Abstract
Inadequate management of household and industrial wastes pose major challenges to human and environmental health. Advances in synthetic biology may help address these challenges by engineering biological systems to perform new functions such as biomanufacturing of high-value compounds from low-value waste streams and bioremediation of industrial pollutants. The current emphasis on microbial systems for biomanufacturing, which often require highly pre-processed inputs and sophisticated infrastructure, is not feasible for many waste streams. Concerns about transgene biocontainment have limited the release of engineered microbes or plants for bioremediation. Engineering animals may provide opportunities for utilizing various waste streams that are not suitable for microbial biomanufacturing while effective transgene biocontainment options should enable in situ bioremediation. Here, we engineer the model insect Drosophila melanogaster to express a functional laccase from the fungus Trametes trogii. Laccase expressing flies reduced concentrations of the endocrine disruptor bisphenol A by more than 50% when present in their growth media. A lyophilized powder made from engineered adult flies retained substantial enzymatic activity, degrading more than 90% of bisphenol A and the textile dye indigo carmine in aqueous solutions. Our results demonstrate that transgenic animals may be used to bioremediate environmental contaminants in vivo and serve as novel production platforms for industrial enzymes. These results support further development of insects, and possibly other animals, as bioproduction platforms and their potential use in bioremediation.
Main Text
Developing economically viable approaches for sustainable waste management and mitigation of environmental pollution are important for ensuring a high quality of life1. At present, over half of all municipal solid waste is either landfilled2, where it is estimated to contribute to 11% of global methane emissions3,4, or openly dumped, where it contaminates drinking water and disrupts natural ecosystems5. Pollution from industrial wastes includes a variety of persistent compounds which have been linked to neurological defects, cancer, and species extinction6–10.
Regulatory changes including prohibiting discharge of industrial pollutants and reducing the landfilling of food waste are helpful, however, the cost of compliance can be substantial11. Generating value from a variety of waste-streams and developing inexpensive methods to treat pollutants in situ will reduce the costs of achieving sustainability targets and help support a circular economy12.
We propose developing solutions by expanding the enzymatic repertoire of select animals to consume waste, degrade xenobiotic chemicals, and produce high-value compounds (Fig. 1). Microbial and fungal enzymes can enable animals to breakdown pollutants in situ. These may include herbivores that degrade pollutants accumulating in plants, fish that secrete enzymes to degrade aquatic pollutants, or invertebrates engineered to interrupt the biomagnification of xenobiotics in food webs. Effective transgene biocontainment safeguards are currently a challenge for plants/microbes13,14, but are possible in animals via surgical sterilization or engineered genetic incompatibilities15. Developing insects as a biomanufacturing platforms for industrial enzymes, lipids, or other molecules is also an unexplored avenue for waste valorisation.
Insects have advantages over bacteria and fungi for processing many waste-streams. Their complex digestive systems, including mastication, allow them to consume a variety of low-value wastes, including municipal organic waste16 and livestock manure17. Crude waste can be fed directly to insects without pre-processing, refinement, and sterilization necessary for microbial fermentation18,19. Some insect species, such as Black soldier flies (Hermetia illucens), which are already used to manage waste have excellent tolerance to bacteria and fungi20 and are easily separated from the organic waste for downstream processing21. Insect biotechnology is readily scalable for mass production, as the infrastructure can be relatively unsophisticated, and the installation costs and land use requirements are low22. Furthermore, utilising organic waste means crops that could otherwise be used for food do not need to be diverted as feedstocks for fermentation, and will not threaten food security.
There is a long history of using lepidopteran insect larvae to make recombinant proteins starting with Bombyx mori expressing human α-interferon23. However, these utilize transient baculoviral systems to infect the larvae. This approach is not practical for large-scale or long-term, multi-generational use. Here we explore the potential of stably engineered insects as platforms for the production of an industrially relevant fungal laccase and bioremediation of polluted sites. Laccases are multicopper oxidases that are present in many taxa. Fungal laccases were selected due to their demonstrated utility for bioremediation of a wide variety of industrial pollutants such as bisphenols24, industrial dyes25, pharmaceuticals26, phenolics27, polycyclic aromatic hydrocarbons28, perfluoroalkyl and polyfluoroalkyl substances (PFAS)29,30, plastics31, pesticides32, and mycotoxin contaminants such as aflatoxins33. Fungal laccases also have diverse industrial applications in the textile, paper and pulp, food, pharmaceutical, chemical synthesis, and forestry industries34–36. They have been applied to treat distillery37, paper and pulp38, and olive mill39 industry wastewater. Ligninolytic enzymes, such as fungal laccases, also show potential application in the delignification of feedstocks for biofuel production40. However, fungal laccases have not yet reached appreciable commercial scale production. This is because the native hosts for laccases often produce low yields and although this has been improved using heterologous hosts, the production levels are still too low for many commercial applications41.
We engineered the common fruit fly (Drosophila melanogaster) to express a laccase from the fungus Trametes trogii. Transgenic flies degraded bisphenol A (BPA), a widespread endocrine disruptor42, when present in their diet. We also demonstrate that aqueous solutions of BPA and indigo carmine (IC), a textile dye and aquatic pollutant43, can be treated with a lyophilized powder from adult transgenic flies to degrade both compounds. Our results describe the first instance of engineering an animal to perform an ecosystem service by heterologous expression of a fungal enzyme and demonstrate the potential for using transgenic strains of insects for enzyme bioproduction.
