Kun Zhu

Acetate is increasingly recognized as a promising feedstock for biorefineries, serving as an alternative carbon source for microbial cell factories. Nevertheless, high concentrations of acetate can inhibit microbial growth and metabolism. In this study, the acetate assimilation capability of Yarrowia lipolytica was progressively enhanced through metabolic network reprogramming, leading to the accumulation of 968.01 mg/L (196.41 mg/g DCW) of squalene. Subsequently, the construction of an orthogonal squalene biosynthetic pathway within subcellular compartments boosted production to 1815.46 mg/L (354.61 mg/g DCW). By engineering a parallel glucose-acetate metabolic pathway to enable compartmentalized squalene synthesis, titers were further elevated to 2957.25 mg/L (371.84 mg/g DCW) in engineered strain YAS13. Upon introducing the engineered MspCrtN variant, the YAS13 was employed as a squalene-producing platform for the biosynthesis of downstream C30 carotenoids, achieving a titer of 150.99 mg/L (18.37 mg/g DCW). Finally, fed-batch fermentation in a 1.5 L bioreactor using a glucose-acetate co-substrate strategy with the engineered strain yielded a maximum C30 carotenoid production of 3028.76 mg/L (59.26 mg/g DCW). Collectively, this work presents an effective metabolic reprogramming approach that coordinates carbon co-utilization to improve bioproduction efficiency, thereby establishing a versatile platform for the synthesis of various high-value terpenoid compounds.

Traditional microbial research has focused on C₄₀ carotenoids. Recent reports showed that novel C₃₀ carotenoid compounds, found in certain bacterial microorganisms, exhibited antioxidant properties similar to or greater than those of their C₄₀ counterparts. However, the biosynthesis yields were relatively low. In this study, an artificial 8-step pathway for de novo mevalonate biosynthesis in Yarrowia lipolytica was constructed, increasing squalene titer to 1235.79 mg/L. This led to the development of a high-production squalene platform strain capable of supplying sufficient precursors for C₃₀ carotenoid biosynthesis. We identified the low substrate conversion rate as the main limitation through a systematic research. The bottleneck was addressed through protein and compartmentalization engineering, resulting in 90.948 mg/L C₃₀ carotenoid production (11.091 mg/g DCW). Ultimately, by incorporating these approaches, we developed strains capable of producing 2686.77 mg/L C₃₀ carotenoids with a cellular content of 35.05 mg/g DCW in bioreactor fermentations, a significant improvement over the initial strain and the highest ever reported for C₃₀ carotenoids.