Abstract
Chlorothricin (CHL), produced by Streptomyces antibioticus DSM 40725 (wild-type strain, WT), belongs to a growing family of spirotetronate antibiotics that have biological activities inhibiting pyruvate carboxylase and malate dehydrogenase. ChlF2, a cluster-situated SARP regulator, can activate the transcription of chlJ, chlC3, chlC6, chlE1, chlM, and chlL to control CHL biosynthesis. Co-expression of chlF2 and chlK encoding type II thioesterase in WT strain under the control of Pkan led to high production of chlorothricin by 840% in comparison with that of WT. Since the inhibitory activity of CHL against several Gram-positive bacteria is higher than des-CHL, combinatorial strategies were applied to promote the conversion of des-CHL to CHL. Over-expression of chlB4, encoding a halogenase, combining with the supplementation of sodium chloride led to further 41% increase of CHL production compared to that of F2OE, a chlF2 over-expression strain. These findings provide new insights into the fine-tuned regulation of spirotetronate family of antibiotics and the construction of high-yield engineered strains.
Highlights
Streptomycetes are the most abundant source of antibiotics widely applied in clinical treatments, animal husbandry, and plant crop protection (Chen et al, 2008; Guo et al, 2010; Zhang et al, 2016)
To detect the biological activities of des-CHL and CHL against Bacillus subtilis CGMCC 1.1630 (B. subtilis) and Staphylococcus aureus CGMCC 1.89 (S. aureus), the purified des-CHL and CHL dissolved in 30 μl methanol were added into the holes with 0.8 cm diameter in LB agar medium containing 1% (v/v) B. subtilis or S. aureus culture, respectively
Streptomyces antibiotic regulatory proteins (SARPs) are a specific family of paralogous proteins that are associated with antibiotic biosynthesis in many streptomycetes
Summary
Streptomycetes are the most abundant source of antibiotics widely applied in clinical treatments, animal husbandry, and plant crop protection (Chen et al, 2008; Guo et al, 2010; Zhang et al, 2016). It is common that antibiotic biosynthesis is specified by the corresponding gene cluster, usually including pathway-specific regulatory genes (Liu et al, 2013; Li and Tan, 2017; Guan et al, 2019). Spirotetronate polyketides have been defined as a new family of microbial metabolites with potent antitumor properties or antibacterial activity, of which chlorothricin (CHL) was discovered as the first member of the spirotetronate family in 1969 (Lacoske and Theodorakis, 2015). Of the spirotetronate/spirotetramate natural products, ChlF2 Activates Chlorothricin Biosynthesis members of this family usually possess a characteristic pentacyclic aglycone comprising a trans-decalin system and a tetronate or tetramate spiro-conjugate, e.g., kijanimicin, pyrrolosporin, tetrocarcin, lobophorin, versipelostatin, nomimicin, and abyssomicin (Igarashi et al, 2012; Li S. et al, 2013; Lacoske and Theodorakis, 2015; Tian et al, 2015). Abyssomicin C and tetrocarcin A exhibit significant activity against antibioticresistant Staphylococcus aureus and Mycobacterium tuberculosis, as well as for the treatment of various lymphomas and solid tumors (Braddock and Theodorakis, 2019)
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