The discovery of new enzymes for industrial application relies on a robust discovery pipeline. Such a pipeline should facilitate efficient molecular cloning, recombinant expression and functional screening procedures. Previously, we have developed a vector set for heterologous expression in Escherichia coli. Here, we supplement the catalogue with vectors for expression in Bacillus. The vectors are made compatible with a versatile cloning procedure based on type IIS restriction enzymes and T4 DNA ligase, and encompass an effective counter-selection procedure and complement the set of vectors with options for secreted expression. We validate the system with expression of recombinant subtilisins, which are generally challenging to express in a heterologous system. The complementarity of the E. coli and Bacillus systems allows rapid switching between the two commonly used hosts without comprehensive intermediate cloning steps. The vectors described are not limited to the expression of certain enzymes, but could also be applied for the expression of other enzymes for more generalized enzyme discovery or development.
Keywords: Bacillus; ccdB; cloning; recombinant DNA technology; subtilisin.