Heterologous expression of microbial nitroreductases for TNT degradation in transgenic animals

Heterologous expression of microbial nitroreductases for TNT degradation in transgenic animals | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Heterologous expression of microbial nitroreductases for TNT degradation in transgenic animals View ORCID Profile Dimitri Perusse , View ORCID Profile Kate Tepper , View ORCID Profile Adam Sychla , View ORCID Profile Samuel J. Beach , View ORCID Profile Michael J. Smanski , View ORCID Profile Maciej Maselko doi: https://doi.org/10.1101/2025.06.11.659045 Dimitri Perusse 1 Department of Biochemistry, Molecular Biology, and Biophysics. University of Minnesota , Saint Paul, MN 55108 2 Biotechnology Institute, University of Minnesota , Saint Paul, MN 55108 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dimitri Perusse Kate Tepper 3 Applied BioSciences, Macquarie University , Sydney, NSW 2109, Australia 4 Australian Research Council Centre of Excellence in Synthetic Biology, Macquarie University , Sydney, NSW 2109, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kate Tepper Adam Sychla 1 Department of Biochemistry, Molecular Biology, and Biophysics. University of Minnesota , Saint Paul, MN 55108 2 Biotechnology Institute, University of Minnesota , Saint Paul, MN 55108 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Adam Sychla Samuel J. Beach 3 Applied BioSciences, Macquarie University , Sydney, NSW 2109, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Samuel J. Beach Michael J. Smanski 1 Department of Biochemistry, Molecular Biology, and Biophysics. University of Minnesota , Saint Paul, MN 55108 2 Biotechnology Institute, University of Minnesota , Saint Paul, MN 55108 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael J. Smanski Maciej Maselko 3 Applied BioSciences, Macquarie University , Sydney, NSW 2109, Australia 4 Australian Research Council Centre of Excellence in Synthetic Biology, Macquarie University , Sydney, NSW 2109, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maciej Maselko For correspondence: maciej.maselko{at}mq.edu.au Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract TNT (2,4,6-trinitrotoluene) from unexploded ordinances is a common environmental pollutant near munitions factories, military training sites, and areas of armed conflict. As physical and chemical approaches for TNT remediation are costly and difficult to scale, in situ bioremediation is an attractive alternative. TNT is a phytoaccumulative pollutant. Herbivores engineered to detoxify TNT are a potentially cost-effective platform to bioremediate large areas of contaminated sites while grazing or browsing. As a first step towards engineering herbivores with metabolic capabilities for TNT degradation, we engineered the animal genetic model, Drosophila melanogaster , to screen a library of microbial nitroreductases that can catalyse the initial degradation steps required for TNT degradation. We find strong, cofactor-dependent activity in fly lysates engineered to express NsfA from Escherichia coli , and NsfI from Enterobacter cloacae . Introduction Pollution from explosives, including TNT, is a widespread problem. According to the Food and Agriculture Organisation of the United Nations, very high levels of pollution from explosives occurs in Southeast Asia, the Middle East, and Africa 1 . Investigative journalism by Propublica in 2017 following freedom of information requests revealed that the US is estimated to spend a billion dollars a year to remediate >40,000 sites that are contaminated with explosives 2 . Explosives used in the current wars in Ukraine and Gaza are expected to leave behind legacies of environmental pollution, which include TNT contamination 3 – 5 . Ukraine is one of the largest exporters of grains in the world, and as of March 2023, over a quarter of their agricultural land is estimated to be contaminated with explosives 3 , 4 . TNT released into the environment is highly recalcitrant to degradation. It can adsorb to the soil matrix and leach into groundwater, where it can persist for many decades to centuries 6 . Some sites contaminated during World War II still present high concentrations of TNT 7 . Natural nitroaromatic compounds are rare, which may explain why TNT is not readily mineralized biologically 8 . TNT toxicity can have broad impacts. In animals, TNT causes hemolysis leading to anaemia and damages blood conditioning organs such the liver, kidneys, and spleen 9 , 10 . Rats exposed to TNT over 2 years developed bladder carcinomas 9 , and it is toxic to the male