Cloning of the Zygosaccharomyces bailii GAS 1 homologue and effect of cell wall engineering on protein secretory phenotype

Abstract

BackgroundZygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a β-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion.ResultsThe GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii Δgas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of ~400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Δgas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1β secretion disruption had no relevant effect.ConclusionsThe data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as already widely reported in literature, our data confirmed that an "always working" solution to the problems related to recombinant protein production can be hardly, if never, found; instead, manipulations have to be finely tuned for each specific product and/or combination of host cell and product.

Advances in molecular tools for the use of Zygosaccharomyces bailii as host for biotechnological productions and construction of the first auxotrophic mutant

“…However, one would imagine that even longer flanking regions could help to raise the frequency of the event, which in our case remained low. Remarkably, we found that the deletion of another, nonessential, gene (ZbGAS1) required only about 400-bp-long flankings (Passolunghi et al, 2010), in agreement with the data of Mollapour & Piper (2001). The targeted gene replacement can be considered a corruption of the repair process of DNA double-strand breaks by homologous recombination, as it leads to permanent loss of the targeted chromosomal region rather than restoration of a damaged sequence (Rothstein, 1983;Langston & Symington, 2005).…”

“…For example, in the nonconventional yeast Hansenula polymorpha, flanking regions of at least 1 Kbp seem to be a key factor in obtaining satisfactory homologous recombination efficiency (about 50%) (Gonzalez et al, 1999). Remarkably, we found that the deletion of another, nonessential, gene (ZbGAS1) required only about 400-bp-long flankings (Passolunghi et al, 2010), in agreement with the data of Mollapour & Piper (2001). Hence, it appears that the length of homologous recombinogenic flanking regions has to be optimized for each individual organism.…”

Random and direct mutagenesis to enhance protein secretion inAshbya gossypii

“…Deletion of GAS1 in S. cerevisiae resulted in an almost 7-fold increase in the level of human hIGF1 29 . Similarly, secreted enzymatic activity was almost doubled when GAS1 was deleted in Z. bailii producing C. rugosa lipase Crl1p or Y. lipolytica protease Xpr2p 31 . In A. fumigatus , an increase in glycoprotein secretion has also been reported for gel2 Δ mutants 46 …”

“…Therefore, there may still be sufficient protein in the wall of the Aggas1A Δ mutant to explain the lack of improved secretion. It is worth noting that also in P. pastoris the disruption of GAS1 had no effect on the secretion of human trypsinogen or human serum albumin, although the amount of R. oryzae lipase released from the cells was doubled 31 . Similarly, GAS1 deletion in Z. bailii did not increase human IL-1b secretion 32 .…”

“…GAS1 was chosen as a target for A. gossypii strain improvement because in yeast it encodes a cross-linking enzyme known to affect the structure and permeability of the cell wall. Cell wall defective strains deleted in the GAS1 gene of S. cerevisiae , 29 P. pastoris 30 and Z. bailii 31 have been reported as super-secreting. Deletion of GAS1 in S. cerevisiae resulted in an almost 7-fold increase in the level of human hIGF1 29 .…”