A Heterologous Push–Pull Metabolic Engineering Strategy Synergistically Enhances Citric Acid Secretion in Yarrowia lipolytica

Citric acid is one of the most effective organic acids for rare earth bioleaching. Yarrowia lipolytica represents a promising chassis due to its inability to produce oxalic acid; however, the metabolic bottlenecks between citrate synthesis and mitochondrial export remain to be systematically addressed. In this study, a heterologous “push–pull” strategy was applied to simultaneously reinforce citrate synthesis and mitochondrial efflux by co-overexpressing YlAMPD (encoding AMP deaminase) and AnYHM2 (encoding a mitochondrial citrate carrier from Aspergillus niger) under the strong constitutive promoters. Three recombinant strains were constructed in Yarrowia lipolytica Po1f: YlAMPD-overexpressing Po1f-pJQ08, AnYHM2-expressing Po1f-pJQ09, and the co-expression strain Po1f-pJQ12. RT-qPCR confirmed successful transcriptional activation of the target genes. Shake-flask fermentation revealed that YlAMPD and AnYHM2 single-gene overexpression strains increased citric acid production by 34.23% and 29.62%, respectively, whereas the co-expression strain achieved a titer of 22.33 g·L−1, representing a 94.51% increase over the control. This enhancement substantially exceeded the arithmetic sum of the individual increases (63.85%), thereby exhibiting a distinct supra-additive effect. This study provides a candidate strain with application potential for rare earth bioleaching and offers a referable metabolic engineering strategy for citric acid production in Yarrowia lipolytica.