Production of secretory cutinase by recombinant Saccharomyces cerevisiae protoplasts

Abstract

Background: 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.

Findings: 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.

Conclusions: 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.

Keywords: Cutinase; Immobilization; Protoplast; Recombinant yeast.

Fig. 1

Time course of the extracellular cutinase activity of cultured S. cerevisiae cells bearing pYES/EXL, protoplasts bearing pYES/EXL, and protoplasts bearing pYES3/CT. All experiments were performed in triplicate. The results are expressed as means; there was less than 5 % deviation in the results. In the cell culture at 72 h, the A ₆₀₀ was 6.0

Fig. 2

Time course of the galactose consumption (a) and extracellular cutinase activity (b) of S. cerevisiae cells and protoplasts (bearing pYES/EXL) immobilized in various kinds of gel in suspension culture. All experiments were performed in triplicate. The results are expressed as means; there was less than 5 % deviation in the results. Symbols: blue triangles with a solid line, cells immobilized in a 2.5 % w/v calcium-alginate gel; green circles with a solid line, cells immobilized in a 3.0 % w/v agarose block; red squares with a solid line, cells entrapped in a 1.92 % w/v calcium-alginate capsule; blue triangles with a dotted line, protoplasts immobilized in a 2.5 % w/v calcium-alginate gel; green circles with a dotted line, protoplasts immobilized in a 3.0 % w/v agarose block; red squares with a dotted line, protoplasts entrapped in a 1.92 % w/v calcium-alginate capsule

Fig. 3

Effect of the osmotic pressure of the medium on the extracellular production of cutinase by S. cerevisiae protoplasts (bearing pYES/EXL) entrapped in a 1.92 % w/v calcium-alginate capsule. All experiments were performed in triplicate. The results are expressed as means; there was less than 5 % deviation in the results