Applications for Bacillus megaterium in synthetic biology and modular protein and peptide secretion

Abstract

The second half of our work describes the development and characterization of a novel system for modular protein/peptide secretion from B. megaterium. Secretion can often lead to increased stability and solubility of recombinant proteins/peptides, as well as a simplified purification scheme. As B. megaterium couples efficient protein secretion with a low-protease extracellular environment it may be an ideal host for the production of peptides, which have been difficult to produce using traditional methods because of their susceptibility to protease degradation. Recombinant proteins/peptides which are not naturally secreted can be secreted from bacterial hosts by fusing a short signal peptide (SP) taken from a naturally secreted protein to their N-terminus. However, recombinant secretion has been an underutilized tool largely due to the development time required to find a match between SP and protein that will provide for efficient secretion. The new system described here consists of a SP, Linker, and Intein (SPLInt) fused to a variable target protein or peptide. The SPLInt system provides efficient passage of a target protein/peptide outside of the cell, followed by intein-mediated cleavage to release the target for extracellular accumulation. We anticipate that these new tools will expand the applications for B. megaterium, and increase its use in both academia and industry.

Bacillus megaterium is a Gram-positive organism that holds great promise as a recombinant host in the biotechnology industry. It displays stable maintenance of plasmids, high-level recombinant expression, efficient protein secretion, and low extracellular protease activity. Here we describe the development of new tools for making B. megaterium a more attractive host for both research and commercial bioprocessing. The first half of this work describes the construction of new synthetic communication systems for regulating gene expression in B. megaterium. As our bioprocessing targets become increasingly complex, a clear focus must be placed on bioprocess efficiency and productivity. Two such approaches include: adding control over expression levels and timing of proteins along a metabolic pathway within a single cell, and splitting up metabolic tasks among multiple populations in a microbial consortia. Here we use the peptide-based agr quorum-sensing (QS) system native to Staphylococcus aureus to build synthetic communication systems in B. megaterium. We describe some of the first synthetic cell-cell communications systems to function from Gram-negative (Escherichia coli) to Gram-positive (B. megaterium) hosts, and between two populations of a Gram-positive host (B. megaterium). Furthermore we use the agr QS machinery to build a synthetic QS system in B. megaterium for population-wide autoinduction of recombinant genes.