Abstract
Genome mining provides exciting opportunities for the discovery of natural products. However, in contrast to traditional bioassay-guided approaches, challenges of genome mining include poor or no expression of biosynthetic gene clusters (BGCs). Additionally, given that thousands of BGCs are now available through extensive genome sequencing, how does one select BGCs for discovery? Synthetic biology techniques can be used for BGC refactoring and activation, whereas resistance-gene-directed genome mining is a promising approach to discover bioactive natural products. Here we report the selection of a BGC by applying a resistance-gene-directed approach, cloning of the silent BGC from Micromonospora sp. B006, promoter exchange, and heterologous expression in Streptomyces coelicolor M1152. While we have yet to identify the encoded compound, we unexpectedly observed induction of a host metabolite, which we hypothesize is due to the presence of a ClpC chaperone gene in the BGC, suggesting that ClpC chaperones may be used for BGC activation.
Highlights
Natural products are an exceptional source of drug leads
A basic local alignment search tool (BLAST)[35] search and subsequent phylogenetic analysis revealed nine genes encoding putative caseinolytic protease (Clp) subunits in the genome of strain B006, i.e., three ClpP proteases (MicB006_0681, MicB006_3937, and MicB006_3938), one ClpX subunit (MicB006_3936), one ClpB subunit (MicB006_0157), as well as four potential caseinolytic protease C (ClpC) adenosine triphosphatase (ATPase) (MicB006_1895, MicB006_4945, MicB006_3122, and MicB006_6016). This biosynthetic gene clusters (BGCs) meets the requirements of target-directed genome mining, that is, it contains an extra copy of a housekeeping gene in association with natural product biosynthetic genes
Freshwater Actinobacteria remain underexplored as a source of natural products
Summary
Natural products are an exceptional source of drug leads. For instance, the vast majority of antibiotics and anticancer agents in the clinic today are natural products or derivatives thereof. Direct TAR cloning of the NRPS-PKS gene cluster from genomic DNA was carried out using a previously reported protocol with minor modifications.[7] Solutions used were prepared according to Kouprina and Larionov.[24] Spheroplast cells were prepared using Zymolyase (100T equivalent; Zymo Research) at a final concentration of 1 mg mL-1. The 20 μL reactions consisted of 0.2 mM of each deoxynucleoside triphosphate (dNTP), 3% dimethylsulfoxide (DMSO), 0.5 μM (each) primer, and 0.02 U μL-1 Phusion HighFidelity DNA polymerase (Thermo Fisher Scientific) in the buffer supplied with the enzyme and using the following thermal cycling parameters: 30 s at 98 °C; 35 cycles of 98 °C for 10 s, 62.8/65 °C for 20 s, and 72 °C for 30 s; and a terminal extension for 5 min at 72 °C.
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