Abstract
Linear plus linear homologous recombination-mediated recombineering (LLHR) is ideal for obtaining natural product biosynthetic gene clusters from pre-digested bacterial genomic DNA in one or two steps of recombineering. The natural product salinomycin has a potent and selective activity against cancer stem cells and is therefore a potential anti-cancer drug. Herein, we separately isolated three fragments of the salinomycin gene cluster (salO-orf18) from Streptomyces albus (S. albus) DSM41398 using LLHR and assembled them into intact gene cluster (106 kb) by Red/ET and expressed it in the heterologous host Streptomyces coelicolor (S. coelicolor) A3(2). We are the first to report a large genomic region from a Gram-positive strain has been cloned using LLHR. The successful reconstitution and heterologous expression of the salinomycin gene cluster offer an attractive system for studying the function of the individual genes and identifying novel and potential analogues of complex natural products in the recipient strain.
Highlights
An emerging idea in cancer biology is that tumors harbor a group of cells, known as cancer stem cells (CSCs), which have the unique ability to regenerate cancers[13,14]
In order to construct a vector for direct cloning of the salinomycin gene cluster, the four fragments each had a 50-bp overlapping sequence, as illustrated in Fig. 1, and were co-electroporated into GB05dir-gyrA4625, a CcdB-resistant E
Red/ET recombineering has traditionally been applied for heterologous expression of biosynthetic pathways to modify the biosynthetic pathways[30]
Summary
An emerging idea in cancer biology is that tumors harbor a group of cells, known as cancer stem cells (CSCs), which have the unique ability to regenerate cancers[13,14]. In vitro data revealed that salinomycin pre-treatment reduced the tumor-seeding ability of cancer cell lines greater than 100-fold over the chemotherapy drug paclitaxel. The compound interferes with potassium transport across mitochondrial membranes, reducing intracellular energy production. Upstream of the PKS genes, the adjacent orf[1], orf[2], and orf[3] do not belong to the salinomycin cluster, but salN and salO encode putative regulatory proteins. Downstream of the PKS genes, orf[18] is predicted to encode a peptidyl carrier protein, and targeted inactivation of orf[18] results in a 50–60% reduction in salinomycin production compared to wild-type[25]. We report the cloning of the 106-kb salinomycin gene cluster (salO-orf18) from the genomic DNA of Streptomyces albus DSM41398 by three rounds of direct cloning followed by assembling. The gene cluster was introduced into S. coelicolor A3(2) for successful heterologous production of salinomycin
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