reproductive system 9 , 10 . In plants, it damages the root system, stunts growth, and reduces seedling emergence 11 – 13 . It is also toxic to organisms important for soil health, such as microorganisms, fungi, and meso- and macro-organisms 12 , 14 . In aquatic environments, TNT can impede algae growth, and reduce survival of invertebrates, amphibians, and fish 13 . A common method to remediate polluted soils is through composting 8 . This process relies on the ability of some microorganisms to degrade TNT into less toxic molecules 8 , 15 . TNT in contaminated soil may also be mineralized into its inorganic chemical constituents by incineration 8 , 16 , 17 . Groundwater is usually treated by filtration over granular activated carbon, or by Fenton processes 16 , 17 . All these processes can be expensive, requiring dedicated infrastructure, or a large-scale and disruptive reshaping of the environment. The US is reported to have spent over $40 billion US dollars on remediating pollution from explosives, and is expecting to pay a further $28 billion US dollars to complete the clean-up 2 . Bioremediation is an attractive economical solution to clean up TNT from large areas with low-concentrations, and where the clean-up of a contaminated site can take a long time. Many plants accumulate TNT from the soil when they grow on contaminated sites 18 . Even if they possess the metabolic capacity to metabolize this chemical 19 , the process is slow and does not complete the break-down of TNT 20 , 21 . However, several enzymes have been characterized that rapidly metabolize TNT 22 . This led French et al ., almost twenty-five years ago to engineer tobacco plants to express PETN reductase from the bacterium Enterobacter cloacae PB2 for phytoremediation 23 . PETN reductase catalyses the removal of one nitro group from TNT 24 , allowing it to be conjugated to glutathione, which facilitates its sequestration into vacuoles or the lignin fraction of cell walls 25 . Following this, multiple enzymes from bacteria or animals have been introduced in different plants to confer the ability to metabolize multiple nitroaromatic pollutants 26 – 31 . Although phytoremediation remains attractive, its deployment at scale is challenging. Phytoremediation requires first removing the native plants present in a contaminated site and then growing the transgenic phytoremediator over many years 28 . Following clean up, the transgenic plants are removed before attempting to re-establish the native flora. Biocontainment strategies need to be robust to avoid the undesired spread of transgenic plants in the area 32 . A more scalable approach would involve engineering large herbivores to break down explosives, including TNT, in their diet ( Figure 1C ). These animals can be easily transported between contaminated sites and they would metabolize the TNT that accumulates in native flora, as they naturally break down plant matter during digestion. Biocontainment of fewer and larger transgenic animals is likely to be more practically achieved relative to phytoremediation approaches by fencing, surgical sterilization, or genetic approaches 33 until TNT levels have dropped below an appropriate threshold. Download figure Open in new tab Figure 1: Engineering animals for TNT bioremediation. A. Chemical transformations involved in TNT bioremediation. Blue arrows and intermediates denote the oxygen-sensitive pathway. Red arrows show spontaneous oxidations. B. Diagram of the genetic constructs used to engineer D. melanogaster to express microbial nitroreductases. C. Functional microbial nitroreductases are first screened using D. melanogaster . D. Herbivores, such as goats and sheep, could subsequently be engineered to detoxify explosives, such as TNT, that accumulate in plants growing on contaminated sites. TNT: 2,4,6-Trinitrotoluene, TNB: 1,3,5-Trinitrobenzene, NARDNT: Nitroanion radical-dinitrotoluene, NDNT: Nitroso-dinitrotoluene, HADNT: Hydrolamino-dinitrotoluene, ATNDT: Azoxy-tetranitroditoluene, ADNT: Amino-dinitrotoluene. Enzymatic approaches for TNT detoxification involve first the removal of the nitro groups or their transformation to facilitate further degradation reactions 8 , 25 . This can be achieved by microbial nitroreductases, which use electrons from NADH or NADPH to catalyse the stepwise reduction of one or more of the nitro-groups of TNT into nitroso-, then hydroxylamino-, and finally amino-group(s) ( Figure 1A ) 8 . Though these breakdown products and their condensation products, azoxynitrotoluenes, remain toxic, reduction of one or more of the nitro groups allows the