Protein misfolding and degradation at the Bacillus membrane

Microorganisms, and the enzymes they produce, have been utilized by civilization for centuries. Outside their biological environment, recombinantly expressed enzymes play a crucial role in a wide range of industrial applications. The food, textile, paper, detergent or pharmaceutical industries have all been drastically transformed by the application of enzymes. In moving towards a more sustainable future, enzymes are a key element in transforming traditional chemical processes towards an environmentally friendly industry, due to their higher efficiencies and milder reaction conditions.
The non-pathogenic Gram-positive bacteria Bacillus subtilis is widely used as an expression host for industrial enzyme production due to its capability of producing and secreting high amounts of proteins into the extracellular media thus making the downstream processing more feasible.
In B. subtilis most secretory proteins are translocated through the widely conserved SecAYEG system. Proteins are exported through the Sec pathway in an unfolded state, and they emerge in an environment highly dominated by the negative charge and the metal cations of the cell wall. Proper and quick folding is essential to avoid undesired interactions with other proteins, the cell membrane or the cell wall. Folding catalysts play a very important role at this stage. If unfolded proteins are not cleared from the cell membrane and allowed to accumulate, they will form aggregates at the membrane-cell wall interface, thus triggering the secretion stress response. The major extracellular folding factor in B. subtilis is the essential protein PrsA. PrsA is a dimeric protein attached to the outer site of the membrane and has two domains: the NC and the PPiase (peptidyl prolyl cis/trans isomerase) domain. When PrsA dimerises, the combined NC domains form a hydrophobic bowl-like crevice. This crevice seems to be highly responsible for the specificity of PrsA and thought to accommodate extracellular unfolded polypeptides in-vivo. The secretion of several proteins has been shown to be PrsA dependent and the overexpression of PrsA have been repeatedly demonstrated to increase the secretion of industrial relevant alpha-amylases. However, only the overexpression of the native PrsA in Bacillus subtilis had been reported to facilitate this increased production.
This work was aimed at exploring the potential secretion boosting capacity of heterologous PrsA candidates. Furthermore, the study set out to map the effects of PrsA overexpression on the secretion stress response coupled with its ability to increase enzymes production.
In this work we show how the co-expression of several heterologous PrsA’s is more beneficial to increase production of certain heterologous amylases than the overexpression of the hosts PrsA. We also demonstrate how the co-expression of certain PrsA’s decreased the secretion stress response triggered by forced amylase production. We have presented the construction of an engineered PrsA with increased capability of supporting amylase secretion and decreasing secretion stress. In addition, we have studied the effect of amylase overproduction in the membrane proteome of Bacillus subtilis, and how this is affected by the co-expression of heterologous PrsA’s. Finally, we present the construction of a novel PrsA library as a tool to find the best foldase for a given secretory enzyme