Xplor® 2—an optimized transformation/expression system for recombinant protein production in the yeast Arxula adeninivorans
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Combining ease of genetic manipulation and fermentation with the ability to secrete and to glycosylate proteins in the basic eukaryotic manner, Arxula adeninivorans provides an attractive expression platform. Based on a redesign of the basic vector, a new Arxula vector system, Xplor® 2, for heterologous gene expression was established, which allows (1) the construction of expression plasmids for supertransformation of A. adeninivorans strains secreting target proteins of biotechnological interest and (2) the integration of small vector cassettes consisting of yeast DNA sequences only. For this purpose, a set of modules including the ATRP1m selection-marker module, expression modules for constitutive expression of the genes phyK (Klebsiella-derived phytase) and IFNα2a (human interferon α), the HARS (Hansenula polymorpha autonomous replication sequence) for autonomous replication and the chaperone module AHSB4 promoter –HpCNE1 gene (calnexin) –PHO5 terminator to improve secretion efficiency were constructed and integrated in various combinations in the basic vector Xplor® 2. After removal of the complete Escherichia coli-based plasmid parts (resistance marker, ColE1 ori and f1(−) origin), the remaining yeast-based linear vector fragment with or without rDNA targeting sequences were transformed as yeast rDNA integrative expression cassettes and yeast integrative expression cassettes (YICs), respectively, and the resulting strains were tested for their capacity to secrete PhyK or IFNα2a. Maximal expression levels were consistently obtained using YICs for transformation irrespective of whether or not they carry HARS and/or calnexin modules. It is recommended that at least 50 such transformants be analyzed to ensure selection of the best transformants.
KeywordsArxula adeninivorans Interferon α Phytase Transformation Xplor® 2 Yeast
The authors are grateful to Dr. P. Hardy for helpful discussions and critical reading of the manuscript and R. Franz and H. Bohlmann for excellent technical assistance. The research work was supported by grants from the German national Ministry of Education and Research (BMBF) within the CLIB2021 cluster (Wettbewerb Industrielle Biotechnologie 2021) and the Fonds der chemischen Industrie (GK).
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