A direct enzymatic evaluation platform (DEEP) to fine‐tuning pyridoxal 5′‐phosphate‐dependent proteins for cadaverine production

Abstract

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.

Decarboxylation in Natural Products Biosynthesis

“…TPP- and PLP-dependent dependent enzymes have been successfully engineered to produce numerous commodity chemicals. TPP-dependent decarboxylases are widely used in constructing artificial biosynthetic pathways for the production of alcohols such as isobutanol, n-pentanol, and 1,2,4-butanetriol, − while PLP-dependent decarboxylases have been exploited to produce commodity chemicals and important precursors such as γ-aminobutyric acid, cadaverine (a biopolymer precursor), and dopamine. − Additionally, given that numerous decarboxylases are involved in the production of alkene and alkanes from abundant fatty acids, there is significant potential for their use in biofuel production.…”

Fine-Tuning Pyridoxal 5′-Phosphate Synthesis in Escherichia coli for Cadaverine Production in Minimal Culture Media

“…The elution buffer used was 0.1 mol/L potassium dihydrogen phosphate (pH 6.6), with an elution time of 0–15 min and a flow rate of 1.0 mL/min. The column was pre-equilibrated at 35 °C, and detection was conducted at a wavelength of 380 nm …”

“…The column was preequilibrated at 35 °C, and detection was conducted at a wavelength of 380 nm. 22 2.8. Statistical Analysis and Reproducibility.…”

“…20 However, excessive intracellular PLP levels can suppress cell growth, 17 necessitating controlled PLP biosynthesis. New strategies, including (i) the use of the Ribo1 riboswitch to regulate the metabolic flux of pdxS/T from Bacillus subtilis 21 or (ii) the establishment of a direct enzyme evaluation platform using CRISPRi to target the pdxB in E. coli MG1655, 22 offer ways to potentially enhance cadaverine production while eliminating the need for external PLP, thereby reducing production costs. Nevertheless, an effective and balanced genetically engineered system for PLP regeneration remains an unrealized goal to date.…”