Overexpression of the ICL1 gene changes the product ratio of citric acid production by Yarrowia lipolytica.
ABSTRACT 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.
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ABSTRACT: The possibility of using glycerol and glycerol-containing waste from biodiesel manu-facture as a carbon and energy source for microbiological production of citric acid has been studied. Acid formation on the selective media had previously been tested in 66 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis and Yarrowia). Under growth limitation by nitrogen, 41 strains (belonging mainly to species Yarrowia lipolytica) produced acids; unlike 25 strains of the genera Debaryomyces, Candida, Pichia, Saccharomyces and Torulopsis. Among the 41 acid-producing strains, mutant strain Yarrowia lipolytica N15 was selected since it was able to produce citric acid presumably in high amounts. The cit-ric acid production by the selected strain was studied in dependence on the medium pH, aeration and concentration of glycerol. Under optimal conditions, the mutant Y. lipolytica N15 produced up to 98 g/L of citric acid when grown in a fermentor with the medium containing pure glycerol, and 71 g/L of citric acid when grown on glycerol-containing waste. The effect of growth phases on physiological peculiarities of the citric acid producer was discussed.
Article: Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes.[show abstract] [hide abstract]
ABSTRACT: BACKGROUND: Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. RESULTS: For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems.Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system.Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. CONCLUSIONS: Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes.Microbial Cell Factories 08/2012; 11(1):106. · 3.55 Impact Factor