LAUSR

Pyridoxal 5′‐phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose‐5‐phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high‐throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP‐dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5′‐phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high‐throughput screening of the best chassis for PLP engineering but also practical in fine‐tuning the quantity and quality of enzymes.