High-level production and secretion of recombinant proteins by the dimorphic yeastArxula adeninivorans

Abstract

The non-conventional dimorphic thermo- and salt-resistant yeast Arxula adeninivorans has been developed as a host for heterologous gene expression. For assessment of the system two model genes have been selected: the GFP gene encoding the intracellular green fluorescent protein, and the HSA gene encoding the secreted human serum albumin. The expression system includes two host strains, namely A. adeninivorans LS3, which forms budding cells at 30 degrees C and mycelia at >42 degrees C, and the strain A. adeninivorans 135, which forms mycelia at temperatures as low as 30 degrees C. For expression control the constitutive A. adeninivorans-derived TEF1-promoter and S. cerevisiae-derived PHO5-terminator were selected. The basic A. adeninivorans transformation/expression vector pAL-HPH1 is further equipped with the Escherichia coli-derived hph gene conferring hygromycin B resistance and the 25S rDNA from A. adeninivorans for rDNA targeting. Transformants were obtained for both budding cells and mycelia. In both cell types similar expression levels were achieved and the GFP was localised in the cytoplasm while more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials on a 200-ml shake flask scale maximal HSA product levels were observed after 96 h of cultivation.

Cloning, Production, and Functional Expression of the Bacteriocin Enterocin A, Produced by Enterococcus faecium T136, by the Yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans

“…The higher production of EntA by P. pastoris X-33EA than by K. lactis GG799EA may be due to promoter differences, to the dosages of the entA gene in their genomes, and/or to differences in the genetic backgrounds of the two strains. On the other hand, the P TEF1 promoter that drives the production of EntA in H. polymorpha KL8-1EA is a strong but constitutive promoter (42). For protein production, inducible systems are often considered superior to constitutive expression systems, since the former enable the achievement of sufficient biomass prior to the initiation of target protein expression and the consequent metabolic burden on the cell (27).…”

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

“…These unusual properties make A. adeninivorans an ideal host for heterologous gene expression as well as a useful source of genes of biotechnological significance (Wartmann et al, 1998(Wartmann et al, , 2002a(Wartmann et al, , 2003a(Wartmann et al, , 2003bBöer et al, 2004Böer et al, , 2005Böer et al, , 2007. Enzymes with wide substrate spectra and high specific activities are of special interest for biotechnological applications.…”

“…Plasmid DNA isolation, restriction fragment isolation, labelling of fragments and Southern transfer were carried out as previously described (Wartmann et al, 2002a).…”

Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans

“…An expression platform based on A. adeninivorans has been developed in recent years, and has been used to synthesize recombinant proteins such as human serum albumin (HSA), lipase, invertase, xylitol dehydrogenase, transaldolase, interleukin-6, α-amylase, acid phosphatase and phytase, and for production of polyhydroxyalkanoates (Wartmann et al, 2002(Wartmann et al, , 2003a(Wartmann et al, , 2003bBöer et al, 2004bBöer et al, , 2005aBöer et al, , 2005bBöer et al, , 2005cBöer et al, , 2007Terentiev et al, 2004;Steinborn et al, 2005Steinborn et al, , 2007El Fiki et al, 2007;Kaur et al, 2007). The system involves the use of vectors with multiple cloning sites for the integration of selection markers, expression cassettes bearing strong constitutive or inducible promoters, autonomously replicating sequences and chaperone cassettes, which are inserted between rDNA-targeting sequences (Steinborn et al, 2007).…”

“…Isolation of plasmid DNA and restriction fragments, labelling of fragments and Southern transfer were carried out as previously described (Wartmann et al, 2002). For RNA isolation, yeast cells were suspended in lysis buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sodium N -lauroylsarcosinate, 1% mercaptoethanol).…”