Figure 1.

Diagram of myxochromide S gene cluster engineering. In the first step, the inverted repeat and MycoMar transposase gene (IR-Tps, i) plus amp (ii) were inserted into the mchS expression plasmid (iii) backbone by triple recombination to delete zeo and kan (iv). The MycoMar Tps plus IR fragment (i) was generated by PCR from the original MycoMar transposon (12). In the second step, the oriT-tet-trpE-cm-xylS section (13) in the backbone was replaced with a right IR and Tn5-kan cassette (v), which included a ribosomal binding site for the mchS gene cluster, by selection for kanamycin resistance. In the third step, the modified plasmid (vi) was electroporated into M. xanthus and kanamycin-resistant colonies were selected. To introduce the gene cluster into P. putida, an additional cassette containing oriT and the tetracycline-resistant gene (oriT-tetR-tet, vii) was inserted between amp and the pUC origin (pUC) to form the final construct (viii) for P. putida expression. See remarks in the Materials and Methods section.