomycetesmentioning

confidence: 99%

Section: Genetic Engineering Of Actinomycetesmentioning

confidence: 99%

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An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

2020

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“…Actinorhodin (Wang et al, 2019) be separated and extracted using multi-rounds of pulsed field gel electrophoresis (PFGE) with different ramped pulse times (Clos and Zander-Dinse, 2019). Compared to mechanical fragmentation methods, the unevenly distributed restriction sites in the genome may cause inherently biased and incomplete library with enzyme methods.…”

Section: Llhrmentioning

confidence: 99%

“…For example, Dai et al (2015) intergrated plasmid pEry-up and pEry-down with the BT1 integrase recognition sites BattP and BattB via single-or double-crossover at both ends of erythromycin BGC, after which genome DNA was carefully isolated and treated with the BT1 integrase to circularize at att recombination sequences as a plasmid via in vitro site-specific recombination. Similarily, iCatch intergrates homing endonucleases I-SceI and PI-PspI recognition sites flanking the region of interest, after which the genome is isolated and digested with I-SceI or PI-PspI and then self-ligated to clone the target BGC in vitro (Wang et al, 2019). Moreover, several groups have developed methods that express recombinases to extract DNA fragments between two integrase recognition sites and circularize the plasmid in vivo (Figure 1D), such as phage φBT1 integrase-mediated site-specific recombination (Du et al, 2015), Cre/loxP plus BAC (Hu et al, 2016).…”

Section: Cloning Strategiesmentioning

confidence: 99%

Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters

Lin

Nielsen

Liu

2020

Front. Bioeng. Biotechnol.

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Since the discovery of penicillin, natural products and their derivatives have been a valuable resource for drug discovery. With recent development of genome mining approaches in the post-genome era, a great number of natural product biosynthetic gene clusters (BGCs) have been identified and these can potentially be exploited for the discovery of novel natural products that can find application as pharmaceuticals. Since many BGCs are silent or do not express in native hosts under laboratory conditions, heterologous expression of BGCs in genetically tractable hosts becomes an attractive route to activate these BGCs to discover the corresponding products. Here, we highlight recent achievements in cloning and discovery of natural product biosynthetic pathways via intact BGC capturing, and discuss the prospects of high-throughput and multiplexed cloning of rational-designed gene clusters in the future.

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In Vitro Packaging Mediated One-Step Targeted Cloning of Natural Product Pathway

et al. 2019

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Direct cloning of natural product pathways for efficient refactoring and heterologous expression has become an important strategy for microbial natural product research and discovery, especially for those kept silent or poorly expressed in the original strains. Accordingly, the development of convenient and efficient cloning approaches is becoming increasingly necessary. Here we presented an in vitro packaging mediated cloning approach that combines CRISPR/Cas9 system with in vitro λ packaging system, for targeted cloning of natural product pathways. In such a scheme, pathways of Tu3010 (27.4 kb) and sisomicin (40.7 kb) were respectively cloned, and stuR was further depicted to positively regulate Tu3010 production. In vitro packaging mediated approach not only enables to activate cryptic pathways, but also facilitates refactoring or interrogating the pathways in conjunction with various gene editing systems. This approach features an expedited, convenient, and generic manner, and it is conceivable that it may be widely adopted for targeted cloning of the natural product pathways.

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iCatch: a new strategy for capturing large DNA fragments using homing endonucleases

Cited by 7 publications

References 33 publications

An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters

In Vitro Packaging Mediated One-Step Targeted Cloning of Natural Product Pathway

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