Cell surface engineering of Bacillus subtilis improves production yields of heterologously expressed α-amylases

Haojie Cao, Auke J. van Heel, Hifza Ahmed, Maarten Mols, Oscar P. Kuipers

Bacillus subtilis is widely used as a cell factory for numerous heterologous proteins of commercial value and medical interest. To explore the possibility of further enhancing the secretion potential of this model bacterium, a library of engineered strains with modified cell surface components was constructed, and the corresponding influences on protein secretion were investigated by analyzing the secretion of α-amylase variants with either low-, neutral- or high- isoelectric points (pI). Relative to the wild-type strain, the presence of overall anionic membrane phospholipids (phosphatidylglycerol and cardiolipin) increased dramatically in the PssA-, ClsA- and double KO mutants, which resulted in an up to 47% higher secretion of α-amylase. Additionally, we demonstrated that the appropriate net charge of secreted targets (AmyTS-23, AmyBs and AmyBm) was beneficial for secretion efficiency as well. In B. subtilis, the characteristics of cell membrane phospholipid bilayer and the pIs of heterologous α-amylases appear to be important for their secretion efficiency. These two factors can be engineered to reduce the electrostatic interaction between each other during the secretion process, which finally leads to a better secretion yield of α-amylases.