| Erythritol was proven to be an effective table sugar alternatives which is non-cariogenic and zero-calories.The yeast Yarrowia lipolytica(Y.lipolytica)is a non-conventional yeast which is widely employed for the erythritol production.The erythrose reductase,catalyzed the D-erythrose to erythritol,was recognized as one of the key enzymes.However the corresponding ER gene(s)still remain unknown in the erythritol-producing yeast Y.lipolytica.In this study,three erythrose reductases genes,ER10,ER25 and ER27,were identified from erythritol-producing Y.lipolytica,and heterolo-geously expressed in Escherichia coli.The three ERs shared<35%amino acid identity with each other.All the three ERs required nicotinamide adenine dinucleotide phosphate(NADP)as cofactor.However,they showed diverse characteristics,ER25 has the biggest catalytic efficiency,and the acidic environment is more beneficial to the activity of the enzyme.Disruptants with ER25 or ER27 still can synthesize erythritol from glucose but with significant decreased production and yield.Interestingly,disruption of the ER10 gene resulted in enhanced erythritol production and yield.Enzymatic characterizations showed that the enzyme encoded by ER10 gene is a bifunctional enzyme,which could also oxidize erythritol to L-erythrulose with NADP~+as cofactor as well as reduce erythrose to erythritol with NADPH as cofactor.Overexpression of one of the three ER genes in Y.lipolytica resulted in a 15.6%higher yield and 31.2%higher productivity than those of the wild type.Based on the function of ER genes characterized in this study,a higher erythritol-producing strain which co-overexpressed ER10,ER25,ER27,glucose 6-P dehydrogenase gene(ZWF1)and gluconate 6-P dehydrogenase gene(GND1)genes was constructed,with a yield of 0.63 g erythritol per g glucose,the highest reported thus far,23.5%higher than that of the control strain. |
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