[Enhancement of salinomycin production and its activity optimization - A review].
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Salinomycin is a monocarboxylic acid polyether antibiotics produced by Streptomyces albus. It has strong inhibiting and killing activity against most gram-positive bacteria and various coccidiums with low adverse impact on environment. In addition, salinomycin can specifically inhibit the growth of a variety of cancer cells and cancer stem cells via targeting to multiple sites, and is a promising anti-tumor drug candidate. To obtain high yield salinomycinproducing strain, conventional mutation techniques and modern molecular genetic methods have been used. Meanwhile, bioactivity and selectivity of salinomycin could be improved by modifying the chemical structure and changing drug delivery methods. Here, we summarize the key strategies for enhancing salinomycin production and review the progresses in optimizing its drug activity and targeting properties. The future research focus is also addressed.Keywords:
Salinomycin
Streptomyces albus
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Abstract Salinomycin is a promising anticancer drug for chemotherapy. A highly productive biosynthetic gene cluster will facilitate the creation of analogs with improved therapeutic activity and reduced side effects. In this study, we engineered an artificial 106‐kb salinomycin gene cluster and achieved efficient heterologous expression in three hosts: Streptomyces coelicolor CH999, S. lividans K4‐114, and S. albus J1074. The six‐operon artificial gene cluster consists of 25 genes from the native gene cluster organized into five operons and five fatty acid β‐oxidation genes into one operon. All operons are driven by strong constitutive promoters. For K4‐114 and J1074 harboring the artificial gene cluster, salinomycin production in shake flask cultures was 14.3 mg L −1 and 19.3 mg L −1 , respectively. The production was 1.3‐fold and 1.7‐fold higher, respectively, than that of the native producer S. albus DSM41398. K4‐114 and J1074 harboring the native gene cluster produced an undetectable amount of salinomycin and 0.5 mg L −1 , respectively. CH999 harboring the artificial gene cluster produced 10.3 mg L −1 of salinomycin, which was 92% of the production by DSM41398. The efficient heterologous expression system based on the 106‐kb multioperon artificial gene cluster established in this study will facilitate structural diversification of salinomycin, which is valuable for drug development and structure–activity studies.
Salinomycin
Gene cluster
Streptomyces albus
Heterologous
Heterologous expression
Myxococcus xanthus
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[Objective] In the salinomycin biosynthetic gene cluster, slnTI and slnTII encode the ABC transporter ATP-binding subunit and trans-membrane subunit, respectively. We speculate that slnTI and slnTII are most likely related to salinomycin export. Here, the role of slnTI and slnTII on salinomycin biosynthesis and resistance was investigated by gene replacement and overexpression. [Methods] Using REDIRECT? technology, the slnTI mutant LJ01 and the slnTII mutant LJ02 were constructed, and further verified by trans-complementation with cloned corresponding genes. SlnTI and SlnTII were overexpressed in the wild-type Streptomyces albus XM211. Also, slnTI and slnTII were introduced into S. lividans 1326, and the resistance to salinomycin of the resulted strains was tested. [Results] Compared with the wild-type strain, salinomycin production in LJ01 and LJ02 decreased by 27.2% and 45.4%, respectively. The transcription of structure gene slnA3 and regulatory gene slnR were both reduced in LJ01 and LJ02. The overexpression of slnTI and slnTII in the wild-type XM211 resulted in a 14.6% enhanced salinomycin production. Correspondingly, the transcription of slnA3 and slnR was also increased. Additionally, heterologous expression of slnTI and slnTII in S. lividans 1326 slightly increased its resistance to salinomycin. [Conclusion] slnTI and slnTII are identified to be related to the production of salinomycin, but not to be the main resistant gene for salinomycin in the producer.