subsequent oxidation of aromatic ring of TNT 8 . This oxidation step can be achieved using various ligninolytic oxidative enzymes from white-rot fungi, to complete the mineralisation of TNT 8 . Though ligninolytic enzymes would be toxic to plants and the biocontainment would be challenging in microbes, animals can be engineered to express functional ligninolytic enzymes to oxidize various aromatic industrial pollutants 34 . Functional variants of nitroreductases could be co-expressed with ligninolytic enzymes in an animal host to complete the detoxification of TNT. As a first step toward herbivore TNT bioremediation, we engineered the animal model D. melanogaster to express six nitroreductases and characterized their activity in vitro . The functional microbial nitroreductases may provide a selection of low risk candidates that can be engineered into long lived herbivores ( Figure 1 ). Results Engineering flies to express a library of microbial nitroreductases Six bacterial nitroreductases previously described in the literature were engineered into D. melanogaster ( Table 1 ). These included: nsfI from E. cloacae 96-3 37 , nfsA from E. coli 38 , pnrA from Pseudomonas putida JRL11 39 , snrA from Salmonella enterica serovar Typhimurium TA1535 40 , mm-nr from Morganella morganii B2 41 , and xenA from P. putida II-B 42 ( Table 1 ). Most of the enzymes are oxygen-insensitive nitroreductases. Each nitro group reduction requires two electrons, leading directly to the first nitroso intermediates 2-nitroso-4,6-dinitrotoluene or 4-nitroso-2,6-dinitrotoluene ( Figure 1a ) 37 – 41 . The enzyme XenA, on the other hand, is an oxygen-sensitive nitroreductase. In this case, the reduction of the nitro moiety into a nitroso is carried out by two successive one-electron transfers with the formation of a nitroanion radical intermediate 8 . This nitroanion radical can be easily quenched by a molecule of dioxygen to regenerate TNT ( Figure 1 ). In most cases, the enzymes accept only NADPH as their electron source, except for NsfI, which can use NADPH and NADH to carry out the successive reductions 37 – 41 . View this table: View inline View popup Download powerpoint Table 1: Nitroreductase genes engineered into D. melanogaster Three of the six microbial nitroreductases have previously been expressed in a eukaryotic host. The genes nsfI, nsfA , and pnrA were engineered into Arabidopsis thaliana 43 , tobacco 29 , aspen 44 , or grasses 45 for the purpose of TNT bioremediation. Transgenic human cell lines involving nsfA were created to activate prodrugs containing nitro groups into cytotoxic chemicals for cancer research 46 . The nitroreductases open reding frames were codon optimised for D. melanogaster and expressed from a truncated alpha tubulin promoter ( Figure 1B ). We selected this promoter as it has been used previously to heterologously express enzymes for prototyping animal bioremediation in flies 34 , 47 . It provides expression likely across a broad set of tissues, and at a moderate level to reduce potential toxic overexpression 48 . The expression vectors were integrated into D. melanogaster strains harboring AttP landing sites using ϕC31 mediated integration 49 . Transgenic D. melanogaster were selected using the mini-white marker 50 . We validated the genomic integration of the expression vectors by PCR and Sanger sequencing (data not shown). We also validated nitroreductase mRNA expression from the truncated alpha tubulin promoter by RT-qPCR. The nitroreductases’ mRNA levels were between 10% and 30% of the housekeeping reference gene EF1 (Supplementary Figure 1). In vitro TNT degradation We tested the library of microbial nitroreductases engineered into D. melanogaster for their ability to degrade TNT in vitro . Triplicates of transgenic fly lysates and w 1118 fly lysate controls were incubated with 50 µM TNT and 300 µM NADH or NADPH as an electron donor for 90 mins. TNT concentrations were measured by HPLC. With NADH as the electron donor, only the fly lysates prepared from adult flies expressing NsfI were able to degrade TNT ( Figure 2A ). At the 90 min timepoint, 5.5% of TNT remained in solution. No significant degradation activity was detected for the other fly lysates compared to the w 1118 control within the allocated experimental time ( Figure 2B ). Download figure Open in new tab Figure 2: TNT degradation in fly lysates Fly lysates and controls were incubated with 50 µM TNT with 300 µM A . NADH or C . NADPH in 25 mM Tris-HCl pH 7.4 for 90 minutes. TNT concentrations were measured using HPLC. n = 3 biologically independent replicates. Statistical