Isolation and Biosynthesis of an Azoxyalkene Compound Produced by a Multiple Gene Disruptant of Streptomyces rochei

Abstract

Streptomyces rochei 7434AN4 predominantly produces lankacidin and lankamycin under normal culture conditions, thus suggesting that other biosynthetic gene clusters for secondary metabolites are silent. To identify the silent metabolites of 7434AN4, we constructed mutant KA57 with multiple disruptions of the transcriptional repressor srrB and the biosynthesis genes for both antibiotics. KA57 accumulated a compound (KA57A) with a strong UV absorption at 235 nm, not detected in the parent strain or other mutants. Various spectroscopic analyses revealed that KA57A is an azoxyalkene compound with the molecular formula C10 H20 N2 O3 and with the R configuration at C-2. Biosynthesis of KA57A was also studied by feeding with labeled acetates, amino acids, and 1-hexylamine. The hexenyl moiety (C1'-C6') was derived from fatty acid, whereas the 3-aminobutan-1,2-diol moiety (C1-C4) was derived from C-2 of acetate (C1) and serine (C2-C4). Incorporation of [1,1-(2) H2 ]-1-hexylamine indicated that C1'-C2' dehydrogenation occurs as the final step of biosynthesis.

SrrB, a Pseudo-Receptor Protein, Acts as a Negative Regulator for Lankacidin and Lankamycin Production in Streptomyces rochei

“…A mutant of the repressor gene ksbC accumulated β-carboline compound kitasetaline in Kitasatospora setae (Aroonsri et al, 2012). To our surprise, azoxyalkene compound KA57-A accumulated in a triple knockout mutant of S. rochei that have mutations on two biosynthetic gene clusters for LC, LM, and srrB (Kunitake et al, 2015). The genome sequence of the S. rochei chromosome has been determined to be 8.36 Mb in size, and at least 35 secondary metabolites gene clusters are coded on the chromosome (Nindita et al, 2019).…”

The genome sequence of Streptomyces rochei 7434AN4, which carries a linear chromosome and three characteristic linear plasmids

“…We isolated an azoxyalkene compound KA57-A (Fig. 1) from a genetically engineered strain KA57, which contains triple mutations on srrB (a tetR -type receptor gene), lkcF-KR1 (a ketoreductase domain 1 of lkcF for lankacidin biosynthesis), and lkmE (a type-II thioesterase gene for lankamycin biosynthesis) coded on pSLA2-L 35 . KA57-A has a unique azoxy group (N = N + -O − ) and its biosynthetic gene ( azx ) cluster was located at nt 2,061,273–2,097,283 of the chromosome by comparison with the BGC for valanimycin 40 .…”

Chemistry and Biology of Natural Azoxy Compounds

“…56 Motivated by its prominent activity, elaiomycin (1) and five structurally related compounds, elaiomycins D−H (2−6), were isolated from Streptomyces sp. Streptomyces chattanoogensis L10 not reported 14,30 azoxybenzene-4,4′-dicarboxylic acid (24) Entomophthora virulenta not reported 31,32 4′-hydroxy-methylazoxybenzene-4-carboxylic acid (25) Entomophthora virulenta insecticidal 31,32 strain HKI0708. 17 All compounds showed weak antimicrobial activity against several tested bacterial strains.…”

“…However, the configuration of the secondary alcohol carbon in 16) exhibited weak inhibition towards the fungus Rhodotorula sp. 24 Jietacins. Jietacins A (17) and B ( 18) are Streptomycesderived natural products containing a unique terminal vinyl azoxy group and a long aliphatic chain.…”

“…While gene clusters have not been reported for the elaiomycins, analysis of the genome of the KA57A producer Streptomyces rochei revealed the presence of homologues for VlmJ and VlmK, the kinase and dehydratase that dehydrate the serine residue. 24 Besides investigations of bacterial azoxy metabolite biosynthesis, the fungal product lyophyllin (35) was studied by labeling experiments. Incorporation of 13 C-labeled N-hydroxy-N′,N′-dimethylurea confirmed it as a precursor, 42 indicating that both fungi and bacteria employed N-hydroxy compounds as an important intermediate.…”