The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10-15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0-6.8 and resulted in production of 127-140 g l(-1) CA with a yield Y (CA) of 0.75-0.82 g g(-1), whereas at pH 5.0, 87 g l (-1) with a yield Y (CA) of 0.51 g g(-1) were produced. The CA-productivity Q (CA) increased from 0.40 g l (-1) h(-1) at pH 5.0 up to 0.73 g l (-1) h(-1) at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene-dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric acid (CA) and isocitric acid (ICA) under an excess of carbon source and several conditions of growth limitation. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether this CA/ICA product ratio can be influenced by gene-dose-dependent overexpression of aconitase (ACO)-encoding gene ACO1, a recombinant Y. lipolytica strain was constructed containing multiple copies of ACO1. The high-level expression of ACO in the ACO1 multicopy integrative transformant resulted in a shift of the CA/ICA product pattern into the direction of ICA. On sunflower oil, a striking increase of the ICA proportion from 35-49% to 66-71% was observed compared to wild-type strains without influencing the total amount of acids (CA and ICA) produced. On glycerol, glucose or sucrose, the ICA proportion increased only moderately from 10-12% to 13-17%. This moderate shift into the direction of ICA was also observed in an icl1-defective strain.

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric (CA) and isocitric (ICA) acids, triggered by growth limitation caused by different factors and an excess of carbon source. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether the CA/ICA product ratio can be influenced by gene-dose-dependent overexpression or by disruption of the isocitrate lyase (ICL)-encoding gene ICL1, recombinant Y. lipolytica strains were constructed, which harbour multiple ICL1 copies or a defective icl1 allele. The high-level expression of ICL in ICL1 multicopy integrative transformants resulted in a strong shift of the CA/ICA ratio into direction of CA. On glycerol, glucose and sucrose, the ICA proportion decreased from 10-12% to 3-6%, on sunflower oil or hexadecane even from 37-45% to 4-7% without influencing the total amount of acids (CA and ICA) produced. In contrast, the loss of ICL activity in icl1-defective strains resulted in a moderate 2-5% increase in the ICA proportion compared to ICL wild-type strains on glucose or glycerol.

Nine potential (fatty) alcohol dehydrogenase genes and one alcohol oxidase gene were identified in Yarrowia lipolytica by comparative sequence analysis. All relevant genes were deleted in Y. lipolytica H222ΔP which is lacking β-oxidation. Resulting transformants were tested for their ability to accumulate ω-hydroxy fatty acids and dicarboxylic acids in the culture medium. The deletion of eight alcohol dehydrogenase genes (FADH, ADH1-7), which may be involved in ω-oxidation, led only to a slightly increased accumulation of ω-hydroxy fatty acids, whereas the deletion of the fatty alcohol oxidase gene (FAO1), which has not been described yet in Y. lipolytica, exhibited a considerably higher effect. The combined deletion of the eight (fatty) alcohol dehydrogenase genes and the alcohol oxidase gene further reduced the formation of dicarboxylic acids. These results indicate that both (fatty) alcohol dehydrogenases and an alcohol oxidase are involved in ω-oxidation of long-chain fatty acids whereby latter plays the major role. This insight marks the first step toward the biotechnological production of long-chain ω-hydroxy fatty acids with the help of the nonconventional yeast Y. lipolytica. The overexpression of FAO1 can be further used to improve existing strains for the production of dicarboxylic acids.