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

Abstract

Background 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).ResultsRelative 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.ConclusionsIn 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.

Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis

“…While membrane engineering for enhanced biological production is a challenging strategy with increasing applications in bacteria (Chen, 2007;Chou, 2007), the approach is unpopular for B. subtilis. A documented study focused on the alteration of the lipid composition of the B. subtilis membrane, leading to enhanced secretion of the industrially important enzyme α-amylase (Cao, Heel, Ahmed, Mols, & Kuipers, 2017). Mutation of clsA or pssA, encoding phosphatidylserine synthase (PssA), significantly increased extracellular amylase production, presumably through creating a more negative membrane charge density (Cao et al, 2017).…”

“…A documented study focused on the alteration of the lipid composition of the B. subtilis membrane, leading to enhanced secretion of the industrially important enzyme α-amylase (Cao, Heel, Ahmed, Mols, & Kuipers, 2017). Mutation of clsA or pssA, encoding phosphatidylserine synthase (PssA), significantly increased extracellular amylase production, presumably through creating a more negative membrane charge density (Cao et al, 2017). In this study, we engineered the cell membrane of an HA-producing strain of B. subtilis to increase the functional expression of heterologously expressed SeHAS and, in turn, improve HA production.…”

EngineeringBacillusfor efficient production of heterologous protein: current progress, challenge and prospect

“…Moreover, their results implied that the secretion efficiency of amylase with low PI (5·70) was higher than those of amylase with PIs at 6·72, 6·41 and 5·88 (Cao et al . ). In addition, deletion of the dlt operon was proven to be an efficient tactic to improve the net negative charge of the cell surface for protein secretion (Hyyrylainen et al .…”

“…O-phosphatidyltransferase gene pssA and cardiolipin synthetase gene clsA improved the cell surface net negative charge, and the improvement of net negative charge enhanced the folding and stability of target protein, which further benefited protein secretion (Cao et al 2017). Moreover, their results implied that the secretion efficiency of amylase with low PI (5Á70) was higher than those of amylase with PIs at 6Á72, 6Á41 and 5Á88 (Cao et al 2017).…”

“…O-phosphatidyltransferase gene pssA and cardiolipin synthetase gene clsA improved the cell surface net negative charge, and the improvement of net negative charge enhanced the folding and stability of target protein, which further benefited protein secretion (Cao et al 2017). Moreover, their results implied that the secretion efficiency of amylase with low PI (5Á70) was higher than those of amylase with PIs at 6Á72, 6Á41 and 5Á88 (Cao et al 2017). In addition, deletion of the dlt operon was proven to be an efficient tactic to improve the net negative charge of the cell surface for protein secretion (Hyyrylainen et al 2000), and the yields of cyclodextrin glycosyltransferase (Craynest et al 2003), rPA (Thwaite et al 2002), a-amylase, nattokinase and b-mannanase were all significantly enhanced in the dlt deletion strains.…”