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In previous research, a thiamine-auxotrophic yeast for alpha-ketoglutaric acid (KGA) overproduction was screened in our laboratory and named Yarrowia lipolytica WSH-Z06 (CCTCC no. M207143). However, the high concentration of by-products (mainly pyruvate) limited its application on an industrial scale. To enhance KGA production and reduce pyruvate (PA) accumulation, the pyruvate carboxylation pathway was regulated. By overexpressing the pyruvate carboxylase genes ScPYC1 from Saccharomyces cerevisiae and RoPYC2 from Rhizopus oryzae in Y. lipolytica WSH-Z06, the yields of KGA in Y. lipolytica-ScPYC1 and Y. lipolytica-RoPYC2 increased by 24.5 and 35.3 %, and the yields of PA decreased by 51.9 and 69.8 % in shake flasks, respectively. These changes in the expression levels and activities of key intracellular enzymes showed that enhancing the pyruvate carboxylation pathway had successfully redistributed the carbon flux from PA to KGA. Finally, by controlling the pH in a 3-L fermenter, the maximum concentration of KGA in Y. lipolytica-RoPYC2 reached 62.5 g L⁻¹ with an evident decrease in PA yield from 35.2 to 13.5 g L⁻¹.

α-Ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid (TCA) cycle and has an important role in the regulation of the balance between carbon and nitrogen metabolism in most microorganisms. In previous research, a thiamine-auxotrophic yeast for α-KG overproduction was screened and named as Yarrowia lipolytica WSH-Z06. To enhance α-KG production and reduce by-product (mainly pyruvate) accumulation, the cofactor metabolism was regulated to redistribute the carbon flux from pyruvate to α-KG. The acetyl-CoA synthetase gene, ACS1, from Saccharomyces cerevisiae and the ATP-citrate lyase gene, ACL, from Mus musculus were expressed to regulate the acetyl-CoA metabolism in Y. lipolytica WSH-Z06. The resultant strains were designated as Y. lipolytica-ACS1 and Y. lipolytica-ACL, respectively. Both of the ACS1 and ACL genes could increase the level of acetyl-CoA and enhance the α-KG production. In a 3-L jar fermenter, the highest yield of α-KG in Y. lipolytica-ACL reached up to 56.5 g L(-1) with an obvious decrease of pyruvate accumulation from 35.1 g L(-1) to 20.2 g L(-1). This study demonstrated that enhancing the acetyl-CoA availability could effectively increase the α-KG production in Y. lipolytica., (Copyright © 2012 Elsevier B.V. All rights reserved.)