Streptomyces rochei
7434AN4 produces two structurally unrelated polyketide antibiotics, lankacidin and lankamycin, and carries three linear plasmids, pSLA2-L (211 kb), -M (113 kb), and -S (18 kb), whose nucleotide sequences were previously reported. The complete nucleotide sequence of the
S. rochei
chromosome has now been determined using the long-read PacBio RS-II sequencing together with short-read Illumina Genome Analyzer IIx sequencing and Roche 454 pyrosequencing techniques. The assembled sequence revealed an 8,364,802-bp linear chromosome with a high G + C content of 71.7% and 7,568 protein-coding ORFs. Thus, the gross genome size of
S. rochei
7434AN4 was confirmed to be 8,706,406 bp including the three linear plasmids. Consistent with our previous study, a
tap-tpg
gene pair, which is essential for the maintenance of a linear topology of
Streptomyces
genomes, was not found on the chromosome. Remarkably, the
S. rochei
chromosome contains seven ribosomal RNA (
rrn
) operons (16S-23S-5S), although
Streptomyces
species generally contain six
rrn
operons. Based on 2ndFind and antiSMASH platforms, the
S. rochei
chromosome harbors at least 35 secondary metabolite biosynthetic gene clusters, including those for the 28-membered polyene macrolide pentamycin and the azoxyalkene compound KA57-A.
should be a good model compound for nanoparticle studies in both photochemistry and photophysics, since it has strict core±cell molecular structure.
ExperimentalPreparation of Zn 4 O(AID) 6 : A solution of 7-azaindole (1.5 mmol) in methanol (20 cm 3 ) was heated to boiling and mixed with a solution of zinc acetate dehydrate (1 mmol in 30 cm 3 methanol). Triethylamine (1 cm 3 ) was then added. The colorless complex Zn 4 O(AID) 6 was obtained after filtration. The crystallography data were collected on an Enraf-Nonius CAD4 four-circle diffractometer.Time-resolved luminescence spectroscopy was recorded by a laser system. The sample excitation was performed with a pulsed, frequency-tripled titanium±sapphire laser, yielding optical pulses with a pulse length of 2 ps and a repetition rate of about 82 MHz. The excitation wavelength can be tuned between 250±300 nm. The PL signal was dispersed with a 0.32 m JobinYvon monochromator and recorded using a Streak camera system with a S20 cathode and a subsequent charge coupled device (CCD) camera. The overall time resolution of the setup is about 5 ps.
Although transcriptional activation of pathwayspecific positive regulatory genes and/or biosynthetic genes is primarily important for enhancing secondary metabolite production, reinforcement of substrate supply, as represented by primary metabolites, is also effective. For example, partial inhibition of fatty acid synthesis with ARC2 (an analog of triclosan) was found to enhance polyketide antibiotic production. Here, we demonstrate that this approach is effective even for industrial high-producing strains, for example enhancing salinomycin production by 40%, reaching 30.4 g/l of salinomycin in an industrial Streptomyces albus strain. We also hypothesized that a similar approach would be applicable to another important antibiotic group, nonribosomal peptide (NRP) antibiotics. We therefore attempted to partially inhibit protein synthesis by using ribosome-targeting drugs at subinhibitory concentrations (1/50∼1/2 of MICs), which may result in the preferential recruitment of intracellular amino acids to the biosynthesis of NRP antibiotics rather than to protein synthesis. Among the ribosome-targeting drugs examined, chloramphenicol at subinhibitory concentrations was most effective at enhancing the production by Streptomyces of NRP antibiotics such as actinomycin, calcium-dependent antibiotic (CDA), and piperidamycin, often resulting in an almost 2-fold increase in antibiotic production. Chloramphenicol activated biosynthetic genes at the transcriptional level and increased amino acid pool sizes 1.5- to 6-fold, enhancing the production of actinomycin and CDA. This "metabolic perturbation" approach using subinhibitory concentrations of ribosome-targeting drugs is a rational method of enhancing NRP antibiotic production, being especially effective in transcriptionally activated (e.g., rpoB mutant) strains. Because this approach does not require prior genetic information, it may be widely applicable for enhancing bacterial production of NRP antibiotics and bioactive peptides.
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