Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis

ABSTRACTBacteria in the genusStreptomycesand its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on theint/attPloci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologousStreptomyceshost.Streptomycesstrains containing the three polyketide synthase geneseryAI,eryAII, anderyAIIIexpressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31int/attPlocus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene,eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.

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Distinct Modified Nucleosides in tRNATrp from the Hyperthermophilic Archaeon Thermococcus kodakarensis and Requirement of tRNA m2G10/m22G10 Methyltransferase (Archaeal Trm11) for Survival at High Temperatures

Journal of Bacteriology ◽

10.1128/jb.00448-19 ◽

2019 ◽

Vol 201 (21) ◽

Cited By ~ 4

Author(s):

Akira Hirata ◽

Takeo Suzuki ◽

Tomoko Nagano ◽

Daishiro Fujii ◽

Mizuki Okamoto ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

High Temperatures ◽

Modified Nucleosides ◽

Liquid Chromatography Mass Spectrometry ◽

Content Type ◽

Hyperthermophilic Archaeon ◽

Thermococcus Kodakarensis ◽

Chromatography Mass Spectrometry ◽

Modified Nucleoside

ABSTRACT tRNA m2G10/m22G10 methyltransferase (archaeal Trm11) methylates the 2-amino group in guanosine at position 10 in tRNA and forms N2,N2-dimethylguanosine (m22G10) via N2-methylguanosine (m2G10). We determined the complete sequence of tRNATrp, one of the substrate tRNAs for archaeal Trm11 from Thermococcus kodakarensis, a hyperthermophilic archaeon. Liquid chromatography/mass spectrometry following enzymatic digestion of tRNATrp identified 15 types of modified nucleoside at 21 positions. Several modifications were found at novel positions in tRNA, including 2′-O-methylcytidine at position 6, 2-thiocytidine at position 17, 2′-O-methyluridine at position 20, 5,2′-O-dimethylcytidine at position 32, and 2′-O-methylguanosine at position 42. Furthermore, methylwyosine was found at position 37 in this tRNATrp, although 1-methylguanosine is generally found at this location in tRNATrp from other archaea. We constructed trm11 (Δtrm11) and some gene disruptant strains and compared their tRNATrp with that of the wild-type strain, which confirmed the absence of m22G10 and other corresponding modifications, respectively. The lack of 2-methylguanosine (m2G) at position 67 in the trm11 trm14 double disruptant strain suggested that this methylation is mediated by Trm14, which was previously identified as an m2G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures. The m22G10 modification might have effects on stabilization of tRNA and/or correct folding of tRNA at the high temperatures. Collectively, these results provide new clues to the function of modifications and the substrate specificities of modification enzymes in archaeal tRNA, enabling us to propose a strategy for tRNA stabilization of this archaeon at high temperatures. IMPORTANCE Thermococcus kodakarensis is a hyperthermophilic archaeon that can grow at 60 to 100°C. The sequence of tRNATrp from this archaeon was determined by liquid chromatography/mass spectrometry. Fifteen types of modified nucleoside were observed at 21 positions, including 5 modifications at novel positions; in addition, methylwyosine at position 37 was newly observed in an archaeal tRNATrp. The construction of trm11 (Δtrm11) and other gene disruptant strains confirmed the enzymes responsible for modifications in this tRNA. The lack of 2-methylguanosine (m2G) at position 67 in the trm11 trm14 double disruptant strain suggested that this position is methylated by Trm14, which was previously identified as an m2G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures.

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Exceptional Production of both Prodigiosin and Cycloprodigiosin as Major Metabolic Constituents by a Novel Marine Bacterium, Zooshikella rubidus S1-1

Applied and Environmental Microbiology ◽

10.1128/aem.01986-10 ◽

2011 ◽

Vol 77 (14) ◽

pp. 4967-4973 ◽

Cited By ~ 64

Author(s):

Jong Suk Lee ◽

Yong-Sook Kim ◽

Sooyeon Park ◽

Jihoon Kim ◽

So-Jung Kang ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Secondary Metabolite ◽

Metabolite Profile ◽

The Yellow Sea ◽

Liquid Chromatography Mass Spectrometry ◽

Bacterial Strains ◽

Content Type ◽

Chromatography Mass Spectrometry ◽

Metabolite Profiles

ABSTRACTA Gram-negative, red-pigment-producing marine bacterial strain, designated S1-1, was isolated from the tidal flat sediment of the Yellow Sea, Korea. On the basis of phenotypic, phylogenetic, and genetic data, strain S1-1 (KCTC 11448BP) represented a new species of the genusZooshikella. Thus, we propose the nameZooshikella rubidussp. nov. Liquid chromatography and mass spectrometry of the red pigments produced by strain S1-1 revealed that the major metabolic compounds were prodigiosin and cycloprodigiosin. In addition, this organism produced six minor prodigiosin analogues, including two new structures that were previously unknown. To our knowledge, this is the first description of a microorganism that simultaneously produces prodigiosin and cycloprodigiosin as two major metabolites. Both prodigiosin and cycloprodigiosin showed antimicrobial activity against several microbial species. These bacteria were approximately 1.5-fold more sensitive to cycloprodigiosin than to prodigiosin. The metabolites also showed anticancer activity against human melanoma cells, which showed significantly more sensitivity to prodigiosin than to cycloprodigiosin. The secondary metabolite profiles of strain S1-1 and two reference bacterial strains were compared by liquid chromatography-mass spectrometry. Multivariate statistical analyses based on secondary metabolite profiles by liquid chromatography-mass spectrometry indicated that the metabolite profile of strain S1-1 could clearly be distinguished from those of two phylogenetically related, prodigiosin-producing bacterial strains.

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Antibiotic Activity of a Paraphaeosphaeria sporulosa-Produced Diketopiperazine against Salmonella enterica

Journal of Fungi ◽

10.3390/jof6020083 ◽

2020 ◽

Vol 6 (2) ◽

pp. 83

Author(s):

Raffaele Carrieri ◽

Giorgia Borriello ◽

Giulio Piccirillo ◽

Ernesto Lahoz ◽

Roberto Sorrentino ◽

...

Keyword(s):

Mass Spectrometry ◽

Salmonella Enterica ◽

Inhibitory Concentration ◽

Animal Husbandry ◽

Antibiotic Activity ◽

Future Application ◽

Liquid Chromatography Mass Spectrometry ◽

In Vivo Experiments ◽

First Time

A diketopiperazine has been purified from a culture filtrate of the endophytic fungus Paraphaeosphaeria sporulosa, isolated from healthy tissues of strawberry plants in a survey of microbes as sources of anti-bacterial metabolites. Its structure has been determined by nuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry (LC–MS) analyses and was found to be identical to cyclo(L-Pro-L-Phe) purified from species of other fungal genera. This secondary metabolite has been selected following bioguided-assay fractionation against two strains of Salmonella enterica, the causal agent of bovine gastroenteritis. The diketopiperazine cyclo(L-Pro-L-Phe), isolated for the first time from Paraphaeosphaeria species, showed minimum inhibitory concentration (MIC) values of 71.3 and 78.6 μg/mL against the two S. enterica strains. This finding may be significant in limiting the use of synthetic antibiotics in animal husbandry and reducing the emergence of bacterial multidrug resistance. Further in vivo experiments of P. sporulosa diketopiperazines are important for the future application of these metabolites.

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