Efficient secretion of Bacillus subtilis lipase A in Saccharomyces cerevisiae by translational fusion to the Pir4 cell wall protein.
Both the secretion and the cell surface display of Bacillus subtilis lipase A (Lip A) in Saccharomyces cerevisiae was investigated using different domains of the cell wall protein Pir4 as translational fusion partners. LipA gene minus its leader peptide was fused inframe in two places of PIR4 to achieve cell wall targeting, or substituting most of the PIR4 sequence, after the signal peptide and the Kex2 processed subunit I of Pir4 to achieve secretion to the growth medium. Expression of the recombinant fusion proteins was investigated in a standard and a glycosylation-deficient strain of S. cerevisiae, grown in selective or rich medium. Fusion proteins intended to be retained at the cell wall were secreted to the growth medium, most likely as result of the degradation of the Pir4 moiety containing the cell wall retention domain, giving low levels of lipase activity. However, the fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 400 IU of lipase activity per milliliter of cell supernatant. This is, to our knowledge, the first report of the efficient production, as a secreted protein, of lipase A of B. subtilis in baker's yeast.
Mormeneo
María
M
Unidad de Microbiología. Facultad de Farmacia, Burjassot, Valencia, Spain.
Andrés
Isabel
I
Bofill
Cristina
C
Díaz
Pilar
P
Zueco
Jesús
J
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism*; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C482291:CIS3 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D008049:Lipase