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Heterologous expression of biosynthetic pathways is an important way to research and discover microbial natural products.  Bacillus subtilis  is a suitable host for the heterologous production of natural products from bacilli and related Firmicutes. Existing technologies for heterologous expression of large biosynthetic gene clusters in  B. subtilis  are complicated. Herein, we present a simple and rapid strategy for direct cloning based heterologous expression of biosynthetic pathways in  B. subtilis  via Red/ET recombineering, using a 5.2 kb specific direct cloning vector carrying homologous sequences to the  amyE  gene in  B. subtilis  and CcdB counterselection marker. Using a two-step procedure, two large biosynthetic pathways for edeine (48.3 kb) and bacillomycin (37.2 kb) from  Brevibacillus brevis  X23 and  B. amyloliquefaciens  FZB42, respectively, were directly cloned and subsequently integrated into the chromosome of  B. subtilis  within one week. The gene cluster for bacillomycin was successfully expressed in the heterologous host, although edeine production was not detectable. Compared with similar technologies, this method offers a simpler and more feasible system for the discovery of natural products from bacilli and related genera.

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