Heterologous expression and genetic engineering of the phenalinolactone biosynthetic gene cluster by using Red/ET recombineering

The heterologous expression of natural product biosynthetic pathways is of increasing interest in biotechnology and drug discovery, This approach enables the production of complex metabolites in more amenable host organisms and provides the basis for the generation of novel analogues through genetic engineering. Here we describe a straightforward strategy for the heterologous expression of the highly complex phenalinolactone biosynthetic pathway, which was recently cloned from Streptomyces sp. Tu6071. The biosynthetic gene cluster comprises at least 11 transcriptional units that harbor 35 genes, which together catalyze the assembly of structurally unique tricyclic terpene glycosides with antibacterial activity. By using Red/ET recombineering, the phenalinolactone pathway was reconstituted from two cosmids and heterologously expressed in several Streptomyces strains. The established expression system now provides a convenient platform for functional investigations of the biosynthetic genes and the generation of novel analogues, by genetic engineering of the pathway in Escherichia coli. Deletion of a modifying gene from the expression construct resulted in a novel, unglycosylated phenalinolactone derivative; this demonstrates the promise of this methodology.