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Increasing the self-resistance levels of Streptomycesis an effective strategy to improve the production of antibiotics. To increase the oxytetracycline (OTC) production in Streptomyces rimosus, we investigated the cooperative effect of three co-overexpressing OTC resistance genes: one gene encodes a ribosomal protection protein (otrA) and the other two express efflux proteins (otrBand otrC). Results indicated that combinational overexpression of otrA, otrB, and otrC(MKABC) exerted a synergetic effect. OTC production increased by 179% in the recombinant strain compared with that of the wild-type strain M4018. The resistance level to OTC was increased by approximately two-fold relative to the parental strain, thereby indicating that applying the cooperative effect of self-resistance genes is useful to improve OTC production. Furthermore, the previously identified cluster-situated activator OtcR was overexpressed in MKABC in constructing the recombinant strain MKRABC; such strain can produce OTC of approximately 7.49 g L−1, which represents an increase of 19% in comparison with that of the OtcR-overexpressing strain alone. Our work showed that the cooperative overexpression of self-resistance genes is a promising strategy to enhance the antibiotics production in Streptomyces.

C–C bond ring cleaving oxygenases represent a unique family of enzymes involved in the B ring cleavage reaction only observed in atypical angucycline biosynthesis. B ring cleavage is the key reaction leading to dramatic divergence in the final structures of atypical angucyclines. Here, we present the crystal structure of AlpJ, the first structure of this family of enzymes. AlpJ has been verified as the enzyme catalyzing C–C bond cleavage in kinamycin biosynthesis. The crystal structure of the AlpJ monomer resembles the dimeric structure of ferredoxin-like proteins. The N- and C-terminal halves of AlpJ are homologous, and both contain a putative hydrophobic substrate binding pocket in the “closed” and “open” conformations, respectively. Structural comparison of AlpJ with ActVA-Orf6 and protein–ligand docking analysis suggest that the residues including Asn60, Trp64, and Trp181 are possibly involved in substrate recognition. Site-directed mutagenesis results supported our hypothesis, as mutation of these residues led to nearly a complete loss of the activity of AlpJ. Structural analysis also revealed that AlpJ possesses an intramolecular domain–domain interface, where the residues His50 and Tyr178 form a hydrogen bond that probably stabilizes the three-dimensional structure of AlpJ. Site-directed mutagenesis showed that the two residues, His50 and Tyr178, were vital for the activity of AlpJ. Our findings shed light on the structure and catalytic mechanism of the AlpJ family of oxygenases, which presumably involves two active sites that might function in a cooperative manner.

The glycerol utilization (gyl) operon is involved in clavulanic acid (CA) production by Streptomyces clavuligerus, and possibly supplies the glyceraldehyde-3-phosphate (G3P) precursor for CA biosynthesis. The gyloperon is regulated by GylR and is induced by glycerol. To enhance CA production in S. clavuligerus, an extra copy of ccaRexpressed from Pgyl(the gylpromoter) was integrated into the chromosome of S. clavuligerusNRRL 3585. This construct coordinated the transcription of CA biosynthetic pathway genes with expression of the gyloperon. In the transformants carrying the Pgyl-controlled regulatory gene ccaR, CA production was enhanced 3.19-fold in glycerol-enriched batch cultures, relative to the control strain carrying an extra copy of ccaRcontrolled by its own promoter (PccaR). Consistent with enhanced CA production, the transcription levels of ccaR, ceas2and claRwere significantly up-regulated in the transformants containing Pgyl-controlled ccaR.

We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerusmutants. The assay is based on a β-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λmax=390 nm) is converted to a red product (λmax=486 nm). Since CA can irreversibly inhibit β-lactamase activity, the level of CA in a sample can be measured as a function of the A390/A486ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L−1and 50 μg L−1to 10 mg L−1, respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerusmutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.

Rapid identification of glycosylation sites of glycoproteins is urgently needed in the study of glycoproteomics. In the present work, a rapid and simple method based on non-specific digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry was described, which can efficiently identify the N-linked glycosylation sites. One-step in-gel digestion of ribonuclease B (RNase B) by proteinase K was employed to generate glycopeptides with short and discrepant peptide composition. When compared with glycopeptides prepared by two-step in-gel digestion using trypsin–proteinase K or trypsin-pronase, the direct proteinase K treatment showed obvious superiority in both glycopeptide recovery and preparation simplicity. Most importantly, it helps to generate greater variety of glycopeptide series with rich information for glycosylation site identification. In addition, binary matrices 5-chloro-2-mercaptobenzothiazole/2,5-dihydroxybenzoic acid were found to form homogeneous microcrystals on the target with the purified glycopeptides, leading to improved detection sensitivity. Thus, the present work provides an optimized solution to speed up the characterization of N-linked glycosylation sites in glycoproteins.

The important and diverse regulatory roles of Ca2+in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.

LC–MS and LC–MS/MS analyses indicated that an enzyme responsible for inactivating the antibiotic etamycin is specific for streptogramins and acts on both type B-I and B-II streptogramin subgroups. No enzymatic activity was detected for other cyclodepsipeptides such as surfactins and viscosin. It was demonstrated using analogs of etamycin that the picolinyl moiety is essential to obtain enzyme-generated ring-opened compounds. Because the picolinyl moiety is also essential for the biological activity of streptogramins, it is proposed that this residue is a distinctive topographic feature in the binding of this group of antibiotics to enzyme active sites. © 1997 John Wiley & Sons, Ltd.

Plasmid pJV4, containing a 2.4-kilobase pair insert of genomic DNA from the chloramphenicol (Cm) producer Streptomyces venezuelaeISP5230, confers resistance when introduced by transformation into the Cm-sensitive host Streptomyces lividansM252 (Mosher, R. H. Ranade, N. P., Schrempf, H., and Vining, L. C.(1990) J. Gen. Microbiol.136, 293-301). Transformants rapidly metabolized Cm to one major product, which was isolated and purified by reversed phase chromatography. The metabolite was identified by nuclear magnetic resonance spectroscopy and mass spectrometry as 3′-O-phospho-Cm, and was shown to have negligible inhibitory activity against Cm-sensitive Micrococcus luteus. The nucleotide sequence of the S. venezuelaeDNA insert in pJV4 contains an open reading frame (ORF) that encodes a polypeptide (19 kDa) with a consensus motif at its NH2terminus corresponding to a nucleotide-binding amino acid sequence (motif A or P-loop; Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J.(1982) EMBO J.1, 945-951). When a recombinant vector containing this ORF as a 1.6-kilobase pair SmaI-SmaI fragment was used to transform S. lividansM252, uniformly Cm-resistant transformants were obtained. A strain of S. lividanstransformed by a vector in which the ORF had been disrupted by an internal deletion yielded clones that were unable to phosphorylate Cm, and exhibited normal susceptibility to the antibiotic. The results implicate the product of the ORF from S. venezuelaeas an enzymic effector of Cm resistance in the producing organism by 3′-O-phosphorylation. We suggest the trivial name chloramphenicol 3′-O-phosphotransferase for the enzyme.