The yeast : a new host for heterologous protein production, secretion and for metabolic engineering applications

Abstract

Molecular tools for the production of heterologous proteins and metabolic engineering applications of the non-conventional yeast Zygosaccharomyces bailii were developed. The combination of Z. bailii's resistance to relatively high temperature, osmotic pressure and low pH values, with a high specific growth rate renders this yeast potentially interesting for exploitation for biotechnological purposes as well as for the understanding of the biological phenomena and mechanisms underlying the respective resistances. Looking forward to these potential applications, here we present the tools required for the production and the secretion of different heterologous proteins, and one example of a metabolic engineering application of this non-conventional yeast, employing the newly developed molecular tools.

Production ofl-Ascorbic Acid by Metabolically EngineeredSaccharomyces cerevisiaeandZygosaccharomyces bailii

“…The S. cerevisiae ALO1 and A. thaliana LGDH genes were also successfully expressed in Z. bailii (Table 2). Z. bailii was selected for this study because it tolerates high-sugar concentrations, acidic environments, and relatively high temperatures and can survive in the presence of high concentrations of chemical preservatives (3). Because of some of these features, fermentation bioprocesses could be performed at very low pH values, simplifying the downstream process and, at the same time, preventing bacterial contamination.…”

“…70191-4). Finally, the coding regions were cloned into the S. cerevisiae expression vectors of the pYX series (R&D Systems, Inc.) or the Z. bailii expression vectors pZ 3 and pAG26 in the following manner. AGD and ALO1 were subcloned into pYX042 (integrative; LEU2 auxotrophic marker and S. cerevisiae triose phosphate isomerase [TPI] promoter) with EcoRI.…”

“…For construction of pAG26TPI-LGDH (autonomous replication sequence-centromeric [ARS-CEN] region; hygromycin resistance marker and TPI promoter), the expression cassette consisting of the TPI promoter, the LGDH gene, and the terminator was cut from pYX022-LGDH with the restriction enzymes AatII and PvuII, blunt ended, cloned into pAG26 (7), cut with ApaI, and blunt ended. The plasmid pZ 3 (ARS-CEN region; G418 resistance marker and TPI promoter) was constructed as described by Branduardi et al (2,3). In short, the ARS-CEN region was cut from Ycplac33 (5) by cutting it with the restriction enzyme ClaI, blunt ending, further cutting with SpeI, and cloning into pYX022, which had been cut with DraIII, blunt ended, and cut with SpeI.…”

“…The resulting plasmid was opened with KpnI and blunt ended to receive the G418 resistance cassette, cut from pFA6-kanMX4 (28) with the restriction enzymes SphI/SacI, and blunt ended. ALO1 and Gulo were cloned into pZ 3 with the restriction enzyme EcoRI, following blunt ending.…”

Improvement of Lactic Acid Production in Saccharomyces cerevisiae by Cell Sorting for High Intracellular pH

“…Further improvements in lactic acid productivity were obtained by using S. cerevisiae strains lacking any pyruvate decarboxylase activity and thus unable to produce ethanol (15). Production of lactic acid was also attempted in nonconventional yeasts, e.g., Kluyveromyces lactis (2,17), Torulaspora delbrueckii (16), and Zygosaccharomyces bailii (4). Recently, it has been shown that the production of lactic acid in S. cerevisiae is strongly dependent on the genetic background of the host and on the source of the heterologous L-lactate dehydrogenase (5).…”