Production of secretory cutinase by recombinant Saccharomyces cerevisiae protoplasts

Hideki Aoyagi, Yoichi Katakura, Akio Iwasaki

During heterologous protein production using recombinant microbes, the protein tends to accumulate in the cell and may not be secreted. Here, we studied the production of secretory cutinase (heterologous protein) by recombinant Saccharomyces cerevisiae protoplasts. Recombinant S. cerevisiae cells (i.e., cells into which the cutinase gene was transferred) secreted trace amounts of cutinase into the broth. Approximately 28 % of the cutinase produced in the cells localized to the cell walls and/or between cell wall and cell membrane (CW). In comparison with cell culture, protoplasts in a static culture secreted measurable amounts of cutinase into the broth. Protoplasts were protected from physical and osmotic stresses by entrapping them in a membrane capsule with a low-viscous liquid-core of 1.92 % w/v calcium-alginate. To increase secretory cutinase production, the entrapped protoplasts were cultivated in a shake flask at low osmotic pressure without disruption. During 60 h of cultivation, the extracellular cutinase activity of the free protoplasts at 29.3 atm and protoplasts entrapped in the capsule at 17.2 atm were 0.13 and 0.39 U/mL, respectively. This is the first report which demonstrates that the efficient production of a secretable enzyme by using protoplasts isolated from recombinant microbes. This system described here is useful to produce products that accumulate in the CW.