Efficient 5-aminolevulinic acid production through reconstructing the metabolic pathway in SDH-deficient Yarrowia lipolytica

Abstract 5-Aminolevulinic acid (5-ALA) is a precursor for the biosynthesis of tetrapyrrole compounds such as chlorophyll a, heme, and vitamin B12, which has been widely used in the fields of agriculture and medicine. 5-ALA can be biosynthesized through C4 pathway that use succinyl-CoA and glycine as precursor, or through C5 pathway that use glutamate as precursor. Yarrowia lipolytica is an important unconventional yeast and has wide applications in metabolic engineering and industrial biotechnology. It possesses strong metabolic flux through tricarboxylic acid cycle, and prominent acid tolerance, which make it an attractive chassis cell for 5-ALA fermentation. However, there are no reports on the 5-ALA production in Y. lipolytica so far. In this study, a series of 5-ALA producing engineered strains were constructed by using succinate dehydrogenase (SDH)-deficient Y. lipolytica. The related genes of C4 and C5 pathway were co-expressed, and the best engineered strain PGC62-IAL (MatA, xpr2–322, axp-2, leu2–270, ura3–302, ΔSdh5::loxP, ΔAch1::loxP, ScPck, ScHemⅠ, StHemA, EcHemL) for efficient ALA production was obtained. Through optimizing culture conditions, the titer of 5-ALA was 1050 mg/L in shake flask fermentation with glycerol as carbon source. Finally, the 5-ALA titer reached 2216.8 mg/L in the fed-batch fermentation, which is the highest 5-ALA titer achieved using yeast globally. Due to accumulation of the by-products including succinic acid and porphyrin compounds, the 5-ALA yield only reached 0.024 g/g glucose. Nevertheless, Y. lipolytica was engineered to produce 5-ALA for the first time, and indicated that SDH-deficient strains can be used as platform for efficient 5-ALA production.