Metabolic engineering of Yarrowia lipolytica Po1f for efficient citric acid production

Abstract

Citric acid (CA) is a key metabolite in the oxidative branch of the TCA cycle. By constructing a reverse glyoxylate shunt (rGS), malate and succinate can be converted into isocitrate via a three-step enzymatic reaction, thereby enhancing CA synthesis. Yarrowia lipolytica , a promising candidate for CA production, was utilized to validate the efficacy of this strategy. Firstly, a linker for the two subunits of malate thiokinase was rationally designed, and the enzyme and malyl-CoA lyase were co-expressed, thereby enabling glyoxylate synthesis from malate. Subsequently, the expression of isocitrate lyase was enhanced via iterative transformation and screening, achieving the effective conversion of glyoxylate and succinate to isocitrate. Consequently, rGS was successfully established in the mitochondria of Y. ipolytica Po1f, and CA titer was increased by 4.2 times compared with the Po1f strain. Then, through four metabolic engineering strategies—such as promoting malate entry into mitochondria and facilitating citrate export from mitochondria—the CA titer was further increased. The final strain Po1f-rGS6 achieved a CA titer 9.1-fold higher than that of the Po1f strain when cultivated on glucose, and 7.9-fold higher when cultivated on glycerol. Finally, CA titer of 83.3 g/L and a yield of 0.62 g/g were achieved by optimizing fermentation conditions. Meanwhile, after adaptive evolution, Po1f-rGS6 also demonstrated excellent production performance in the hydrolysate of corn stover. This study demonstrates the feasibility of establishing rGS in Y. lipolytica Po1f for enhancing CA production, thereby offering new insights into the future application of Y. lipolytica for CA and related metabolite production.