Central carbon metabolism of the biocontrol yeast Pichia anomala

The biocontrol yeast Pichia anomala prevents mould damage of moist cereal grain during malfunctioning airtight storage but it can also spoil food and feed. This thesis focuses on the physiology and metabolism of P. anomala, in particular during oxygen limitation, a condition relevant to airtight storage of cereal grain. P. anomala grew under strictly anaerobic conditions, at temperatures between 3 and 37 degrees, pH values between 2.0 and 12.4, low water activity (0.85), and on many different nutrients. Accumulation of low-molecular compounds in living cells was analysed by HR MAS-NMR. Glycerol, arabitol, and trehalose accumulation increased with reduced oxygen availability, indicating a role during oxygen-limited growth. Regulation of the central aerobic and hypoxic metabolism of P. anomala was investigated under controlled fermentor conditions. Oxygen limitation induced alcoholic fermentation as well as activity of the key fermentative enzymes, ADH and PDC. Metabolic flux analysis revealed that the TCA pathway operated as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Hypoxic conditions also increased the production of ethyl acetate, an ester involved in the biocontrol activity of P. anomala. Genes encoding the ADH and PDC enzymes were cloned, PaADH1, PaADH2, and PaPDC1, and their expression was analysed with real-time RT-PCR. PaADH1 and PaPDC1 were expressed during aerobic growth on glucose and ethanol and were up-regulated in response to oxygen limitation. PaADH2 expression was low during these growth conditions, i.e.