Salinomycin
Streptomyces albus
Gene cluster
Wild type
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[Objective] This study aimed to explore the mutagenesis effects of N+ ion beam irradiation on Streptomyces albus and obtain high-yield salinomycin-producing strain.[Method] Streptomyces albus strain S-11-04 was mutated with different doses of N+ implantation.The effects of low energy N+ implantation on the survival rate,colony shape and salinomycin-producing ability were investigated.[Result] The results showed that low energy N+ implantation can efficiently improve the positive mutation rate of Streptomyces albus;13 mutant strains with high yield of salinomycin were isolated;mutant strain N3-6 has good inheritance and fermentation stability with four continuous generations,and the titres of salinomycin were increased by 41% in the shake-flask culture and 20.5% in mass production compared with the control.[Conclusion] N+ ion beam irradiation is an effective method to obtain high-yield salinomycin-producing Streptomyces albus strain.
Salinomycin
Streptomyces albus
Strain (injury)
Mutation Breeding
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Summary Salinomycin, an FDA‐approved polyketide drug, was recently identified as a promising anti‐tumour and anti‐viral lead compound. It is produced by Streptomyces albus , and the biosynthetic gene cluster ( sal ) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini‐cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter P kasO* or P neo . The pks operon was not included in the mini‐cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini‐cluster was introduced into the wild‐type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3‐fold and 5.1‐fold compared with that of the wild‐type strain respectively. Intriguingly, mini‐cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini‐cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.
Salinomycin
Gene cluster
Streptomyces albus
Ribosomal binding site
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The results of research show that oxadin has mutagenous effect on the tested actinomyces strains, in particular on Streptomyces albus--producer of the polyether antibiotic salinomycin. The colonies grown up after spores treatment with oxadin, have been tested for auxotrophy, morphological mutations and biosynthesis of the antibiotic salinomycin. Treatment of Streptomyces albus strain with 10% oxadin for 10 or 20 min provided the isolation of highly active strains. Dispersion analysis data revealed statistically significant differences between the control (non treated) and the oxadin-treated Streptomyces albus populations.
Salinomycin
Streptomyces albus
Strain (injury)
Isolation
Auxotrophy
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Abstract Linear plus linear homologous recombination-mediated recombineering (LLHR) is ideal for obtaining natural product biosynthetic gene clusters from pre-digested bacterial genomic DNA in one or two steps of recombineering. The natural product salinomycin has a potent and selective activity against cancer stem cells and is therefore a potential anti-cancer drug. Herein, we separately isolated three fragments of the salinomycin gene cluster ( sal O -orf 18) from Streptomyces albus ( S. albus ) DSM41398 using LLHR and assembled them into intact gene cluster (106 kb) by Red/ET and expressed it in the heterologous host Streptomyces coelicolor ( S. coelicolor ) A3(2). We are the first to report a large genomic region from a Gram-positive strain has been cloned using LLHR. The successful reconstitution and heterologous expression of the salinomycin gene cluster offer an attractive system for studying the function of the individual genes and identifying novel and potential analogues of complex natural products in the recipient strain.
Streptomyces albus
Salinomycin
Recombineering
Gene cluster
Heterologous expression
Cloning (programming)
Heterologous
genomic DNA
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[Objective] This study aimed to explore the mutagenesis effects of N+ ion beam implantation on Streptomyces albus and obtain high-yield salinomycin-producing mutant strain. [Method] Streptomyces albus strain S-11-04 was mutated with different doses of N+ implantation. The effects of low energy N+ implantation on the survival rate, colony morphology and salinomycin-producing ability were investigated. [Result] The results showed that low energy N+ implantation can efficiently improve the positive mutation rate of Streptomyces albus; 13 mutant strains with high yield of salinomycin were isolated; to be specific, mutant strain N3-6 has relatively good genetic stability with four continuous generations, and the titres of salinomycin were increased by 41% in the shake-flask culture and 20.5% in mass production compared with the control. [Conclusion] N+ ion beam irradiation is an effective method to obtain high-yield salinomycin-producing Streptomyces albus strain.
Salinomycin
Streptomyces albus
Mutation Breeding
Strain (injury)
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Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.
Salinomycin
Streptomyces albus
Gene cluster
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Streptomyces albus
Salinomycin
Sigma factor
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