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Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters and is an important industrial enzyme. In the present study, transgenic Arxula adeninivorans strains were optimised for tannase production. Various plasmids carrying one or two expression modules for constitutive expression of tannase were constructed. Transformant strains that overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter produce levels of up to 1,642 U L(-1) when grown in glucose medium in shake flasks. The effect of fed-batch fermentation on tannase productivity was then investigated in detail. Under these conditions, a transgenic strain containing one ATAN1 expression module produced 51,900 U of tannase activity per litre after 142 h of fermentation at a dry cell weight of 162 g L(-1). The highest yield obtained from a transgenic strain with two ATAN1 expression modules was 31,300 U after 232 h at a dry cell weight of 104 g L(-1). Interestingly, the maximum achieved yield coefficients [Y(P/X)] for the two strains were essentially identical.

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 IFNalpha2a (human interferon alpha), 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 IFNalpha2a. 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.

Yeasts provide attractive expression platforms. They combine ease of genetic manipulations and the option for a simple fermentation design of a microbial organism with the capabilities of an eukaryotic organism to secrete and to modify a protein according to a general eukaryotic scheme. For platform applications, a range of yeast species has been developed during the last decades. We present in the following review a selection of established and newly defined expression systems. The review is concluded by the description of a wide-range vector system that allows the assessment of the selected organisms in parallel for criteria like secretion or appropriate processing and modification in a given case.