Engineering the Cad pathway in Escherichia coli to produce glutarate from L-lysine.

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-1. 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.