Mitochondrial and plasma‑membrane transport‑based engineering enables industrial‑level isocitric acid production in Yarrowia lipolytica

Abstract Background Isocitric acid (ICA) has recently attracted increasing interest because of its nutraceutical relevance and emerging biomedical potential. However, despite its value, industrial exploitation of ICA is still limited owing to the lack of cost‑effective and selective microbial production processes. Mitochondrial transporters play a pivotal role in controlling the pool sizes and fluxes of tricarboxylic acid (TCA) cycle intermediates by mediating their selective exchange across the inner mitochondrial membrane. Recently, we identified the mitochondrial carriers Yl Yhm2 and Yl Sfc1 in Yarrowia lipolytica as the specific transporters responsible for citric acid (CA) and ICA efflux, respectively, providing for the first time direct control over the selective secretion of these two organic acids. Building on this discovery, a transporter‑based metabolic engineering strategy was developed to establish a robust and highly selective ICA‑overproducing microbial platform. Results Starting from a strain lacking the mitochondrial citrate carrier YlYHM2 and overexpressing the mitochondrial ICA exporter YlSFC1 , ICA secretion was further enhanced through overexpression of the plasma‑membrane exporter YlCEX1 . To further increase mitochondrial carbon availability feeding the TCA cycle, we overexpressed transporters capable of mediating net carbon import into mitochondria, including the dicarboxylate carriers YlOAC1 and YlDIC1 , as well as the pyruvate carrier complex subunits YlMPC1 / YlMPC2 , either individually or in combination. Among these, YlOAC1 overexpression resulted in the most pronounced improvement, enabling production of 58.4 ± 2.4 g/L ICA with a molar yield of 0.62 mol/mol glucose in shake‑flask cultures. Fed‑batch bioreactor cultivation of this strain under nitrogen‑limited conditions yielded 152.76 ± 3.2 g/L ICA with a process selectivity of 95% (ICA/CA = 20), representing the highest titers and selectivity reported to date for a ICA producer. Conclusions These results clearly demonstrate that precise manipulation of mitochondrial and plasma‑membrane transport processes constitutes a powerful and scalable strategy for redirecting carbon flux toward ICA production. This work establishes Y. lipolytica as a robust microbial chassis for industrial ICA manufacturing and highlights selective transport engineering as an effective complementary strategy alongside conventional enzyme centered metabolic engineering approaches, extending and reinforcing previously established roles of mitochondrial carriers in metabolic engineering.