Construction of a new recombinant protein expression system in the basidiomycetous yeast Cryptococcus sp. strain S-2 and enhancement of the production of a cutinase-like enzyme

Abstract

Yeast host-vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host-vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host-vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.

Simultaneous bioethanol distillery wastewater treatment and xylanase production by the phyllosphere yeast Pseudozyma antarctica GB-4(0)

“…When cultivated in flask with modified FMM with 8% xylose, P antarctica GB-4(0) produces a 33-kDa unknown protein (Figure 1 b, indicated by the arrow). Productions of some xylanases are reported to have been induced by xylose (Furukawa et al 2009 ; Gielkens et al 1999 ; Masaki et al 2012 ; Dobberstein and Emeis 1989 ). Among the Pseudozyma species found to have the ability to secrete xylanases are P. hubiensis NCIM3574 (Adsul et al 2009 ) and P. brasiliensis sp.…”

“…A basidiomycetous yeast, Cryptococcus sp. strain S-2 (Masaki et al 2012 ) and a yeast-like fungus, Aureobasidium pullulans (Dobberstein and Emeis 1989 ) secrete xylanases from xylose. Endo- β -xylanases (EC 3.2.1.8), which hydrolyze xylopyranosyl linkages of β -1,4-xylan, the principal type of hemicellulose, are useful for saccharizing lignocellulosic biomass materials for the production of bioethanol and other useful chemicals (Biely 1985 ).…”

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment

“…Next, we developed a transformation method for P. antarctica using lithium acetate treatment. Pseudozyma and other basidiomycetous yeast Cryptococcus strains have been typically transformed by electroporation (Marchand et al ., ; Masaki et al ., ). Lithium acetate treatment with protoplast preparation was previously adapted for U. maydis transformation (Bej and Perlin, ).…”

Codon optimization enables the Zeocin resistance marker’s use in the ascomycete yeast <i>Debaryomyces occidentalis</i>

“…strain S-2 with slight modifications (Masaki et al, 2012). Briefly, cells were cultured overnight in 60 ml of YM medium (1.0% D-glucose, 0.5% peptone, 0.3% yeast extract and 0.3% malt extract) in a 200-ml baffled Erlenmeyer flask at 25∞C with vigorous aeration in a shaker at 180 rpm.…”