Cycloheximide resistance in the Lipomycetaceae revisited.

Cycloheximide (CYH) is a heterocyclic, glutarimide antibiotic that is a potent inhibitor of protein biosynthesis in most eukaryotes. This study demonstrated that yeasts from all species of the Lipomycetaceae, with the exception of Dipodascopsis spp., can grow in the presence of up to 5 g.L(-1) CYH -- a concentration that is five times higher than the accepted "highest" concentration of 1 g.L(-1) used in physiological tests for yeast identification. Lipomycetaceous yeasts are known to utilize heterocyclic nitrogen-containing compounds such as thymine as sole nitrogen source. CYH contains a glutarimide ring, which is chemically similar to thymine. We investigated the possibility that CYH resistance in the Lipomycetaceae might be due to an ability to degrade CYH and use it as the sole nitrogen source. However, we were unable to demonstrate significant growth on CYH as sole nitrogen source. When thymine was used as positive control, we could demonstrate its utilization as sole nitrogen source.

Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications.

The alkane-assimilating yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates such as n-alkanes, fatty acids, fats and oils for which it has specific metabolic pathways. An overview of the oxidative degradation pathways for alkanes and triglycerides in Y. lipolytica is given, with new insights arising from the recent genome sequencing of this yeast. This includes the interaction of hydrophobic substrates with yeast cells, their uptake and transport, the primary alkane oxidation to the corresponding fatty alcohols and then by different enzymes to fatty acids, and the subsequent degradation in peroxisomal beta-oxidation or storage into lipid bodies. Several enzymes involved in hydrophobic substrate utilisation belong to multigene families, such as lipases/esterases (LIP genes), cytochromes P450 (ALK genes) and peroxisomal acyl-CoA oxidases (POX genes). Examples are presented demonstrating that wild-type and genetically engineered strains of Y. lipolytica can be used for alkane and fatty-acid bioconversion, such as aroma production, for production of SCP and SCO, for citric acid production, in bioremediation, in fine chemistry, for steroid biotransformation, and in food industry. These examples demonstrate distinct advantages of Y. lipolytica for their use in bioconversion reactions of biotechnologically interesting hydrophobic substrates.

The use of Yarrowia lipolytica for the expression of human cytochrome P450 CYP1A1.

Cytochromes P450 constitute a superfamily of haem-thiolate mono-oxygenases that are involved in the oxidative metabolism of lipophilic subtrates. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. We have expressed human cytochrome P450 CYP1A1 under the POX2 promoter (pPOX2-CYP1A1) in Y. lipolytica, with or without overproduction of Y. lipolytica CPR expressed under the ICL promoter (pICL-CPR) or the POX2 promoter (pPOX2-CPR). Activity of cytochrome CYP1A1 was analysed by conversion of hydroxyresorufin to resorufin. Strain JMY330 and JMY330-CPR present no activity, the monocopy cytochrome CYP1A1 integrant JMY331 and JMY331-CPR derivatives present an average activity of 32.0 pM/min/dw and 48.3 and 64.6 pM/min/dw for pICL-CPR and pPOX2-CPR, respectively. Increase of CPR expression resulted in about two-fold higher activity. The multicopy 1A1 integrant JMY339 and JMY339-CPR derivatives present an activity of 129 pM/min/dw and 815-1845 pM/min/dw, respectively. Increase of CPR expression resulted in 6.3-12.8-fold higher activity, depending on the CPR transformant. We observed a 50-fold increase of activity between the monocopy integrant JMY331 as compared to the multicopies integrant JMY339-CPR in which CPR was overexpressed.

Acetate enhances citric acid production by Yarrowia lipolytica when grown on sunflower oil.

It was discovered that the addition of 10 g/l acetate to a medium containing 30 g/l sunflower oil caused a drastic increase in citric acid production by Yarrowia lipolytica UOFS Y-1701 i.e. from 0.5 g/l in the absence of acetate to 18.7 g/l in the presence of acetate. Similarly, the ratio of citric acid:isocitric acid increased significantly from 1.7:1 in the absence of acetate to 3.7:1 in the presence of acetate after 240 h of growth.