Metabolic engineering of Escherichia coli for 2,4-dinitrotoluene degradation.

2,4-Dinitrotoluene (2,4-DNT) as a common industrial waste has been massively discharged into the environment with industrial wastewater. Due to its refractory degradation, high toxicity, and bioaccumulation, 2,4-DNT pollution has become increasingly serious. Compared with the currently available physical and chemical methods, in situ bioremediation is considered as an economical and environmentally friendly approach to remove toxic compounds from contaminated environment. In this study, we relocated a complete degradation pathway of 2,4-DNT into Escherichia coli to degrade 2,4-DNT completely. Eight genes from Burkholderia sp. strain were re-synthesized by PCR-based two-step DNA synthesis method and introduced into E. coli. Degradation experiments revealed that the transformant was able to degrade 2,4-DNT completely in 12 h when the 2,4-DNT concentration reached 3 mM. The organic acids in the tricarboxylic acid cycle were detected to prove the degradation of 2,4-DNT through the artificial degradation pathway. The results proved that 2,4-DNT could be completely degraded by the engineered bacteria. In this study, the complete degradation pathway of 2,4-DNT was constructed in E. coli for the first time using synthetic biology techniques. This research provides theoretical and experimental bases for the actual treatment of 2,4-DNT, and lays a technical foundation for the bioremediation of organic pollutants.

Dual metabolic pathways co-determine the efficient aerobic biodegradation of phenol in Cupriavidus nantongensis X1.

Phenol, as an important chemical raw material, often exists in wastewater from chemical plants and pollutes soil and groundwater. Aerobic biodegradation is a promising method for remediation of phenolic wastewater. In this study, degradation characteristics and mechanisms of phenol in Cupriavidus nantongensis X1 were explored. Strain X1 could completely degrade 1.5 mM phenol within 32 h and use it as the sole carbon source for growth. The optimal degradation temperature and pH for phenol by strain X1 were 30 °C and 7.0. The detection of 3-oxoadipate and 4-hydroxy-2-oxopentanoate indicated that dual metabolic pathways coexist in strain X1 for phenol degradation, ortho- and meta-pathway. Genome and transcriptome sequencing revealed the whole gene clusters for phenol biomineralization, in which C12O and C23O were key enzymes in two metabolic pathways. The ribosome proteins were also involved in the regulation of phenol degradation. Meanwhile, the degradation activities of enzyme C23O was 188-fold higher than that of C12O in vitro, which indicated that the meta-pathway was more efficient than ortho-pathway for catechol degradation in strain X1. This study provides an efficient strain resource for phenol degradation, and the discovery of dual metabolic pathways provides new insight into the aerobic biological metabolism and bioremediation of phenol.

Complete pentachlorophenol biodegradation in a dual-working electrode bioelectrochemical system: Performance and functional microorganism identification.

Bioelectrochemical system (BES) can effectively promote the reductive dechlorination of chlorophenols (CPs). However, the complete degradation of CPs with sequential dechlorination and mineralization processes has rarely achieved from the BES. Here, a dual-working electrode BES was constructed and applied for the complete degradation of pentachlorophenol (PCP). Combined with DNA-stable isotope probing (DNA-SIP), the biofilms attached on the anodic and cathodic electrode in the BES were analyzed to explore the dechlorinating and mineralizing microorganisms. Results showed that PCP removal efficiency in the dual-working BES (84% for 21 days) was 4.1 and 4.7 times higher than those of conventional BESs with a single anodic or cathodic working electrode, respectively. Based on DNA-SIP and high-throughput sequencing analysis, the cathodic working electrode harbored the potential dechlorinators (Comamonas, Pseudomonas, Methylobacillus, and Dechlorosoma), and the anodic working enriched the potential intermediate mineralizing bacteria (Comamonas, Stenotrophomonas, and Geobacter), indicating that PCP could be completely degraded under the synergetic effect of these functional microorganisms. Besides, the potential autotrophic functional bacteria that might be involved in the PCP dechlorination were also identified by SIP labeled with 13C-NaHCO3. Our results proved that the dual-working BES could accelerate the complete degradation of PCP and enrich separately the functional microbial consortium for the PCP dechlorination and mineralization, which has broad potential for bioelectrochemical techniques in the treatment of wastewater contaminated with CPs or other halogenated organic compounds.

