State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, China.
Appl Microbiol Biotechnol. 2021 May;105(9):3587-3599. doi: 10.1007/s00253-021-11275-1. Epub 2021 Apr 27.
For the efficient industrial production of glutarate, an important C5 platform chemical that is widely used in the chemical and pharmaceutical industries, a five-enzyme cascade pathway was designed and reconstructed in vitro to synthesize glutarate from L-lysine. Then, the imbalanced enzyme expression levels of L-lysine decarboxylase from Escherichia coli (EcCA), putrescine aminotransferase (KpcPA) and γ-aminovaleraldehyde dehydrogenase (KpcPD) from Klebsiella pneumoniae, and the poor catalytic efficiency of KpcPA were identified as the rate-limiting bottlenecks. To this end, ribosome binding site regulation was employed to coordinate the enzyme molar ratio of EcCA:KpcPA:KpcPD at approximately 4:8:7 (the optimum ratio obtained in vitro), and volume scanning and hydrophobicity scanning were applied to increase KpcPA activity toward cadaverine from 15.89 ± 0.52 to 75.87 ± 1.51 U·mg. Furthermore, the extracellular accumulation of 5-aminovalerate (5AVA) was considerably reduced by overexpressing gabP encoding the 5AVA importer. Combining these strategies into the engineered strain Glu-02, 77.62 g/L glutarate, the highest titer by E. coli to date, was produced from 100 g/L L-lysine in 42 h, with a yield and productivity of 0.78 g/g L-lysine and 1.85 g/L/h, respectively, at a 5-L scale. The results presented here provide a novel and potential enzymatic process at industrial-scale to produce glutarate from cheaper amino acids. KEY POINTS: • The bioconversion of l-lysine to glutarate using the Cad pathway was first achieved. • Enhancing the conversion efficiency of the Cad route maximizes glutarate in E. coli. • Achieving the highest titer of glutarate by E. coli to date.
为了高效地工业化生产戊二酸，这是一种在化学和制药行业广泛应用的重要 C5 平台化学品，设计并重建了一个包含 5 种酶的级联途径，以从 L-赖氨酸合成戊二酸。然后，鉴定出大肠杆菌（EcCA）L-赖氨酸脱羧酶、阴沟肠杆菌（KpcPA）腐胺转氨酶和γ-氨基戊醛脱氢酶（KpcPD）的酶表达水平不平衡，以及 KpcPA 的催化效率较差，是限速瓶颈。为此，通过核糖体结合位点调控，协调 EcCA:KpcPA:KpcPD 的酶摩尔比约为 4:8:7（体外获得的最佳比例），并进行体积扫描和疏水性扫描，将 KpcPA 对尸胺的酶活从 15.89 ± 0.52 U·mg 提高到 75.87 ± 1.51 U·mg。此外，通过过表达编码 5-氨基戊酸（5AVA）转运体的 gabP，大大减少了 5-氨基戊酸的胞外积累。将这些策略结合到工程菌株 Glu-02 中，从 100 g/L L-赖氨酸在 42 h 内生产出 77.62 g/L 戊二酸，达到了大肠杆菌迄今为止的最高产量，L-赖氨酸的得率和生产强度分别为 0.78 g/g L-赖氨酸和 1.85 g/L/h，在 5-L 规模下。本文的结果为利用更廉价的氨基酸在工业规模上生产戊二酸提供了一种新的、有潜力的酶法工艺。
Cad 途径首次用于 l-赖氨酸到戊二酸的生物转化。
提高 Cad 途径的转化效率可使大肠杆菌中戊二酸的产量最大化。
实现了迄今为止大肠杆菌戊二酸的最高产量。
