谷氨酸棒状杆菌基于甘氨酸代谢调控的5-氨基乙酰丙酸合成研究

5-Aminolevulinic acid (ALA), a non-proteinogenic amino acid, is widely applied in medicine and agriculture areas. In recent years, ALA biosynthesis has attracted much attention, and Corynebacterium glutamicum has become one of the optimal hosts because of its bio-safety and robustness. Previously, we engineered this bacteria to produce ALA. Employing a two-stage fermentation strategy, 14.7 g/L ALA was produced, representing the highest titer reported. However, glycine, one of the substrates, was exogenously added, which increases the cost. In this sense, it’s worthy to efficiently produce ALA using glucose as the sole carbon source for production cost reduction and process simplication. In this project, we intend to realize de-novo glycine biosynthesis through introduction of the glyoxylate transamination pathway, and construct an ALA production pathway using glucose as the sole carbon source. Then, we will increase the titer and productivity of ALA using metabolic regulation methods, which would lay the foundation for construction of an efficient ALA producer strain. Meanwhile, we intend to reveal the regulation mechanism of glyoxylate cycle, central metabolic pathway and C4 anaplerotic pathway through transcriptomic analysis, metabolite analysis and metabolic engineering methods, providing theoretical basis for biosynthesis of other related chemicals.

5-氨基乙酰丙酸（ALA）是一种非蛋白质氨基酸，在医药和农业领域应用广泛。近年来，ALA的生物合成引起了研究者的广泛关注，其中谷氨酸棒状杆菌凭借其生物安全性和优良的抗逆性成为理想宿主之一。先前研究中，我们构建了一株谷氨酸棒状杆菌生产菌株，采用双阶段发酵策略，ALA产量达14.7g/L，为目前报道的最高产量。然而，发酵过程仍然依赖甘氨酸的外源添加，成本较高。因此，实现以葡萄糖为唯一碳源的ALA高效生产对于降低生产成本、简化生产流程意义重大。本项目拟通过乙醛酸转氨途径的引入实现甘氨酸从头合成，构建葡萄糖为唯一碳源的ALA合成途径，结合代谢流调控手段进一步增加ALA的产量和生产速率，为高产ALA工程菌株的构建奠定基础；同时利用转录组学、代谢产物分析、代谢工程改造等手段研究乙醛酸循环、中心代谢途径、C4回补途径的调控规律，为其它相关化学品的生物合成提供理论依据。

5-氨基乙酰丙酸（ALA）是一种非蛋白质氨基酸，在医药和农业领域应用广泛。近年来，ALA的生物合成引起了研究者的广泛关注，然而多数发酵过程依赖甘氨酸的外源添加，成本较高。因此，实现以葡萄糖为唯一碳源的ALA高效生产意义重大。本项目通过谷氨酸：乙醛酸转氨酶（GGAT）的引入实现了甘氨酸从头合成，构建了葡萄糖为唯一碳源的ALA合成途径。GGAT同源蛋白筛选表明，拟南芥来源的活力更高。随后我们进行了菌株基因工程改造并调节了关键酶GGAT的表达强度，结果敲除aceB并弱化icd，高表达GGAT的情况下，ALA积累较高，达到1.12 g/L。我们优化了CGXII培养基，以尿素作为氮源提高了经济性，同时使野生型谷氨酸棒状杆菌的生物量积累提高54%，转录组数据表明培养基成分变化对细菌碳代谢、阳离子运输和能量合成造成了影响。当前我们正在进行ALA合成酶的突变筛选，改造ppc、pyc、gltA等靶点以及优化发酵条件，期望进一步增加ALA的产量，为高产ALA工程菌株的构建奠定基础，同时揭示相关代谢途径的调控规律。