Sequential deletion of all the polyketide synthase and nonribosomal peptide synthetase biosynthetic gene clusters and a 900-kb subtelomeric sequence of the linear chromosome of Streptomyces coelicolor

Zhou, Min; Jing, Xinyun; Xie, Pengfei; Chen, Weihua; Wang, Tao; Xia, Haiyang; Qin, Zhongjun

doi:10.1111/j.1574-6968.2012.02609.x

Cited by 57 publications

(49 citation statements)

References 34 publications

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“…Strain ZM12 was derived from S. coelicolor , probably the best-studied model Streptomyces , through deleting all ten native PKS and NRPS gene clusters present in its genome. [16] HPLC showed that this strain produced very few metabolites (Figure S2A). However, upon introducing pSETHSAF3, both compounds 2 and 3 were produced as the main metabolites in strain ZM12, in addition to a number of minor peaks.…”

mentioning

confidence: 99%

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

Chen

Ding

et al. 2014

Angew Chem Int Ed

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Antifungal HSAF (heat‐stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single‐module polyketide synthase–nonribosomal peptide synthetase (PKS‐NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two “clean” strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl‐coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene–PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS‐NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide–peptide natural products.

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confidence: 99%

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

Chen

Ding

et al. 2014

Angew Chem Int Ed

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“…PCR-targeted gene replacement procedure of Gust et al (2003) is also very useful for generating large deletions of chromosomal segments, including ten secondary metabolite biosynthetic gene clusters from S. coelicolor chromosome (Zhou et al 2012). The targeted recombination strategy adds an alternative approach for genome shuffling in Streptomyces genomes.…”

Section: Application For Identification Of Essential Regions and Genomentioning

confidence: 99%

A highly efficient targeted recombination system for engineering linear chromosomes of industrial bacteria <i>Streptomyces</i>

Pan

Chen²

2018

J. Gen. Appl. Microbiol.

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Soil bacteria Streptomyces are the most important producers of secondary metabolites, including most known antibiotics. These bacteria and their close relatives are unique in possessing linear chromosomes, which typically harbor 20 to 30 biosynthetic gene clusters of tens to hundreds of kb in length. Many Streptomyces chromosomes are accompanied by linear plasmids with sizes ranging from several to several hundred kb. The large linear plasmids also often contain biosynthetic gene clusters. We have developed a targeted recombination procedure for arm exchanges between a linear plasmid and a linear chromosome. A chromosomal segment inserted in an artificially constructed plasmid allows homologous recombination between the two replicons at the homology. Depending on the design, the recombination may result in two recombinant replicons or a single recombinant chromosome with the loss of the recombinant plasmid that lacks a replication origin. The efficiency of such targeted recombination ranges from 9 to 83% depending on the locations of the homology (and thus the size of the chromosomal arm exchanged), essentially eliminating the necessity of selection. The targeted recombination is useful for the efficient engineering of the Streptomyces genome for large-scale deletion, addition, and shuffling.

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“…Later, the construction of S. coelicolor derivatives containing sequential deletions of all the 10 PKS and NRPS biosynthetic gene clusters and a 900-kb subtelomeric sequence (total ca. 1.22 Mb, 14% of the genome) was performed to generate a further minimized S. coelicolor strain 43 . Recently, the CRISPR-Cas system has emerged as a popular tool for genome editing 44, 45 .…”

Section: Host Engineering: Minimized Genomes and Engineered Hostsmentioning

confidence: 99%

New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters

2016

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Natural product scaffolds remain a major source and inspiration for human therapeutics. However, generation of a natural product in the post-genomic era often requires reconstruction of the corresponding biosynthetic gene cluster in a heterologous host. In the burgeoning fields of synthetic biology and metabolic engineering, a significant amount of efforts has been devoted to develop DNA assembly techniques with higher efficiency, fidelity, and modularity, and heterologous expression systems with higher productivity and yield. Here we describe recent advances in DNA assembly and host engineering and highlight their applications in natural product discovery and engineering.

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Sequential deletion of all the polyketide synthase and nonribosomal peptide synthetase biosynthetic gene clusters and a 900-kb subtelomeric sequence of the linear chromosome of Streptomyces coelicolor

Cited by 57 publications

References 34 publications

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

A highly efficient targeted recombination system for engineering linear chromosomes of industrial bacteria <i>Streptomyces</i>

New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters

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