Screening of a thiamine-auxotrophic yeast for α-ketoglutaric acid overproduction

Aims: To obtain a thiamine-auxotrophic yeast strain that overproduces alpha-ketoglutaric acid (alpha-KG) from glycerol and to investigate nutrient effects on alpha-KG production. Methods and Results: Yeast strain WSH-Z06, a thiamine auxotroph that gave high yields of alpha-KG from glycerol, was obtained by screening for ampicillin/kanamycin resistance and thiamine auxotrophy. The strain was identified as Yarrowia lipolytica based on physiological, chemical, and phylogenetic analysis. The ability of the strain to convert glycerol to alpha-KG was analysed by investigating the effects of nutritional factors, including thiamine, riboflavin, nitrogen sources, and calcium ion. Conclusions: Thiamine and calcium ion concentration had the greatest effect on alpha-KG accumulation. Under optimal conditions, a yield of 39 center dot 2 g l-1 alpha-KG was obtained from 100 g l-1 glycerol, with 16 center dot 84 g l-1 pyruvate as a by-product. Significance and Impact of the Study: The current work provides a method for screening for an alpha-KG overproducer. Nutrients have a significant impact on alpha-KG production in the yeast strain presented here. The alpha-KG-overproducing yeast strain Y. lipolytica WSH-Z06 is a promising parent strain for further metabolic engineering to lower by-product accumulation and accelerate glycerol utilization.