Nonactin biosynthesis: the potential nonactin biosynthesis gene cluster contains type II polyketide synthase-like genes

Nonactin is the parent compound of a group of highly atypical polyketide metabolites produced by Streptomyces griseus subsp. griseus ETH A7796. In this paper we describe the isolation, sequencing, and analysis of 15? omitted?559 bp of chromosomal DNA, containing the potential nonactin biosynthesis gene cluster, from S. griseus subsp. griseus ETH A7796. Fourteen open reading frames were observed in the DNA sequence. Significantly, type II polyketide synthase (PKS) homologues were discovered in an apparent operon structure, which also contained the nonactate synthase gene (nonS), clustered with the tetranactin resistance gene. The deduced products of two of the genes (nonK and nonJ) are quite unusual ketoacyl synthase (KAS) alpha and KASbeta homologues. We speculate that nonactic acid, the polyketide precursor of nonactin, is synthesized by a type II PKS system.     

Increase in the toxic effects of Tl+ on isolated rat liver mitochondria in the presence of nonactin

The effects of Tl(+) ions on isolated rat liver mitochondria were studied in the presence of nonactin, a cyclic ionophore. Nonenergized rat liver mitochondria were increasingly swollen at an elevated concentration of Tl(+) in the 160 mOsm medium containing 0-150 mM sucrose and 0-75 mM TlNO(3) or 0-50 mM Tl acetate. On the contrary, mitochondria in experiments with nonactin were contracted in the medium with 5-25 mM Tl(+) and were swollen only in the medium with 50-75 mM TlNO(3) or 50 mM Tl acetate. State 4 respiration along with swelling of succinate-energized mitochondria followed contraction after their deenergization was further enhanced at increasing concentration of Tl acetate in a medium containing nonactin. Regardless of the presence of nonactin, State 3 and 2,4-dinitrophenol (DNP)-stimulated respiration and the monoamine oxidase (MAO) activity were not affected in the medium with 0-25 mM Tl acetate and sucrose. DNP-stimulated respiration decreased and the MAO activity somewhat increased in the medium containing 50 mM Tl acetate and nonactin. Uptake of (86)Rb(+) by energized mitochondria in the presence of valinomycin was considerably decreased when Tl(+) and nonactin were simultaneously present in the medium. An increase of the toxic effect of Tl(+) on rat liver mitochondria in the presence of nonactin is accounted for by disruption of mitochondria due to their more extensive swelling and uncoupling of mitochondria, resulting in the stimulation of State 4 and depletion of their energy store.     

Process analysis of macrotetrolide biosynthesis during fermentation by means of direct infusion LC-MS

The optimization of the biosynthetic pathways is highly attractive for the large-scale preparation of macrotetrolides, because overall yields in the chemical synthesis of compounds like nonactin have been very low. A key success factor determining the outcome of such optimizations is the adequate process analysis for the envisioned product. The analytical methods for process control involved in the past spectrophotometric and chromatographic measurements. LC-MS offers a modern approach to obtain more detailed data than the spectrophotometric and chromatographic measurements used in the past. In this work, a fast and versatile analytical LC-MS method has been set up, which has proven of much value for the in-process analysis of macrotetrolides during fermentation and which has allowed rapid large-scale bioprocess development.