Uncovering production of specialized metabolites by Streptomyces argillaceus: Activation of cryptic biosynthesis gene clusters using nutritional and genetic approaches

Abstract

Sequencing of Streptomyces genomes has revealed they harbor a high number of biosynthesis gene cluster (BGC), which uncovered their enormous potentiality to encode specialized metabolites. However, these metabolites are not usually produced under standard laboratory conditions. In this manuscript we report the activation of BGCs for antimycins, carotenoids, germicidins and desferrioxamine compounds in Streptomyces argillaceus, and the identification of the encoded compounds. This was achieved by following different strategies, including changing the growth conditions, heterologous expression of the cluster and inactivating the adpAa or overexpressing the abrC3 global regulatory genes. In addition, three new carotenoid compounds have been identified.

Fig 1. Gene organization of antimycins (anta), isorenieratene (crta), germicidines (gcsA) and desferrioxamine (desa) biosynthesis gene clusters in S. argillaceus.

Genes are shown to scale. Information about gene functions is shown in S2 to S5 Tables.

Fig 2. Chemical structures of compounds identified in S. argillaceus cultures in this work.

Fig 3. Production of specialized metabolites by S. argillaceus.

(A) antimycins (peaks 1 to 8): UPLC chromatogram at 230 nm of S. argillaceus grown in R5A (black) and in SM30 (red) media; (B) carotenoids: (left) Erlenmeyer flasks containing cultures of S. albus-pKC505 and S. albus-pKC505-C25, and (right) HPLC chromatograms at 450 nm showing production of carotenoids (peaks 9 to 15) of S. albus-pKC505 (black) and S. albus-pKC505-C25 (red) in SM17; (C) germicidins (peaks 16 and 17): UPLC chromatogram at 290 nm of S. argillaceus ΔadpAa (red) and S. argillaceus wild type (black). M, mithramycin; (D) desferrioxamine B (peak 18): HPLC chromatogram at 210 nm of S. argillaceus-pHJLAbrC3c (red) and S. argillaceus-pHJL401c (black). Asterisks correspond to increased production of unknown compounds.

Fig 4. Generation of mutant S. argillaceus ΔadpAa.

(A) Scheme representing the replacement event for generation of mutant ΔadpAa. WT, wild type strain; aac(3)IV, apramycin resistance gene; (B) PCR analysis of ΔadpAa mutant. PCR products from the wild type (WT) strain and from ΔadpAa mutant strain (M), using oligonucleotides SC3-F and adpAaCR. λ, Pst-digested Lambda DNA.