Aims: To explore the potential to enhance secretion of heterologous proteins in Bacillus subtilis by engineering cell factors affecting extracytoplasmic protein folding and degradation. Methods and Results: Bottleneck components affecting the extracytoplasmic phase of protein secretion were genetically engineered and their effects on the secretion of 11 industrially interesting heterologous proteins were studied by Western blotting and enzymatic assays. Overproduction of PrsA lipoprotein enhanced the secretion of a-amylase of Bacillus stearothermophilus (fourfold) and pneumolysin (1AE5-fold). Increasing the net negative charge of the cell wall because of lack of the D D-alanine substitution of anionic cell wall polymers enhanced the secretion of pneumolysin c. 1AE5-fold. Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion. Pertussis toxin subunit, S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases. Conclusions: Secretion of heterologous proteins can be enhanced by engineering components involved in late stages of secretion in a protein-dependent manner. Significance and Impact of the Study: The study revealed both possibilities and limitations of modulating the post-translocational phase of secretion as a means to improve the yield of heterologous proteins.

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