Efficient secretion of Bacillus subtilis lipase A in Saccharomyces cerevisiae by translational fusion to the Pir4 cell wall protein

Abstract

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.

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

“…In our group, fusion with the cell wall protein Pir4 [10] has been used for the expression of Bacillus sp. BP-7 xylanase A [11], Bacillus subtilis lipase A [13] and the VP8* fragment of the rotavirus spike protein [12], all of them using this particular yeast strain as host.…”

“…IL-1 β was expressed in S. cerevisiae BY4741 using the signal peptide and pro domain of the Pir4 cell wall protein [10], previously used to express and secrete into the medium a Bacillus sp.BP-7 xylanase A [11], the VP8* fragment of rotavirus capsid protein [12] and Bacillus subtilis lipase A [13]. …”

Construction of a Highly Active Xylanase Displaying Oleaginous Yeast: Comparison of Anchoring Systems

“…Similarly, previous studies, in which ScPir4 has been linked to Bacillus sp. proteins, lipase A or xylanase A for display in S. cerevisiae , revealed that while 100% of ScPir4-xylanase was cell-bound, only 40% of the lipase A appeared to be anchored to the cell wall [3], [4]. Moreover, when a ScPir4 variant was used, which lacks the aforementioned functionally important Cys residues, only 12% of either protein was found to be cell bound.…”

“…This allows the fusion protein to form disulphide bonds with Aga1, another α-agglutinin subunit, which is in turn covalently linked to the cell wall through GPI anchor. The third system, called Protein Internal Repeat (or Pir)-CWP, is unrelated to the former and permits the covalent linkage of fusion proteins both to cell wall β-1,3 glucans and to structural proteins via disulfide bonds [3], [4]. Finally, an alternative, but rarely employed system consists of the non-covalent adsorption of an enzyme to the cell surface via interactions with a Chitin Binding Module (CBM) [5].…”

Expression of human interleukin-1β in Saccharomyces cerevisiae using PIR4 as fusion partner and production in aerated fed-batch reactor

“…Pir4 has already been successfully used as a fusion partner for the targeting of other proteins of interest such as xylanase A from Bacillus sp. BP-7 (Andrés et al 2005), the VP8* fragment from the rotavirus spike protein (Andrés et al 2006) and lipase A from Bacillus subtilis (Mormeneo et al 2008) either to the cell wall or to the culture medium.…”