Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

Abstract

This study focused on the growth of Saccharomyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP) 2 and (WP) 4 , respectively. Different cultivation and strategies were tested in a 2-L shake¯ask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (speci®c cell activity) of 4.1 Umg )1 with activity per protein broth (speci®c activity) of 266 Umg )1 , whereas cutinase-(WP) 2 was secreted with a speci®c cell activity of 2.1 Umg )1 with a speci®c activity of 200 Umg )1 , and cutinase-(WP) 4 with a speci®c cell activity of 0.7 Umg )1 with a speci®c activity of 15 Umg )1 . The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake¯ask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture). ã

Prediction of retention time of cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatography

“…In general small differences (RMSE < 0.022) between observed and estimated retention times was obtained when applying both the average surface hydrophobicity and the tagged surface hydrophobicity definitions. However, the methodology based on tagged hydrophobic definition (Φtagged) proposed by Simeonidis et al [28], which assumes that amino acids in the tag have a fully exposed surface, proved to be more adequate, since it presented a lower divergence between predicted and experimental retention times (RMSE < 0.022) for the two tagged cutinases evaluated in three different resins. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of cutinases tagged with hydrophobic peptides in HIC.…”

“…(fusion peptide composed of four tryptophan residues interspersed with four proline residues) producing Saccharomyces cerevisiaeMM01strains (Mata, leu2-3, ura3, gal1: URA3, MAL-8, MAL3, SUC3), containing the expression vectors pUR7320, pUR807, and pUR806, respectively, were constructed and provided by Unilever Research Laboratory, Vlaardingen, The Netherlands within the European Union project: Integrated bioprocess design for large scale production and isolation of recombinant proteins [28] (BIO4-CT96-0435).…”

Modeling and optimization of cutinase production by recombinant Escherichia coli based on statistical experimental designs

“…Cutinase can be produced by both fungi and bacteria. Up to now, most studies have focused on the selection of cutinase-producing strains [12], process optimization of microbial cutinase production [13][14][15][16], and cutinase extraction and characterization [17][18][19]. Calado et al developed a fed-batch culture strategy for enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strain [14].…”