Abstract
Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning “plug-and-play” approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.
Highlights
Bacilli are ubiquitous low G1C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules
Their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare
While sophisticated molecular biology techniques have been firmly established to connect biosynthetic gene clusters to encoded natural product molecules in Actinobacteria and Proteobacteria through the use of model expression hosts such as Streptomyces coelicolor and Escherichia coli, Firmicutes, which contain the natural product rich genus Bacillus, are lacking comparable molecular tools to support the heterologous expression of large natural product pathways[3,4]
Summary
Bacilli are ubiquitous low G1C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules Their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. We adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis After validating this direct cloning ‘‘plug-and-play’’ approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. We report the design and implementation of a versatile vector to support the direct capture of Bacillus biosynthetic gene clusters from genomic DNA by transformation-associated recombination (TAR)[5] in yeast and heterologous expression in the model host Bacillus subtilis. This work represents a very useful approach to interrogate the function of biosynthetic gene clusters in Bacillus through heterologous biosynthesis
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call