Real-time detection of lysine decarboxylase activity and cellular energy pool under inducible CadA expression system in engineered Escherichia coli

Abstract

BackgroundBiocatalysts hold significant promise in the production of 1,5-diaminopentane (cadAverine) due to its wide-ranging applications. However, the process of screening recombinant cells expressing lysine decarboxylase (LdcC or CadA) activity via high-performance liquid chromatography is labor-intensive and time-consuming. MethodsThis study has devised a straightforward colorimetric assay to quantify lysine decarboxylase activity. It utilizes the pH indicator bromocresol purple (BCP). As cadAverine is produced from l-lysine, it elevates the reaction pH, leading to a corresponding change in BCP color. Significant findingsReal-time monitoring of CadA activity was conducted using an inducible T7 promoter system, built upon the CadA/-CadB co-expression framework within Escherichia coli BL21(DE3). Analysis of Michaelis-Menten kinetics revealed that pyridoxal-5′-phosphate (PLP) was efficient in substrate conversion compared to pyridoxal (PL) and pyridoxine (PN) as cofactors. Supplementation of PN with ATP successfully restored Vmax to levels comparable with PLP. However, following freezer storage of biocatalysts, insufficient PLP generation from PN was observed due to variations in the cellular metabolic pool and ATP levels. The optimal conditions for CadA/B activity were achieved with 7 μM IPTG induction at 32 °C for 8 h, resulting in a maximum specific activity of 18.2 ± 1.1 mmol/min/g dried cell weight. The BCP-based colorimetric assay showcased its efficacy in rapidly detecting and quantifying CadA/B activity, thereby facilitating high-throughput screening of recombinant libraries.