Dataset Information

Enhancement of himastatin bioproduction via inactivation of atypical repressors in Streptomyces hygroscopicus.

ABSTRACT: Three atypical regulatory genes, hmtABD have been discovered within the himastatin biosynthetic gene cluster (BGC) in Streptomyces hygroscopicus ATCC 53653 and the roles of their products have been identified. HmtA and HmtD do not show any structurally distinct features characteristic of regulatory function yet were shown to play important repressive and stimulatory roles, respectively, related to himastatin biosynthesis. HmtB encodes a conserved acetylglutamate kinase; new member of this family serves as repressor of secondary metabolism. Through repressive networks engineering, the limiting functions of HmtA and HmtB along with the activating functions of HmtD in the himastatin BGC have been identified for the first time by gene activation, qPCR, RT-PCR and HPLC studies of selected mutant strains; two of these mutant strains (?hmtA and ?hmtB) produced himastatin in titers (19.02?±?1.2??g/mL, 9.9 folds and 30.40?±?0.83??g/mL, 15.8 folds) far exceeding those of the wild-type (WT) producer. Overall, this work provides significant insight into secondary metabolic regulatory mechanisms in Streptomyces. These efforts also highlight and validate a new strategy enabling expanded exploitation of cyclopeptidic natural products such as himastatin that demonstrate exciting antimicrobial and antitumor potentials.

SUBMITTER: Xie Y

PROVIDER: S-EPMC6328088 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Enhancement of himastatin bioproduction via inactivation of atypical repressors in Streptomyces hygroscopicus.

Xie Yunchang Y Li Qinglian Q Qin Xiangjing X Ju Jianhua J Ma Junying J

Metabolic engineering communications 20190105

Three atypical regulatory genes, hmtABD have been discovered within the himastatin biosynthetic gene cluster (BGC) in Streptomyces hygroscopicus ATCC 53653 and the roles of their products have been identified. HmtA and HmtD do not show any structurally distinct features characteristic of regulatory function yet were shown to play important repressive and stimulatory roles, respectively, related to himastatin biosynthesis. HmtB encodes a conserved acetylglutamate kinase; new member ...[more]

PMID: 30671346

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Project description:Malaria is one of the life-threatening diseases in the world. The spread of resistance to antimalarial drugs is a major challenge, and resistance to artemisinin has been reported in the Southeast Asian region. In the previous study, the active compound of Streptomyces hygroscopicus subsp. Hygroscopicus (S. hygroscopicus), eponemycin, has been shown to have antimalarial effects. To further analyze the effects of other active compounds on the Plasmodium parasite, identifying and analyzing the effectiveness of compounds contained in S. hygroscopicus through instrumentation of liquid chromatography/mass spectrometry (LC/MS) and in silico studies were very useful. This study aimed at identifying other derivative compounds from S. hygroscopicus and screening the antimalarial activity of the compound by assessing the binding affinity, pharmacokinetic profile, and bond interaction. The derivative compounds were identified using LC/MS. Protein targets for derivative compounds were found through literature studies, and the results of identification of compounds and protein targets were reconstructed into three-dimensional models. Prediction of pharmacokinetic profiles was carried out using Swiss ADME. Screening of protein targets for the derivative compound was carried out using the reverse molecular docking method. Analyzing bond interaction was done by LigPlot. One compound from S. hygroscopicus, i.e., 6,7-dinitro-2-[1, 2, 4]triazole-4-yl-benzo[de]isoquinoline-1,3-dione, was successfully identified using LC/MS. This compound was an isoquinoline derivative compound. Through literature studies with inclusion criteria, thirteen protein targets were obtained for reverse molecular docking. This isoquinoline derivative had the potential to bind to each protein target. The pharmacokinetic profile showed that this compound had the drug-likeness criteria. Conclusion. 6,7-Dinitro-2-[1, 2, 4]triazole-4-yl-benzo[de]isoquinoline-1,3-dione has antimalarial activity as shown by reverse molecular docking studies and pharmacokinetic profiles. The best inhibitory ability of compounds based on bond affinity is with adenylosuccinate synthetase.

Project description:Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII). The glnB gene was cloned from S. hygroscopicus DNA by complementation in an Escherichia coli glutamine auxotrophic mutant (glnA). glnB was subcloned in Streptomyces plasmids by insertion into pIJ486 (pMSG3) and pIJ702 (pMSG5). Both constructions conferred resistance to the tripeptide form of phosphinothricin (bialaphos) and were able to complement a glutamine auxotrophic marker in S. coelicolor. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of S. lividans(pMSG5) revealed a highly overexpressed 40-kilodalton protein. When GS was purified from this strain, it was indistinguishable in apparent molecular mass from the 40-kilodalton protein. The nucleic acid sequence of the cloned region contained an open reading frame which encoded a protein whose size, amino acid composition, and N-terminal sequence corresponded to those of the purified GS. glnB had a high G + C content and codon usage typical of streptomycete genes. A comparison of its predicted amino acid sequence with the protein data bases revealed that it encoded a GSII-type enzyme which had previously been found only in various eucaryotes (47 to 50% identity) and nodulating bacteria such as Bradyrhizobium spp. (42% identity). glnB had only 13 to 18% identity with eubacterial GSI enzymes. Southern blot hybridization experiments showed that sequences similar to glnB were present in all of the five other Streptomyces species tested, as well as Frankia species. These results do not support the previous suggestion that GSII-type enzymes found in members of the family Rhizobiaceae represent a unique example of interkingdom gene transfer associated with symbiosis in the nodule. Instead they imply that the presence of more than one gene encoding GS may be more common among soil microorganisms than previously appreciated.

Dataset Information

Enhancement of himastatin bioproduction via inactivation of atypical repressors in Streptomyces hygroscopicus.

Publications

Enhancement of himastatin bioproduction via inactivation of atypical repressors in <i>Streptomyces hygroscopicus</i>.

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