The potential of Pseudozyma yeastlike epiphytes for the production of heterologous recombinant proteins

Abstract

Although Basidiomycetes represent the most evolved class of fungi, they have been neglected with regard to recombinant gene expression. In this work, basidiomycetous yeasts belonging to Pseudozyma spp. were studied with respect to their amenability to heterologous protein production. Single plasmid or cotransformation experiments routinely afforded 100 to 200 independent transformants for the two tested species of Pseudozyma. Green fluorescent protein (GFP) was expressed in the correctly folded conformation, as demonstrated by fluorescence microscopy, and hen egg white lysozyme (HEWL) was expressed in its active form, as revealed by its lytic activity on Micrococcus lysodeikticus cells. Protease analysis established that Pseudozyma spp. contained equivalent or less extracellular protease activity than yeasts and far less protease activity than ascomycetous filamentous fungi in similar culture conditions. This proteolytic activity was inhibited by over 97% with a combination of PMSF and Pepstatin A. N-glycosylation patterns of native Pseudozyma flocculosa secreted proteins were comprised of one or a few short glycan chains that possess a classic eukaryotic structure typical of higher fungi and animal cells. This is the first report of a Basidiomycete that possesses multiple intrinsic characteristics necessary for use as a heterologous gene expression system.

Usefulness of Heterologous Promoters in thePseudozyma flocculosaGene Expression System

“…The GPD promoter from U. maydis was obtained by amplifying genomic DNA using a forward primer (5 0 -GCCTCGAGAGATCTTGCTGATAGGCA-GGTTT-3 0 ) and a reverse primer (5 0 -GCGGCGCCTA-TGGAAGAGTGTTTTGGTTT-3 0 ), resulting in a 590-bp fragment with XhoI and NarI restriction sites (underlined). Following digestion with XhoI and NarI, the fragment was ligated with a 4.8-kb fragment, which was obtained by digesting plasmid pCPF.GFP containing the sGFP(S65T) gene 1) with XhoI and ClaI to replace the hsp70 promoter in this plasmid. The resulting plasmid, pGFP.gpd, comprising the GPD promoter from U. maydis, the GFP coding region, and the hsp70 terminator from U. maydis, was designed for use in co-transformation experiments in P. flocculosa with plasmid pSceI-hyg (provided by Dr. J. Kronstad, University of British Columbia, Canada), which harbored a selectable marker cassette.…”

“…Following digestion with KpnI and ClaI, the fragment was ligated with a 7.8-kb fragment, which was obtained by digesting plasmid pSPF.GFP 1) with the same enzymes to produce plasmid p-glu.GFP.hsp. Since this plasmid contained both the expression cassette (-glucosidase pro::GFP::hsp70ter) and the selectable marker cassette, it was used in single plasmid transformation of P. flocculosa.…”

“…and P. flocculosa (Traquair, Shaw and Jarvis) Boekhout and Traquair in particular, have been reported to be amenable to recombinant protein production, and they possess several attributes that make them attractive hosts for such an endeavor. 1) For instance, P. flocculosa displays both a yeastlike (conidial) and a filamentous (pseudomycelial) morphology, and it belongs to the most evolved class of fungi, the basidiomycetes.…”

“…1) These results, combined with low levels of secreted proteases, no hypermannosylation of secreted native proteins, and high biomass production in liquid culture, have made Pseudozyma spp. a potentially versatile expression system for recombinant protein production, but very little information is available concerning molecular tools and promoter sequences that might be useful in the expression of heterologous proteins in this host.…”

“…Other features of P. flocculosa also appeared to influence gene expression by the hsp70 promoter. 1) For example, the log phase (exponential growth) is considered as the most active in terms of gene expression with this particular promoter. Contrary to what is generally recognized in yeasts (e.g., Saccharomyces spp.…”