The Identification and Heterologous Expression of the Biosynthetic Gene Cluster Encoding the Antibiotic and Anticancer Agent Marinomycin

Abstract

With the rise in antimicrobial resistance, there is an urgent need for new classes of antibiotic with which to treat infectious disease. Marinomycin, a polyene antibiotic from a marine microbe, has been shown capable of killing methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF), as well as having promising activity against melanoma. An attractive solution to the photoprotection of this antibiotic has been demonstrated. Here, we report the identification and analysis of the marinomycin biosynthetic gene cluster (BGC), and the biosynthetic assembly of the macrolide. The marinomycin BGC presents a challenge in heterologous expression due to its large size and high GC content, rendering the cluster prone to rearrangement. We demonstrate the transformation of Streptomyces lividans using a construct containing the cluster, and the heterologous expression of the encoded biosynthetic machinery and production of marinomycin B.

Keywords: MRSA; VREF; actinomycete; antibiotic; biosynthetic gene cluster; heterologous expression; marine natural product; melanoma; synthetic biology.

Figure 1

Marinomycin (1), and SIA7248 (2). Key differences between the metabolites are highlighted in red.

Figure 2

Comparison of the very similar marinomycin and SIA7248 BGCs. (Top) the validated HMGS cassette (marJ) contained in the predicted BGC for marinomycin. (Bottom) known BGC for SIA7248. Links with arrows show genes common to both biosynthetic pathways.

Figure 3

Ion-extracted LC-MS trace of the cultures of marinomycins’ production of WT and mutant strains. Black: WT CNQ 140; red: Δhmgs, the hmgs knock-out mutant (CNQ 140/hmgs⁻ (hmgs::aac(3)IV-oriT), no marinomycins produced); orange: Δhmgs/hmgs, a genetic complementation strain, restoring the production of marinomycins.

Scheme 1

(Top) BGC for the production of Marinomycin A. (Bottom) Proposed biosynthetic pathway for the production of Marinomycin. KS, DH and KR in module 2 and the first ACP in module 12, indicated by grey shadowed circles, are inactive based on bioinformatics analyses (details of domains’ analysis can be found in SI Section S8, Figures S15–S20). UN in module 3 is tentatively referred to as a UN domain, because no function can be predicted for this whole domain using classical PKS domain bioinformatics analysis. Key differences between SIA7248 and marinomycin are highlighted in red.

Figure 4

Grey: the natural producer “Marinispora” CNQ-140 extracted supernatant EIC shows marinomycins A, B, and C. White: the heterologous host S. lividans extracted supernatant EIC shows marinomycins B and C. Top grey: natural producer, marinomycin B, retention time (RT) 2.94 min, m/z 995.50 [M−H]⁻. Bottom white: heterologous host S. lividans, marinomycin B, RT: 2.99 min m/z 995.50 [M−H]⁻. Details can be checked in SI Section S5, Figures S13 and S14.