analysis of A . and C . were performed at end-point in B . and D ., respectively. The statistical analysis was conducted using one-way ANOVA using Dunnett’s method compared to controls, where ns = not statistically significant (p > 0.05). E. HPLC chromatograms for NsfI fly lysates incubated with 300 µM NADH and 50 µM TNT over a 90-minute time course in comparison to pure standards for TNT and its putative breakdown products 2-ADNT and 4-ADNT. w 1118 = D. melanogaster genetic background for the engineered strains, Fly/NsfA = D. melanogaster engineered to express nsfA from E. coli , Fly/PnrA = D. melanogaster engineered to express pnrA from P. putida , Fly/SnrA = D. melanogaster engineered to express snrA from S. enterica , Fly/Mm-nr = D. melanogaster engineered to express a nitroreductase from M. morganii , and Fly/NsfI = D. melanogaster engineered to express nsfI from E. cloacae . According to previous studies 38 – 42 , most of the enzymes selected for this experiment use NADPH as the electron donor to reduce the nitro moieties of TNT. Using NADPH as an electron donor, we observed degradation of TNT in extracts from flies expressing NsfA and NsfI, with 19% and 30% of the TNT remaining after 90 min, respectively ( Figure 2C ). The difference between degradation by NsfA/I and the w 1118 control was statistically significant ( Figure 2D ). NsfA and NsfI were not statistically different from each other ( Figure 2D ). Fly lysates prepared from flies expressing PnrA show a slight decrease in TNT concentration in later timepoints ( Figure 2C ), but the result is not statistically significant compared to the w 1118 control ( Figure 2D ). The other enzymes, SnrA, Mm-nr, and XenA, and the w 1118 control did not show TNT degradation within the allocated experimental time ( Figure 2C ). TNT degradation products The HPLC chromatogram peaks corresponding to TNT and its expected amino-dinitrotoluene degradation products were annotated by comparison to standards ( Figure 2E ). We observed a small peak corresponding to 4-amino-dinitrotoluene (4-ADNT), though it was not equivalent to the initial amount of TNT. We observed a small and large peak of uncharacterized compounds with a retention time of 64 minutes concomitant with TNT degradation. We postulate that these are isomers of azoxynitrotoluene degradation products ( Figure 1A ). According to the degradation pathway, the hydroxylamino intermediates can spontaneously dimerize in oxygenated conditions, particularly in vitro , to form azoxynitrotoluene molecules ( Figure 1A ) 8 . We did not confirm this identity by NMR or spectroscopic analysis and do not have authentic standards for the azoxynitrotoluene compounds. Discussion This research is the first step towards engineering long lived herbivores, such as goats and sheep, to bioremediate large areas of phytoaccumulative pollutants, such as TNT. Using D. melanogaster as a research tool to screen a library of nitroreductases, we identified NsfI and NsfA are capable of catalysing the first step of TNT degradation in vitro to remove the electron withdrawing nitro-groups of TNT. These enzymes could be combined with the ligninolytic enzymes we characterised previously in D. melanogaster to oxidize the TNT intermediates and complete the detoxification of TNT. Future experiments will involve engineering this enzyme pathway into short lived herbivores, such as rabbits, to prototype animal TNT bioremediation from contaminated plant material and to characterise the breakdown products of the enzyme pathway. Herbivore bioremediation could add unique capabilities to the field of bioremediation. The herbivores can be readily transported between sites and immediately process contaminated native plants. Robust transgene biocontainment can be achieved in herbivores such as goats and sheep, using industry standard surgical sterilization. The animals may also be fenced into specific areas, and/or tracked via GPS. Animals may be engineered to express ligninolytic oxidative enzymes to complete the detoxification of TNT. Though microbes commonly metabolize TNT to the toxic 2-ADNT and 4-ADNT intermediates in aerobic soils, engineering microbes to express oxidative enzymes from white-rot fungi is limited by effective transgene biocontainment strategies required for their wild release 51 . Engineering plants to express ligninolytic oxidative enzymes is challenging as these enzymes will also degrade lignin in plants 52 . A major disadvantage of using herbivores for TNT bioremediation is that TNT predominantly accumulates in the root material, with less than 25% of TNT in the aerial