Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.
Background Nematodes are parasitic animals that cause over 100 billion US dollars loss in agricultural business. The whole-genomes of two Streptomyces strains, Streptomyces spectabilis KCTC9218T and Streptomyces sp. AN091965, were sequenced. Both strains produce spectinabilin, an antinematode drug. Its secondary metabolism was examined to aid the development of an efficient nematicidal drug-producing host strain. Results The whole-genome sequences of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 were analyzed using PacBio and Illumina sequencing platforms, and assembled using hybrid methodology. The total contig lengths for KCTC9218T and AN091965 were 9.97 Mb and 9.84 Mb, respectively. A total of 8,374 and 8,054 protein-coding genes, as well as 39 and 45 secondary metabolite biosynthetic gene clusters were identified in KCTC9218T and AN091965, respectively. 18.4 ± 6.45 mg/L and 213.89 ± 21.30 mg/L of spectinabilin were produced by S. spectabilis KCTC9218T and Streptomyces sp. AN091965, respectively. Pine wilt disease caused by nematode was successfully prevented by lower concentration of spectinabilin injection than that of abamectin recommended by its manufacturer. Production of multiple antinematode drugs, including spectinabilin, streptorubin B, and undecylprodigiosin was observed in both strains using high-resolution liquid chromatography mass spectrometry (LC–MS) analysis. Conclusions Whole-genome sequencing of spectinabilin-producing strains, coupled with bioinformatics and mass spectrometry analyses, revealed the production of multiple nematicidal drugs in the KCTC9218T and AN091965 strains. Especially, Streptomyces sp. AN091965 showed high production level of spectinabilin, and this study provides crucial information for the development of potential nematicidal drug producers.
The xiamycins are bioactive indolosesquiterpenoids that have been isolated from actinobacterial strains belonging to the Streptomyces genus. The overexpression of orf2011, which encodes the LuxR family regulator in a marine Streptomyces strain (HK18) isolated from a hypersaline saltern, significantly increased the production of xiamycin dimers, namely the previously reported dixiamycins A and C (3 and 4), compared to the wild-type strain. In addition, the engineered strain produced new members of the xiamycin family (lipoxiamycins A and B), which possessed a lipophilic chain linked to the indolosesquiterpenoid core structure by a N–O bond. The transcription analysis of the N-hydroxylase-encoding xiaH by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that the transcription level of xiaH responsible for the formation of a nitroxyl radical was increased by the overexpression of orf2011, which is located outside the xiamycin biosynthetic gene cluster. The structures of these compounds were determined by full spectroscopic analysis, and the connectivity between the lipophilic chain and the indolosesquiterpenoid moiety was confirmed in both lipoxiamycins A and B (1 and 2) by MS/MS analysis. Moreover, the absolute configurations of these compounds were established using quantum mechanics-based electronic circular dichroism and DP4 calculations. Finally, it was demonstrated that lipoxiamycin A (1) displayed inhibitory activity against lipopolysaccharide-induced NO production at an IC50 of 9.89 ± 0.92 µM in RAW 264.7 cells.
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