Discovery of an Inducible Toluene Monooxygenase That Cooxidizes 1,4-Dioxane and 1,1-Dichloroethylene in Propanotrophic Azoarcus sp. Strain DD4

Abstract

Cometabolic degradation plays a prominent role in bioremediation of commingled groundwater contamination (e.g., chlorinated solvents and the solvent stabilizer 1,4-dioxane [dioxane]). In this study, we untangled the diversity and catalytic functions of multi-component monooxygenases in Azoarcus sp. DD4, a gram-negative propanotroph that is effective in degrading dioxane and 1,1-dichloroethylene (1,1-DCE). Using a combination of knockout mutagenesis and heterologous expression, a toluene monooxygenase (MO) encoded by the tmoABCDEF gene cluster was unequivocally proved as the key enzyme responsible for the cometabolism of both dioxane and 1,1-DCE. Interestingly, in addition to utilizing toluene as a primary substrate, this toluene MO can also oxidize propane into 1-propanol. Expression of this toluene MO in DD4 appears inducible by both substrates (toluene and propane) and their primary hydroxylation products (m-cresol, p-cresol, and 1-propanol). These findings coherently explain why DD4 can grow on propane and express toluene MO for active co-oxidation of dioxane and 1,1-DCE. Furthermore, upregulation of tmo transcription by 1-propanol underlines the implication potential of using 1-propanol as an alternative auxiliary substrate for DD4 bioaugmentation. The discovery of this toluene MO in DD4 and its degradation and induction versatility renders broad applications spanning from environmental remediation and water treatment to biocatalysis in green chemistry. Importance Toluene MOs have been well recognized given their robust abilities to degrade a variety of environmental pollutants. Built upon previous research efforts, this study ascertained the untapped capability of a toluene MO in DD4 for effective co-oxidation of dioxane and 1,1-DCE, two of the most prevailing yet challenging groundwater contaminants. This report also aligns the induction of a toluene MO with non-toxic and commercially accessible chemicals (e.g., propane and 1-propanol), extending its implication values in the field of environmental microbiology and beyond.

Combined Omics Approach Reveals Key Differences between Aerobic and Microaerobic Xylene-Degrading Enrichment Bacterial Communities: Rhodoferax─A Hitherto Unknown Player Emerges from the Microbial Dark Matter

“…It is important to note that the same tmo gene cluster was described in case of the propanothrophic Azoarcus sp. strain DD4, hinting at the spread of this gene cluster among toluene degraders of the Burkholderiales by horizontal gene transfer. Around 20 kilobases downstream of the gene cluster coding for Tmo, another cluster was found that coded for a multicomponent phenol hydroxylase (mPH) and a complete meta -cleavage pathway (mPH1 cluster).…”

Network And Meta-Omics Reveal The Key Degrader And Cooperation Patterns In An Efficient 1,4-Dioxane-Degrading Microbial Community

“…Metatranscriptomic sequencing revealed that among the multiple monooxygenase genes detected in the culture, including toluene monooxygenase, 4hydroxyphenylacetate 3-monooxygenase, nitronate monooxygenase, salicylate 1-monooxygenase and alkanesulfonate monooxygenase genes, the most abundant monooxygenase genes from ZM13 were identi ed as toluene monooxygenase (Additional le 1: Table S7 and Figure S9), which belong to Group SDIMOs. A recent study rstly proved the 1,4-dioxane degradation function of toluene monooxygenase in a propanotrophic strain DD4 [41]. In addition, the network analysis demonstrated the important role of ZM13 as a bottleneck, serving as a bridge that connected different parts of the community.…”

Emerging investigator series: environment-specific auxiliary substrates tailored for effective cometabolic bioremediation of 1,4-dioxane

“…In DD4, though 1-propanol can induce the expression of TMO, it is the product of the oxidation of propane by TMO, which thus doesn't compete with dioxane for the active sites of TMO. 30 Further, alcohol substrates are miscible with water and can be easily operated for field injection and monitoring. However, 1-propanol and other alcohol substrates are less selective to the inocula and thus more suited for low biomass environments dominated by oligotrophic microbiomes that respond poorly to the addition of auxiliary substrates, such as deep bedrock aquifers.…”

“…The primers and other materials used in the RT-qPCR procedures are described in our previous publication. 30 Differential gene expression was quantified using the 2 −ΔΔ C q method 32 with tmoA as the target gene and 16S rRNA as the housekeeping gene. The expression fold change was calculated with the following formula:ΔΔ C q,Targetgene = ( C q,Targetgene − C q,Housekeepinggene ) Treatment − ( C q,Targetgene − C q,Housekeepinggene ) Control …”

“…Expression of this TMO appeared inducible by both propane and its primary oxidation product, 1-propanol, implying their potential as auxiliary substrates for cometabolic bioremediation. 30 Furthermore, unlike the majority of dioxane degrading actinomycetes, 31 DD4 has the tmo gene on its chromosome. 29 This is important as it precludes the loss of the essential dioxane degradation genes when the culture is fed with liquid alcohols and other non-selective substrates that are readily biodegradable.…”