Design of a recombinant Pseudomonas strain with enhanced degradation viability towards catecholic compounds by reconstituting EDO-hydrolysis pathway

Catecholic compounds caused consequential environmental pollution and posed severe threats to human health. Despite microbial biodegradation of phenolic aromatics providing a naturally evolved solution, the poor degradation viability of natural microbes decisively restricts their practical application for bioremediation. Herein, we demonstrated an effective strategy that the biodegradation capability of a natural Pseudomonas strain P. putida (Pp18) towards catecholic pollutants can be significantly enhanced in the folds of 2–12 with extended catecholic scopes. Based on the analysis of two-mode metabolic pathways of catecholics, the strategy started from screening of Pseudomonas strains, followed by constructing and improving Extradiol dioxygenase (EDO)-hydrolysis pathway with home-discovered EDO Tcu3516 as a starting point. Together with 2-hydroxy-6-oxohexa-2,4-dienoate hydrolase (HIU89), EDO Tcu3516 were co-expressed and further integrated with endogenous enzymes to improve the metabolic efficiency of EDO-hydrolysis mode. The dual-expression strain Tcu-HIU-pV-Pp18 demonstrated greatly enhanced degradation viability towards mono-and di- hydric phenols, e.g. o -cresol and 3-methylcatechol, in a fold of 8 compared to the parent strain. Furthermore, 5 EDO mutants from engineering of Tcu3516 were tested for construction, transformation and co-expression in Pp18, which the strain of NA (mutant V212N/D285A of Tcu3516)-HIU-pV-Pp18 demonstrated further improved biodegrading variability, excellent robustness and resilience to withstand high-concentration toxic catecholic pollutants.