Enhanced propanethiol biodegradation by an optimized propanethiol oxidoreductase in microbial cells within an electrode bioreactor

Propanethiol oxidoreductase (PTO) is a novel thiol-oxidizing enzyme from Pseudomonas putida S-1 that utilizes thiols as the sole nutrition source. This enzyme has shown application potential in the bioremediation of thiols, its substrate selectivity, however, has not yet been elucidated. This study reveals that PTO exhibits catalytic activity towards ethanethiol, propanethiol, and butanethiol, but not methanethiol or phenylmethanethiol, indicating unique substrate specificity. Through directed evolution and semi-rational design, we engineered a PTO mutant (A54V&G316T) with twice the specific activity towards propanethiol than the wild type (from 17.3 to 40.7 μg/mg/h). Applying voltage in electrode bioreactors enhanced the microbial degradation of propanethiol, with the mutant PTO further accelerating this process. Both non-native expression in E. coli and native expression in engineered P. putida S-1 demonstrated the mutant PTO's effectiveness in increasing PT removal rates. The PT degradation efficiency of engineered P. putida S-1 increases by 3-fold compared to the wild-type in the first 5 hours. These findings highlight the potential of combining metabolic and electrochemical engineering to enhance bioremediation of toxic compounds. The engineered PTO mutant improves PT degradation efficiency and broadens its application in practical bioremediation strategies.