parts of the plant, depending on the species 18 . The TNT accessible to the herbivores will therefore be low, and would significantly increase the time it takes to remediate a site. Another major disadvantage is that any pollutant present in plant material is in equilibrium with the soil, and to remediate a pollutant from the soil will also take a long time 28 . These disadvantages may be acceptable in large contaminated areas where no other economical remediation option is available and time is not an important factor. Where duration is an important factor, it could be addressed by combining herbivore bioremediation with phytoremediators engineered to hyperaccumulate TNT into the aerial parts of the plant. Previously we showed that animals engineered to express mercury detoxification enzymes were resistant to greater methylmercury exposure 47 . We expect that animals engineered for TNT detoxification will be similarly resistant to greater TNT exposure. They may therefore remediate a site for wildlife that may be harmed by TNT in the contaminated area, without harm to themselves. Future studies to determine TNT toxicity in wildtype and engineered herbivores will be critical. Materials and Methods Fly expression plasmid assembly The nitroreductase library enzyme sequences ( Table 1 ) were codon optimized (Integrated DNA Technologies (IDT) codon optimization tool) for expression in D. melanogaster. The D. melanogaster Kozak consensus sequence: “AATCTTACAAA” was added immediately preceding the start codon. At least 20 bp of homology arms to the destination plasmid (pMC-1-1-1) 34 were added upstream and downstream. The sequences were ordered as gBlocks from IDT and assembled (NEBuilder® HiFi DNA assembly master mix (New England Biolabs (NEB))) into the pMC-1-1-1 expression plasmid linearized with NotI-HF. The plasmids were verified by Sanger sequencing. See Supplementary Table S3 and S4 for plasmid descriptions and sequences, and Supplementary Table S2 for primers used in this study. D. melanogaster husbandry, strains, and transgenesis All lines were maintained at 25°C and 12 h light/dark cycle and 60–70% relative humidity. The flies were kept in vials containing standard cornmeal/agar diet Bloomington formula (Genesee Scientific, 66–121), 0.05 M propionic acid (Sigma, 402907), and 0.1% tegosept (Genesee Scientific, 20–258). w 1118 D. melanogaster strains were used to control for genetic background. w 1118 are WT flies that have the mini-white gene knocked out, resulting in white eyes. The plasmids containing the different nitroreductases were sent to Bestgene Inc. (Chino Hills, CA) for ϕC31-mediated integration and outcrosses to balancer strains. Homozygous fly lines for the nitroreductase constructs were generated by selecting against balancer phenotypic markers. Homozygous NfsA flies could not be generated and heterozygous flies were used in the assays. See Supplementary Table S1 for fly strains used in this study. Validation of transgene genomic integration Five frozen adult flies were homogenized using a motorized pestle in 500 µL 10 mM Tris-HCl pH 8 (Roche, #10812846001), 1 mM ethylenediaminetetraacetate (EDTA) (Millipore Sigma, #ED-500G), 25 mM sodium chloride (Macron Fine Chemicals, #7581-06), and 200 g/mL Proteinase K (New England Biolabs, #P8107S). The mix was then incubated at 37°C for 30 min, followed by 3 min at 95°C to inactivate the Proteinase K. The tube was centrifuged at 17,900 rcf for 5 min and 0.5 μL of the supernatant was collected for PCR analysis. The gDNA in solution was used as the template with a universal set of primers flanking the nitroreductase locus (Supplementary Table S2) and the region of interest was amplified with CloneAmpTM Hi-Fi PCR Premix (Takara Bio USA, #639298). The DNA thermocycling conditions were: initial denaturation step at 98°C for 3 min; 35 cycles of 98°C for 10 s, 70°C for 15 s, and 72°C for 30 s; and a final extension step at 72°C for 5 min. The PCR products were analyzed by agarose gel electrophoresis and Sanger sequencing (ACGT, Inc.). Validation of transgene mRNA expression RNA was extracted from the flies using the TRIzol reagent (Thermo Fisher Scientific, #15596018) following the manufacturer instructions. Three frozen adult flies from the same line were homogenized with a motorized pestle in 0.2 mL TRIzol. An additional 0.8 mL of TRIzol was added. The RNA isolated was resuspended in nuclease-free water (Takara Bio USA, #9012) and then treated with DNase using the TURBO DNA-free kit (Invitrogen, #AM2238). RT-qPCR was carried out from the isolated RNA (100 