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.

5-氨基乙酰丙酸(5-ALA)是叶绿素a、血红素、维生素B12等四吡咯化合物生物合成的前体，在农业和医药领域有着广泛的应用。5-ALA可以通过以琥珀酰辅酶A和甘氨酸为前体的C4途径或以谷氨酸为前体的C5途径生物合成。Yarrowia lipolytica是一种重要的非常规酵母，在代谢工程和工业生物技术中有着广泛的应用。它通过三羧酸循环具有强大的代谢通量，以及突出的耐酸性，使其成为用于 5-ALA 发酵的有吸引力的底盘细胞。然而，没有关于解脂耶氏酵母中5-ALA 产生的报道迄今为止。在这项研究中，使用琥珀酸脱氢酶 (SDH) 缺陷型解脂耶氏酵母构建了一系列产生 5-ALA 的工程菌株。C4和C5通路相关基因共表达，最佳工程菌株PGC62-IAL（MatA、xpr2-322、axp-2、leu2-270、ura3-302、ΔSdh5::loxP、ΔAch1:: loxP , ScPck , ScHemⅠ , StHemA , EcHemL) 以获得有效的 ALA 生产。通过优化培养条件，在以甘油为碳源的摇瓶发酵中5-ALA的滴度为1050mg/L。最后，补料分批发酵的 5-ALA 滴度达到 2216.8 mg/L，这是全球使用酵母达到的最高 5-ALA 滴度。由于包括琥珀酸和卟啉化合物在内的副产物的积累，5-ALA 产量仅达到 0.024 g/g 葡萄糖。然而，解脂耶氏酵母首次被设计为生产 5-ALA，并表明 SDH 缺陷菌株可用作高效生产 5-ALA 的平台。