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Twenty-four Hansenula polymorpha transformants were passaged and stabilised in glucose medium and screened in glycerol medium for recombinant phytase in shaken test tubes. The cultivations were performed under either limited or non-limited oxygen supply. Maximum oxygen transfer capacities of test tubes were assessed by sulfite oxidation. Oxygen-limited glucose cultures resulted in a partially anaerobic metabolism and formation of 4.1 g ethanol l(-1), which was subsequently aerobically metabolised. Non-limited oxygen supply led to overflow metabolism and to accumulation of 2.1 g acetic acid l(-1), reducing the biomass yield. The use of glycerol in the screening main cultures prevented by-product formation irrespective of oxygen supply. Preculturing in glucose medium under non-limited oxygen supply resulted in a 20-h lag phase of the screening main culture. This lag phase was not observed when preculturing was performed under oxygen limitation. Phytase activity was on average 25% higher in cultures passaged, stabilised and screened under limited oxygen supply than in cultures under non-limited oxygen supply.

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo-and thermotolerance can be addressed for the definition of culture conditions. Hansenula polymorpha and Arxula adeninivorans and their competitive environmentIn the last three decades a wide range of recombinant proteins, especially pharmaceuticals, have been produced based on heterologous gene expression in bacterial organisms, mammalian cells and several yeasts and fungi [1][2][3]. Production processes had to be developed that employ platforms which meet both, the demand for efficient mass production and criteria of safety and authenticity of the produced compounds. In this respect yeasts offer considerable advantages over alternative microbial and mammalian cell systems in providing low-cost screening and production systems for authentically processed and modified proteins. The organisms meet safety prerequisites in that they do not harbour pyrogens, pathogens or viral inclusions [4,5]. Recent engineering of yeast hosts with

Screening cultures are usually non-monitored and non-controlled due to a lack of appropriate measuring techniques. A new device for online measurement of oxygen transfer rate (OTR) in shaking-flask cultures was used for monitoring the screening of Hansenula polymorpha. A shaking frequency of 300 rpm and a filling volume of 20 ml in 250-ml flasks ensured a sufficient oxygen transfer capacity of 0.032 mol (l h)(-1) and thus a respiration not limited by oxygen. Medium buffered with 0.01 mol phosphate l(-1) (pH 6.0) resulted in pH-inhibited respiration, whereas buffering with 0.12 mol phosphate l(-1) (pH 4.1) resulted in respiration that was not inhibited by pH. The ammonium demand was balanced by establishing fixed relations between oxygen, ammonium, and glycerol consumption with 0.245+/-0.015 mol ammonium per mol glycerol. Plate precultures with complex glucose medium reduced the specific growth rate coefficient to 0.18 h(-1) in subsequent cultures with minimal glycerol medium. The specific growth rate coefficient increased to 0.26 h(-1) when exponentially growing precultures with minimal glycerol medium were used for inoculation. Changes in biomass, glycerol, ammonium, and pH over time were simulated on the basis of oxygen consumption.

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