ng) using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs, #E3005L) in a 12 μL reaction total volume following the manufacturer instructions. Fold changes for RT-qPCR were determined by the ΔΔCT method 35 . Expression of the nitroreductases were normalized to D. melanogaster gene Elongation Factor 1 alpha100 (EF1) 36 . See Supplementary Table S2 for primers used in this study. Chemicals and stock solution preparation A slurry of 30% TNT in water was obtained from the US department of defence. A portion of the 30% TNT slurry was frozen at -80°C overnight and was lyophilized to remove the water from the sample. A 0.5 mM TNT solution was prepared in 25 mM Tris buffer solution at pH 7.4. A TNT analytical standard was purchased from Millipore Sigma (#ERT-022S-1.2ML). Analytical standards for 2-amino-dinitrotoluene (2-ADNT) and 4-amino-dinitrotoluene (4-ADNT) were purchased from Millipore Sigma (#ERA-017-100MG and #ERA-018-100MG, respectively). Stock solutions of 2-DANT and 4-DANT (1 mg/mL) were prepared by dissolving each compound in acetonitrile (Fisher Scientific, A998-4). Serial dilutions were prepared from the stock solutions at 100, 50, 25, 10, 5, 2.5, 1, 0.5, and 0.1 µg/mL. See Supplementary Figures S2-S5 for the TNT, 2-ADNT, and 4-ADNT HPLC calibration curves. Solutions of 3 mM NADH and NADPH (Millipore Sigma, #481913-500MG and Neta Scientific, #RPI-N20120-0.1, respectively) were made in 25 mM tris buffer solution at pH 7.4. The solutions were prepared immediately before experiments and kept on ice. In vitro degradation of TNT by adult fly lysates To prepare fly lysate, 20 frozen adult flies were homogenized with a motorized pestle in 450 µL of 50 mM tris-HCl buffer pH 7.4. Each tube was incubated for 15 min at room temperature. Each tube was centrifuged at 17,000 rcf at room temperature for 10 min, and 350 μL of the supernatant was transferred into a clean microcentrifuge tube. The solution was centrifuged again at 17,000 rcf at room temperature for 10 min and 275 μL of supernatant was transferred into another clean tube. After a third centrifuge at the same speed, 200 μL of supernatant was transferred to another tube and diluted up to 1 mL in 25 mM Tris buffer pH 7.4. To prepare the in vitro enzyme assay, 720 μL of the fly lysate prepared above was added to 90 μL of the 0.5 mM TNT stock solution. Then 90 μL aliquots of the enzyme-TNT solution were added into an 8-strip PCR tubes (Genesee, 27-426F). To start the enzymatic reaction, 10 μL of either 3 mM NADH or NADPH stock solutions was added. In the negative control, the electron donor solution was replaced by 10 μL of 25 mM Trizma buffer pH 7.4. The reaction mixes were incubated at room temperature for various durations as described in the results. To stop the reactions, the strip tube was incubated at 80°C for 5 min. TNT degradation analysis by high performance liquid chromatography (HPLC) Samples (100 µL) were introduced into inserts (VWR, #97051-402) for 2 mL HPLC vials (ChromTech, #CTV-1209GSA). The HPLC analyses were performed on an Ultimate 3000 Rapid Separation system using either a Phenomenex Luna C18 column (4.6 × 150 mm, 3 µm) (#00F-4114-E0) or an Agilent Eclipse XDB-C18 column (4.6 × 100 mm, 3.5 µm) (#961967-902). The sample injection volume was 10 µL. All chromatography was performed at a flow rate of 1.2 mL/min using Milli-Q water filtered on 0.22 µm filter (Millipore Sigma, JGWP04700) (solvent A) and acetonitrile (Fisher Scientific, A998-4) (solvent B) with the different gradients depending on the column. Gradient for the Luna column: 0-65 min, 10% to 90% solvent B; 65 - 75 min, 95% solvent B; 77 - 81 min, 90% to 10% solvent B. Gradient for the Eclipse column: 0 - 10 min, 10% to 30% solvent B; 10 - 12.5 min, 30% solvent B; 12.5 - 25 min, 30% to 95% solvent B; 25 - 27 min, 95% solvent B, 27 - 28 min, 95% to 10% solvent B, 28 - 30 min 10% solvent B. Elution compounds were detected at λ = 254 nm. Figures Figures were made using GraphPad Prism 10, Excel, Python, Biorender, and Adobe Illustrator. Author Contributions K.T. and M.M. conceived of the study. K.T., D.P., and S.B. performed the experiments. All authors contributed to experimental designs and data analysis. All authors contributed to writing the manuscript. Co-first authors DP and KT have editorial approval to list their name in the first positions on their respective CVs. Acknowledgements This research was funded by the International Technology Center Pacific (ITC-PAC) under Contract No. FA520923C0014 (M.M.). D.P. was supported by a fellowship from the MnDRIVE at the University of Minnesota. 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