Creation of Environmentally Friendly Super "Dinitrotoluene Scavenger" Plants.

Pervasive environmental contamination due to the uncontrolled dispersal of 2,4-dinitrotoluene (2,4-DNT) represents a substantial global health risk, demanding urgent intervention for the removal of this detrimental compound from affected sites and the promotion of ecological restoration. Conventional methodologies, however, are energy-intensive, susceptible to secondary pollution, and may inadvertently increase carbon emissions. In this study, a 2,4-DNT degradation module is designed, assembled, and validated in rice plants. Consequently, the modified rice plants acquire the ability to counteract the phytotoxicity of 2,4-DNT. The most significant finding of this study is that these modified rice plants can completely degrade 2,4-DNT into innocuous substances and subsequently introduce them into the tricarboxylic acid cycle. Further, research reveals that the modified rice plants enable the rapid phytoremediation of 2,4-DNT-contaminated soil. This innovative, eco-friendly phytoremediation approach for dinitrotoluene-contaminated soil and water demonstrates significant potential across diverse regions, substantially contributing to carbon neutrality and sustainable development objectives by repurposing carbon and energy from organic contaminants.

Optimization and reconstruction of two new complete degradation pathways for 3-chlorocatechol and 4-chlorocatechol in Escherichia coli.

Chlorinated aromatic compounds are a serious environmental concern because of their widespread occurrence throughout the environment. Although several microorganisms have evolved to gain the ability to degrade chlorinated aromatic compounds and use them as carbon sources, they still cannot meet the diverse needs of pollution remediation. In this study, the degradation pathways for 3-chlorocatechol (3CC) and 4-chlorocatechol (4CC) were successfully reconstructed by the optimization, synthesis, and assembly of functional genes from different strains. The addition of a 13C-labeled substrate and functional analysis of different metabolic modules confirmed that the genetically engineered strains can metabolize chlorocatechol similar to naturally degrading strains. The strain containing either of these artificial pathways can degrade catechol, 3CC, and 4CC completely, although differences in the degradation efficiency may be noted. Proteomic analysis and scanning electron microscopy observation showed that 3CC and 4CC have toxic effects on Escherichia coli, but the engineered bacteria can significantly eliminate these inhibitory effects. As core metabolic pathways for the degradation of chloroaromatics, the two chlorocatechol degradation pathways constructed in this study can be used to construct pollution remediation-engineered bacteria, and the related technologies may be applied to construct complete degradation pathways for complex organic hazardous materials.

Synergistic Enzyme Mixtures to Realize Near-Complete Depolymerization in Biodegradable Polymer/Additive Blends.

Embedding catalysts inside of plastics affords accelerated chemical modification with programmable latency and pathways. Nanoscopically embedded enzymes can lead to near-complete degradation of polyesters via chain-end mediated processive depolymerization. The overall degradation rate and pathways have a strong dependence on the morphology of semicrystalline polyesters. Yet, most studies to date focus on pristine polymers instead of mixtures that contain additives and other components despite their nearly universal use in plastic production. Here, additives are introduced to purposely change the morphology of polycaprolactone (PCL) by increasing the bending and twisting of crystalline lamellae. These morphological changes immobilize chain ends preferentially at the crystalline/amorphous interfaces and limit chain-end accessibility by the embedded processive enzyme. This chain-end redistribution reduces the polymer-to-monomer conversion from >95% to less than 50%, causing formation of highly crystalline plastic pieces, including microplastics. By synergizing both random chain scission and processive depolymerization, it is feasible to navigate morphological changes in polymer/additive blends and to achieve near-complete depolymerization. The random scission enzymes in the amorphous domains create new chain ends that are subsequently bound and depolymerized by processive enzymes. Present studies further highlight the importance to consider how the host polymer's morphologies affect the reactions catalyzed by embedded catalytic species.

