Improving Protein Secretion in Bacteria

The major goals of this study are to investigate the genetic requirements for secretion of proteins via the alternative ESX secretion system in Bacillus subtilis and to evaluate the physical and molecular properties allowed for substrates. Secreted proteins are a major resource for industrial purposes, and the Bacilli are utilized abundantly for this purpose. Most protein secretion utilizes the housekeeping secretory (Sec) pathway. Cellular homeostasis, particularly under stress conditions, relies heavily on proteins that are natural substrates of the Sec pathway. Overproduction of heterologous proteins generates significant stresses on the cells and produces a direct competition with the endogenous housekeeping substrates of the Sec system. Moreover, the proteins secreted by the Sec system are exported as unfolded proteins, making them vulnerable to proteolytic cleavage in the extracellular environment. Work from our laboratory revealed that the alternative secretion system (ESX) can export stable, folded proteins and protein complexes and thus has the potential to circumvent several major challenges currently faced in industrial enzyme production. Our hypothesis is that additional genetic and molecular factors may be identified and defined to increase quantity and quality of protein secretion. We will pursue the following objectives related to this hypothesis:1. Dissect genetic requirements of the ESX secretion system in Bacillus subtilis1.1 Examine the genetic differences that contribute to differential secretion yield between B. subtilis strains.1.2 Identify genetic elements that contribute to ESX secretion in B. subtilis2. Determine the range of proteins that can be accommodated by the ESX system2.1 Examine the size of proteins substrates permissible for secretion by ESX.2.2 Assess the folding state of proteins secreted by ESX.2.3 Examine the need for unfolding and polypeptide threading during protein secretion.