Figure 2 | Scientific Reports

Figure 2

From: Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET recombineering

Figure 2

General scheme of direct cloning and heterologous expression of PKS/NRPS gene clusters in B. subtilis via Red/ET recombineering.

In step 1, the digested genomic DNA of the producer strain and linear cloning vector carrying the 70-bp homologous arm with target gene cluster were co-transferred in E. coli GB05-dir to directly clone the target gene cluster into the integration vector by RecET-mediated LLHR. In an optional step (if necessary), some of the cloned gene clusters were further modified by Redγβα-mediated LCHR(linear plus circular homologous recombination), for example, to delete homologous region or key genes, exchange promoters, and add extra genes or elements. In step 2, if the cloned gene clusters did not require further modification, the cloned gene cluster would be directly integrated into the amyE gene locus of the heterologous host B. subtilis by natural competence transformation to express the candidate secondary metabolites.

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