Biosynthesis of ilamycins featuring unusual building blocks and engineered production of enhanced anti-tuberculosis agents

Abstract

Tuberculosis remains one of the world's deadliest communicable diseases, novel anti-tuberculosis agents are urgently needed due to severe drug resistance and the co-epidemic of tuberculosis/human immunodeficiency virus. Here, we show the isolation of six anti-mycobacterial ilamycin congeners (1-6) bearing rare L-3-nitro-tyrosine and L-2-amino-4-hexenoic acid structural units from the deep sea-derived Streptomyces atratus SCSIO ZH16. The biosynthesis of the rare L-3-nitrotyrosine and L-2-amino-4-hexenoic acid units as well as three pre-tailoring and two post-tailoring steps are probed in the ilamycin biosynthetic machinery through a series of gene inactivation, precursor chemical complementation, isotope-labeled precursor feeding experiments, as well as structural elucidation of three intermediates (6-8) from the respective mutants. Most impressively, ilamycins E₁/E₂, which are produced in high titers by a genetically engineered mutant strain, show very potent anti-tuberculosis activity with an minimum inhibitory concentration value ≈9.8 nM to Mycobacterium tuberculosis H37Rv constituting extremely potent and exciting anti-tuberculosis drug leads.Tuberculosis (TB) remains one of the world's deadliest communicable diseases, novel anti-TB agents are urgently needed due to severe drug resistance and the co-epidemic of TB/HIV. Here, the authors show that anti-mycobacterial ilamycin congeners bearing unusual structural units possess extremely potent anti-tuberculosis activities.

Fig. 1

Structures of ilamycins. Compounds 1–6 were isolated from S. atratus SCSIO ZH16 wild-type strain, compounds 6–8 were isolated from engineered mutant strains

Fig. 2

HPLC analyses of fermentation broths. (i) wild-type S. atratus SCSIO ZH16; (ii) ΔilaS mutant; (iii) ΔilaO mutant; (iv) ΔilaG mutant; (v) ΔilaE mutant; (vi) ΔilaD mutant n; (vii) ΔilaF mutant; (viii) ΔilaC mutant; (ix) ΔilaH mutant; (x) ΔilaD mutant fed with AHA; (xi) ΔilaD mutant fed with 4-HA; (xii) ΔilaE mutant fed with AHA; (xiii) ΔilaE mutant fed with 4-HA; (xiv) ΔilaE mutant fed with 2,4-HDA; (xv) ΔilaM mutant; (xvi) ΔilaN mutant; (xvii) ΔilaL mutant; (xviii) ΔilaM mutant fed with 3-NO₂-tyr; (xix) ΔilaN mutant fed with 3-NO₂-tyr; (xx) ΔilaR mutant; the peaks labeled with asterisks are not ilamycin analogs judged by HPLC–DAD–UV analysis

Fig. 3

Biosynthetic gene cluster and proposed biosynthetic pathway of ilamycins. a Organization of the ilamycin gene cluster. b Biosynthetic pathway of ilamycins. c The prenylation of Trp. d The nitration of Tyr. e The biosynthesis of L-AHA unit. f The post-tailoring biosynthetic steps en route to ilamycins. A adenylation, C condensation, T thiolation, MT methylation, TE thioesterase, KS keto synthases, ACP acyl carrier protein, AT acyl transferase, AT-L acyl transferase-like protein, DH dehydratase, KR keto reductase, ER enoyl reductase