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.

Citation

Jiaping Wang, Cong Gao, Xiulai Chen, Liming Liu. Engineering the Cad pathway in Escherichia coli to produce glutarate from L-lysine. Applied microbiology and biotechnology. 2021 May;105(9):3587-3599

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PMID: 33907891

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