Engineering the α-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP⁺-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica

• Published in: Applied microbiology and biotechnology 2014-03, Vol.98 (5), p.2003-2013
• Main Authors: Yovkova, Venelina, Otto, Christina, Aurich, Andreas, Mauersberger, Stephan, Barth, Gerold
• Format: Article
• Language: English
• Subjects: alpha-ketoglutaric acid; Biomedical and Life Sciences; Bioreactors; Biotechnological Products and Process Engineering; Biotechnology; byproducts; carbon; engineering; Gene Dosage; Gene Expression; gene overexpression; genes; glycerol; Glycerol - metabolism; isocitrate dehydrogenase; Isocitrate Dehydrogenase - genetics; Isocitrate Dehydrogenase - metabolism; Ketoglutaric Acids - metabolism; Life Sciences; malates; Metabolic Engineering; Microbial Genetics and Genomics; Microbiology; pyruvate carboxylase; Pyruvate Carboxylase - genetics; Pyruvate Carboxylase - metabolism; pyruvic acid; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; succinic acid; Yarrowia - enzymology; Yarrowia - genetics; Yarrowia - metabolism; Yarrowia lipolytica
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-013-5369-9

To establish and develop a biotechnological process of α-ketoglutaric acid (KGA) production by Yarrowia lipolytica, it is necessary to increase the KGA productivity and to reduce the amounts of by-products, e.g. pyruvic acid (PA) as major by-product and fumarate, malate and succinate as minor by-products. The aim of this study was the improvement of KGA overproduction with Y. lipolytica by a gene dose-dependent overexpression of genes encoding NADP⁺-dependent isocitrate dehydrogenase (IDP1) and pyruvate carboxylase (PYC1) under KGA production conditions from the renewable carbon source raw glycerol. Recombinant Y. lipolytica strains were constructed, which harbour multiple copies of the respective IDP1, PYC1 or IDP1 and PYC1 genes together. We demonstrated that a selective increase in IDP activity in IDP1 multicopy transformants changes the produced amount of KGA. Overexpression of the gene IDP1 in combination with PYC1 had the strongest effect on increasing the amount of secreted KGA. About 19 % more KGA compared to strain H355 was produced in bioreactor experiments with raw glycerol as carbon source. The applied cultivation conditions with this strain significantly reduced the main by-product PA and increased the KGA selectivity to more than 95 % producing up to 186 g l⁻¹ KGA. This proved the high potential of this multicopy transformant for developing a biotechnological KGA production process.