Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

Kim, Seung Young; Ju, Kou San; Metcalf, William W.; Evans, Bradley S.; Kuzuyama, Tomohisa; Donk, Wilfred A. van der

doi:10.1128/aac.06478-11

Cited by 61 publications

(75 citation statements)

References 59 publications

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“…E. coli strains were grown in LB broth at 37°C with standard antibiotic concentrations. WM4489, a DH10B derivative engineered to control copy-number of oriV-based plasmids (40), was used as the host strain for all plasmids generated in this study (Table S2). Plasmid copy number was increased by adding sterile rhamnose to a final concentration of 10 mM.…”

Section: Methodsmentioning

confidence: 99%

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Nayak

Metcalf

2017

Proc. Natl. Acad. Sci. U.S.A.

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121

112

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Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeon Methanosarcina acetivorans. Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon, Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJdependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homologydirected repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.he CRISPR (clustered regularly interspaced palindromic repeats) array and associated cas genes are widespread in microbial genomes (1), where they confer acquired immunity to phage and foreign DNA elements (2). The type IIA system from Streptococcus pyogenes is especially well characterized and has been widely applied as a remarkably effective genome-editing tool (3). During genome editing, heterologous expression of the RNA-guided DNA endonuclease Cas9 and a chimeric singleguide (sg) RNA, comprised of a 20-bp spacer that targets the chromosome and an 80-bp scaffold that binds Cas9, leads to a lethal double-strand break (DSB) at all target sites within the genome that are flanked by a 3′ NGG protospacer adjacent motif (PAM) (4) (Fig. S1). In eukaryotes, the nonhomologous endjoining (NHEJ) repair pathway can mend the DSB by generating simple insertions or deletions at the sgRNA target site, thus preventing additional rounds of Cas9-mediated cleavage (3, 5). Alternatively, the native homology-dependent repair (HDR) pathway can repair the fatal DSB, so long as a repair template that modifies or removes the sgRNA target site is provided, again preventing additional rounds of Cas9-mediated cleavage (3, 5) (Fig. S1). Appropriately ...

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Section: Methodsmentioning

confidence: 99%

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Nayak

Metcalf

2017

Proc. Natl. Acad. Sci. U.S.A.

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121

112

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“…Presumably, these enzymes act to provide autoresistance to the producers. Another such kinase, originally called FosC and found in the fosfomycin producer P. syringae, is actually an ortholog of FomA (Kim et al 2012). …”

Section: Fosfomycin-modifying Enzymesmentioning

confidence: 99%

Fosfomycin: Mechanism and Resistance

Silver¹

2017

Cold Spring Harb Perspect Med

231

169

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“…Because this initial reaction is thermodynamically unfavorable, net synthesis of phosphonates requires a thermodynamically favorable step immediately following PepM. Initial studies identified phosphonopyruvate decarboxylase (Ppd) as the enzyme providing this driving force; however, recent data show that other driving reactions exist (31,32). Subsequent biosynthetic steps diverge to create a wide range of C-P biomolecules.…”

mentioning

confidence: 99%

Diversity and abundance of phosphonate biosynthetic genes in nature

Doroghazi

Janga

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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157

204

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Phosphonates, molecules containing direct carbon-phosphorus bonds, compose a structurally diverse class of natural products with interesting and useful biological properties. Although their synthesis in protozoa was discovered more than 50 y ago, the extent and diversity of phosphonate production in nature remains poorly characterized. The rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate, catalyzed by the enzyme PEP mutase (PepM), is shared by the vast majority of known phosphonate biosynthetic pathways. Thus, the pepM gene can be used as a molecular marker to examine the occurrence and abundance of phosphonate-producing organisms. Based on the presence of this gene, phosphonate biosynthesis is common in microbes, with ∼5% of sequenced bacterial genomes and 7% of genome equivalents in metagenomic datasets carrying pepM homologs. Similarly, we detected the pepM gene in ∼5% of random actinomycete isolates. The pepM-containing gene neighborhoods from 25 of these isolates were cloned, sequenced, and compared with those found in sequenced genomes. PEP mutase sequence conservation is strongly correlated with conservation of other nearby genes, suggesting that the diversity of phosphonate biosynthetic pathways can be predicted by examining PEP mutase diversity. We used this approach to estimate the range of phosphonate biosynthetic pathways in nature, revealing dozens of discrete groups in pepM amplicons from local soils, whereas hundreds were observed in metagenomic datasets. Collectively, our analyses show that phosphonate biosynthesis is both diverse and relatively common in nature, suggesting that the role of phosphonate molecules in the biosphere may be more important than is often recognized.

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Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

Cited by 61 publications

References 59 publications

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Fosfomycin: Mechanism and Resistance

Diversity and abundance of phosphonate biosynthetic genes in nature

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