Construction of an Escherichia coli strain to degrade phenol completely with two modified metabolic modules.

Phenol is a common water pollutant because of its broad industrial applications. Biological method is a promising alternative to conventional physical and chemical methods for removing this toxic pollutant from the environment. In this study, two metabolic modules were introduced into Escherichia coli, the widely used host for various genetic manipulations, to elucidate the metabolic capacity of E. coli for phenol degradation. The first module catalysed the conversion of phenol to catechol, whereas the second module cleaved catechol into the three carboxylic acid circulating intermediates by the ortho-cleavage pathway. Phenol was completely degraded and imported into the tricarboxylic acid cycle by the engineered bacteria. Proteomics analysis showed that all genes in the phenol degradation pathway were over-expressed and affected cell division and energy metabolism of the host cells. Phenol in coking wastewater was degraded powerfully by BL-phe/cat. The engineered E. coli can improve the removal rate and shorten the processing time for phenol removal and has considerable potential in the treatment of toxic and harmful pollutants.

Biodegradation of butyronitrile and demonstration of its mineralization by Rhodococcus sp. MTB5.

A nitrile utilizing bacterium Rhodococcus sp. MTB5 was previously isolated in our laboratory by the enrichment culture technique. It is able to utilize butyronitrile as sole carbon, nitrogen, and energy source. Maximum butyronitrile degrading property of this strain has been investigated. Results reveal that 100, 98, and 88 % degradation was achieved for 2, 2.5, and 3 % butyronitrile, respectively. The strain is capable of growing in as high as 5 % butyronitrile concentration. A two-step pathway involving nitrile hydratase (NHase) and amidase was observed for the biodegradation of butyronitrile. Complete degradation (mineralization) of butyronitrile with the help of metabolite feeding experiment was reported. The significance of this investigation was the capability of the strain to completely degrade and its ability to grow on higher concentrations of butyronitrile. These potential features make it a suitable candidate for practical field application for effective in situ bioremediation of butyronitrile contaminated sites.

Complete biodegradation of chlorpyrifos by engineered Pseudomonas putida cells expressing surface-immobilized laccases.

The long-term abuse use of chlorpyrifos-like pesticides in agriculture and horticulture has resulted in significant soil or water contamination and a worldwide ecosystem threat. In this study, the ability of a solvent-tolerant bacterium, Pseudomonas putida MB285, with surface-displayed bacterial laccase, to biodegrade chlorpyrifos was investigated. The results of compositional analyses of the degraded products demonstrate that the engineered MB285 was capable of completely eliminating chlorpyrifos via direct biodegradation, as determined by high-performance liquid chromatography and gas chromatography-mass spectrometry assays. Two intermediate metabolites, namely 3,5,6-trichloro-2-pyridinol (TCP) and diethyl phosphate, were temporarily detectable, verifying the joint and stepwise degradation of chlorpyrifos by surface laccases and certain cellular enzymes, whereas the purified free laccase incompletely degraded chlorpyrifos into TCP. The degradation reaction can be conducted over a wide range of pH values (2-7) and temperatures (5-55 °C) without the need for Cu(2+). Bioassays using Caenorhabditis elegans as an indicator organism demonstrated that the medium was completely detoxified of chlorpyrifos by degradation. Moreover, the engineered cells exhibited a high capacity of repeated degradation and good performance in continuous degradation cycles, as well as a high capacity to degrade real effluents containing chlorpyrifos. Therefore, the developed system exhibited a high degradation capacity and performance and constitutes an improved approach to address chlorpyrifos contamination in chlorpyrifos-remediation practice.