Azinothricin

Background: Polyoxypeptin A was isolated from a culture broth of Streptomyces sp. MK498-98 F14, which has a potent apoptosis-inducing activity towards human pancreatic carcinoma AsPC-1 cells. Structurally, polyoxypeptin A is composed of a C₁₅ acyl side chain and a nineteen-membered cyclodepsipeptide core that consists of six unusual nonproteinogenic amino acid residues (N-hydroxyvaline, 3-hydroxy-3-methylproline, 5-hydroxypiperazic acid, N-hydroxyalanine, piperazic acid, and 3-hydroxyleucine) at high oxidation states. Results: A gene cluster containing 37 open reading frames (ORFs) has been sequenced and analyzed for the biosynthesis of polyoxypeptin A. We constructed 12 specific gene inactivation mutants, most of which abolished the production of polyoxypeptin A and only ΔplyM mutant accumulated a dehydroxylated analogue polyoxypeptin B. Based on bioinformatics analysis and genetic data, we proposed the biosynthetic pathway of polyoxypeptin A and biosynthetic models of six unusual amino acid building blocks and a PKS extender unit. Conclusions: The identified gene cluster and proposed pathway for the biosynthesis of polyoxypeptin A will pave a way to understand the biosynthetic mechanism of the azinothricin family natural products and provide opportunities to apply combinatorial biosynthesis strategy to create more useful compounds.

The azinothricin family comprises several cyclic hexadepsipeptides with diverse pharmacological bioactivities, including antimicrobial, antitumoral, and apoptosis induction. In this work, using a genome mining approach, a biosynthetic gene cluster encoding an azinothricin-like compound was identified from the Streptomyces sp. s120 genome sequence (pop BGC). Comparative MS analysis of extracts from the native producer and a knockout mutant led to the identification of metabolites corresponding to the pop BGC. Furthermore, regulatory elements of the BGC were identified. By overexpression of an LmbU-like transcriptional activator, the production yield of 1 and 2 was increased, enabling isolation and structure elucidation of polyoxyperuin A seco acid (1) and polyoxyperuin A (2) using high-resolution mass spectrometry and NMR spectroscopy. Compound 1 exhibited a low antibiotic effect against Micrococcus luteus, while 2 showed a strong Gram-positive antibiotic effect in a micro-broth-dilution assay.

(R)-2-Hydroxybutyric acid ((R)-2-HBA) is an important building block for azinothricin family of antitumour antibiotics and biodegradable poly(2-hydroxybutyric acid). However, optically active (R)-2-HBA could not be produced through microbial fermentation or chemical synthesis. Several biocatalytic methods have been reported for the production of (R)-2-HBA. Those processes used expensive and scarce substrates and would not be suitable for practical application. In this work, Gluconobacter oxydans DSM 2003 was confirmed to have the ability to produce (R)-2-HBA from 1,2-butanediol, a non-toxic and inexpensive compound that had a great potential for biotechnological processes. Under the optimal conditions, the biocatalytic process produced (R)-2-HBA at a high concentration (18.5 g l(-1)) and a high enantiomeric excess (99.7%). The biocatalysis process introduced in this study may provide a technically and economically interesting route for production of (R)-2-HBA.