Diazaquinomycin Biosynthetic Gene Clusters from Marine and Freshwater Actinomycetes

Abstract

Tuberculosis is an infectious disease of global concern. Members of the diazaquinomycin (DAQ) class of natural products have shown potent and selective activity against drug-resistant Mycobacterium tuberculosis. However, poor solubility has prevented further development of this compound class. Understanding DAQ biosynthesis may provide a viable route for the generation of derivatives with improved properties. We have sequenced the genomes of two actinomycete bacteria that produce distinct DAQ derivatives. While software tools for automated biosynthetic gene cluster (BGC) prediction failed to detect DAQ BGCs, comparative genomics using MAUVE alignment led to the identification of putative BGCs in the marine Streptomyces sp. F001 and in the freshwater Micromonospora sp. B006. Deletion of the identified daq BGC in strain B006 using CRISPR-Cas9 genome editing abolished DAQ production, providing experimental evidence for BGC assignment. A complete model for DAQ biosynthesis is proposed based on the genes identified. Insufficient knowledge of natural product biosynthesis is one of the major challenges of productive genome mining approaches. The results reported here fill a gap in knowledge regarding the genetic basis for the biosynthesis of DAQ antibiotics. Moreover, identification of the daq BGC shall enable future generations of improved derivatives using biosynthetic methods.

Figure 1.

Structures of diazaquinomycin (DAQ) natural products and related compounds. The bacterial and environmental source is indicated below each structure.

Figure 2.

Diazaquinomycin BGCs from Streptomyces sp. F001 and Micromonospora sp. B006. Homologous genes are connected by grey areas. Genes are color-coded by proposed function as shown.

Figure 3.

Deletion of the putative daq biosynthetic gene cluster abolishes DAQ production in Micromonospora sp. B006. (A) Design of CRISPR-Cas9 genome editing. (B) PCR using primers oJB172/174; expected fragment lengths are 21.5 kb (wt), and 3.5 kb (mutants). (C) PCR using primers oJB140/174; expected fragment lengths are 2 kb (wt), and no band (mutants). (D) HPLC chromatograms, extracted at 280 nm, highlighting the m/z values for DAQ peaks, along with their overlaid UV spectra shown in full scale. The m/z values for DAQ peaks were obtained by UHPLC-QTOFMS (Figure S3). wt, wild type.

Figure 4.

Proposed pathway for biosynthesis of the diazaquinomycin central core. The first four steps involving DaqJ, DaqG, DaqH and DaqI and leading to 3-hydroxy-anthranilic acid are according to the proposal for diazepinomicin biosynthesis by McAlpine et al. See text for further details.

Figure 5.

Proposed pathway for biosynthesis of diazaquinomycin’s side rings. Proposed model to explain the molecular basis for the distinct DAQ derivatives produced by Micromonospora sp. B006 and Streptomyces sp. F001. We hypothesize that DaqN_B006 preferentially accepts malonyl-ACP (or malonyl-CoA) and a branched-chain fatty acyl-CoA or acetyl-CoA monomer as substrates, whereas DaqN_F001 preferentially accepts malonyl-ACP (or malonyl-CoA) and a straight-chain fatty acyl-CoA monomer. DaqU, which is present only in strain F001 would preferentially accept methylmalonyl-ACP (or methylmalonyl-CoA). CoA, coenzyme A. ACP, acyl carrier protein.

Figure 6.

Proposed final steps in diazaquinomycin biosynthesis.