Optimization of the Culture Medium for Efficient Nervonic Acid Production by Engineered Rhodosporidium toruloides

Nervonic acid (NA), a very long-chain monounsaturated fatty acid with critical roles in neurological health and brain development, has garnered increasing interest for microbial production via metabolic engineering. Previously, the laboratory constructed a recombinant strain using Rhodosporidium toruloides as the chassis, which is capable of synthesizing NA. However, the lack of systematic medium optimization remains a major bottleneck for enhancing NA titers in the engineered strains. This study aimed to develop an economical and efficient fermentation medium for NA biosynthesis using the engineered strain in shake-flask cultures. Through one-factor-at-a-time optimization, glucose and corn steep liquor (CSL) were identified as the optimal carbon and organic nitrogen sources, respectively. Further analysis revealed that precise concentrations of glucose (120 g/L), CSL (10 g/L), and ammonium sulfate (1.1 g/L) were critical for balancing cell growth and lipogenesis. Notably, inorganic salts (CaCl₂, MgSO₄·7H₂O, KH₂PO₄) were dispensable in the presence of CSL, which inherently supplied essential trace elements. Maintaining the initial pH at its natural value (4.3) proved optimal, as artificial pH adjustments severely impaired lipid accumulation. Under the optimized conditions, the NA titer reached 7.5 g/L, representing a 29.3% increase over the initial medium (5.8 g/L) and establishing the highest reported shake-flask titer for microbial NA production. This work not only provides a cost-effective medium formulation by eliminating inorganic salts and utilizing low-cost CSL but also offers strategic insights for scaling up NA biosynthesis in bioreactors. The findings underscore the importance of tailored nutrient optimization in aligning metabolic engineering advancements with bioprocess practicality.