CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

Abstract

1.AbstractThe Gram-positive bacteriumBacillus subtilishas long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacteriumBacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the host’s intrinsic natural competence and homologous recombination capabilities. By supplying our reporter strains with a linearized, low copy number plasmid containing homology regions left and right of the reporter genes and an antibiotic resistance marker, we could show an up to 3.6-fold highergfp(green fluorescent protein) expression and up to 1.3-fold highermPLC(mature phospholipase C) expression after successful recombination and thus circularization of our plasmid. Furthermore, the plasmid-bornegfpexpression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher.