非蛋白质氨基酸5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA)是血红素、叶绿素、维生素B12等四吡咯化合物合成的前体物质,在医药和农业等领域具有广泛的应用价值。该研究是旨在无需添加甘氨酸的条件下高效生产5-ALA。为此,在大肠杆菌中引入异源谷氨酸乙醛酸氨基转移酶和5-氨基乙酰丙酸合酶,构建一条非自然C4代谢途径。通过过表达异柠檬酸裂解酶、敲除苹果酸合酶,强化乙醛酸途径,使5-ALA产量提升至577.3 mg/L。随后,通过敲除琥珀酸辅酶A合成酶增加前体物质积累,敲除原卟啉原Ⅲ氧化酶抑制5-ALA的下游代谢,并过表达磷酸烯醇式丙酮酸激酶补充草酰乙酸、加强三羧酸循环。最终,优化菌株ALA-6的5-ALA产量达到1.74 g/L。在进一步优化异丙基-β-D-硫代半乳糖苷诱导浓度和筛选最适培养基后,在乳糖自诱导培养基中实现了1.9 g/L的5-ALA产量。该研究为无需外源添加甘氨酸高效生产5-ALA提供参考。
The non-protein amino acid 5-aminolevulinic acid (5-ALA), a precursor for synthesizing tetrapyrrole compounds such as heme, chlorophyll, and vitamin B12, exhibits broad application value in medicine and agriculture.This study aimed to efficiently produce 5-ALA without glycine supplementation.To achieve this, this study introduced heterologous glutamate glyoxylate aminotransferase and 5-aminolevulinic acid synthase into Escherichia coli, constructing an unnatural C4 metabolic pathway.Overexpressing isocitrate lyase and knocking out malate synthase enhanced the glyoxylate pathway, increasing 5-ALA production to 577.3 mg/L.Subsequently, the precursor supply was boosted by knocking out succinyl-CoA synthetase, while downstream 5-ALA metabolism was blocked by deleting protoporphyrinogen Ⅲ oxidase.Furthermore, phosphoenolpyruvate carboxykinase was overexpressed to replenish oxaloacetate and strengthen the tricarboxylic acid cycle.The optimized strain ALA-6 ultimately produced 1.74 g/L 5-ALA, which reached 1.9 g/L after optimizing IPTG induction and medium selection (lactose auto-induction).This study provides a strategy for glycine-independent 5-ALA production.
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