Genetic Manipulation of Secondary Metabolite Production in Actinomycetes

Abstract

<p>The world is facing a public health threat due to increasing emergence of antibiotic resistance in pathogens. <em>Streptomyces </em>the soil-dwelling, Gram-positive, filamentous bacteria belonging to the family actinomycetes, are proven to be rich sources of natural antibiotics. Genome sequencing of <em>Streptomyces coelicolor, </em>a model organism of this genus, has revealed that in addition to the five antibiotics characterized so far, it possesses abundant genetic architecture of unexpressed biosynthetic or cryptic clusters for secondary metabolite production. The reason for their silence appears to be the poor understanding of their specific activation stimuli. In <em>Streptomyces coelicolor,</em> a pleiotropic regulator belonging to the two-component system family, <em>afsQ1</em>, has shown to activate the production of actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA). The aim of this research was to employ the genetically engineered <em>afsQ1</em> allele (named <em>afsQ1*</em>), which mimics the phosphorylated active form and obviates the need for specific external stimulus, and screen for novel antibiotic production. In this study, <em>afsQ1* </em>was introduced in various wild actinomycete isolates from the Wright Actinomycetes Collection (WAC) by conjugation and the resulting mutants were screened for antibiotic production. Two out of six WAC strains showed <em>afsQ1*- </em>induced antimicrobial activity. Interestingly, we were able to purify two antibiotic compounds, namely 1082 [M+2H]²⁺<strong> </strong>and 782 [M+H]⁺<strong> </strong>from the strain WAC00263. 1082 [M+2H]²⁺,<strong> </strong>a potentially novel antimicrobial peptide, exhibited activity against a wide range of Gram-positive bacteria including resistant pathogens such as vancomycin-resistant <em>Enterococcus</em> ATCC# 51299, a clinical isolate of methicillin resistant <em>Staphylococcus aureus</em>, and a clinical isolate of <em>S. aureus</em> BM3002. Moreover, it also showed activity against an opportunistic Gram-negative multi-drug resistant pathogen <em>Acinetobacter baumannii</em> B0098426R and a virulent strain of the fungus <em>Cryptococcus neoformans </em>H99<em>. </em>The second newly expressed molecule, 782 [M+H]⁺<strong> </strong>was not as potent as 1082 [M+2H]²⁺,<strong> </strong>so<strong> </strong>far only exhibited antimicrobial activity against the Gram-positive laboratory strains <em>Bacillus subtilis</em> #168 and <em>Micrococcus luteus</em>. These results reiterate that the technique of heterologous expression of the pleiotropic regulator, <em>afsQ1*</em>, in diverse actinomycetes is an excellent tool to induce novel antimicrobial production.</p>