Results
Screening for fungal laccase activity in transgenic D. melanogaster
D. melanogaster and other insects express endogenous laccases involved in immune defense44. However, it was unknown if an insect could heterologously express a functional fungal laccase or if its expression would be toxic. We were also uncertain which tissues would provide the optimal expression of a functional laccase for either in vitro or in vivo activity. We selected a short tubulin promoter, previously used to express a toxic gene45, with the goal of achieving moderate expression across many tissues. The native fungal signal peptides were replaced with the D. melanogaster larval cuticle protein 946 signal peptide to facilitate extracellular secretion.
We generated homozygous transgenic strains expressing laccases from two fungal species, T. trogii and Polyporus brumalis, known to have a broad substrate range and high redox potential (Table 1). Fly lysates from both were assayed for oxidation of the chromogenic laccase substrate 2,2’-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). The engineered D. melanogaster strain Dm/Tt.Lcc1, which expresses a laccase from T. trogii, oxidized ABTS (Fig. 2a). There was no activity observed in the wild-type or from the engineered Dm/Pb.Lac1 strain expressing a laccase from P. brumalis (Fig. 2a). Moving forward, we focused on characterizing Dm/Tt.Lcc1.
We next determined if Dm/Tt.Lcc1 flies are capable of degrading bisphenol A (BPA), an endocrine disruptor and emerging environmental contaminant found in many consumer products47. BPA has been shown to be a substrate of T. trogii laccase when expressed from its native host48. We incubated lysates from flies with 25 µg/mL BPA for 21 hours and measured BPA concentrations by liquid-chromatography mass spectrometry (LC-MS). Lysates from Dm/Tt.Lcc1 reduced BPA concentrations by 95% compared to the no-fly controls and the wild-type fly lysate (Fig. 2b-c).
In vivo laccase activity
We evaluated the potential of laccase expressing insects to oxidize substrates present in their environment by secreted enzymes. Flies were reared on a minimal media to avoid binding of substrates to insoluble components present in standard media. The minimal media included 1 mM CuSO4 to supply the laccase co-factor. First, we reared 3 adult males and 3 adult females on minimal diet including 1 mg/mL ABTS for 2 days, after which they were removed. Larvae of the mated flies were grown for an additional 5 days. Dm/Tt.Lcc1 flies oxidized ABTS as indicated by blue colorization of the fly media, however, no oxidation was observed with wild-type flies (Fig. 3a). We controlled for the possibility that the ABTS oxidation was due to laccase that may have been leaking from dead larvae by incubating 6 male flies in ABTS media. After 11 days, only the Dm/Tt.Lcc1 males had oxidized ABTS (Fig. 3b). This result indicates that the oxidation was due to enzyme secreted into the media.
Next, we reared 5 male and 5 female flies in a minimal diet containing 25 µg/mL BPA. Adults were removed after three days. On day 10, BPA was extracted from their diet and quantified by Ultra-High-Performance Liquid Chromatography coupled with fluorescence detection (UHPLC-FLD). Both Dm/Tt.Lcc1 and wild-type flies had reduced BPA concentrations by 36% and 45%, respectively, compared to no-fly controls (Fig. 3c). There was no significant difference between Dm/Tt.Lcc1 and wild-type indicating that the reduction in BPA concentration was not due to the activity of the laccase transgene. The reduction in BPA concentration for wild-type flies relative to the no-fly control may be in part due to BPA absorbed by the adult flies which were removed after three days. Another possibility is that BPA detoxification pathways have been characterized in mammals54 and it is possible D. melanogaster also contains endogenous pathways capable of detoxifying BPA.
Some laccase substrates, such as ABTS, can function as redox mediators to facilitate the oxidation of additional compounds55. When in vivo BPA degradation was tested in the presence of ABTS, Dm/Tt.Lcc1 flies effectively reduced BPA concentration by 68% and 56% compared to the no-fly control and wild-type, respectively (Fig. 3d). It is not clear why ABTS was necessary for the in vivo assay and not the in vitro lysate assays. Adding lysate directly to the media also required ABTS for efficient degredation, suggesting that there is some component of the diet which may be interfering (Fig. S1).
Lyophilized insect powder laccase activity
We next examined the potential of insects as platforms to produce a lyophilized laccase which can be easily stored and transported before use. A crude lyophilized powder from Dm/Tt.Lcc1 was compared to a commercially available purified laccase from Trametes versicolor using ABTS as a substrate. The lyophilized Dm/Tt.Lcc1 displayed a total laccase activity of 1.4 U/g ± 0.1 SEM dry weight, while the commercial purified laccase displayed a specific activity of 75.3 U/g ± 6 SEM dry weight.
We next tested if Dm/Tt.Lcc1 lyophilized whole-fly powder had activity against indigo carmine, a textile industry dye and pollutant43. Incubating 5 mg/mL of Dm/Tt.Lcc1 lyophilized powder in water containing 100 mg/L indigo carmine resulted in 90% decolorization after 48 h; however, WT fly lyophilized powder also decolorized 55% of the dye (Fig. 4a). We observed that insoluble material in the powder appeared to adsorb the dye. However, incubating dye using supernatant from centrifuged lyophilized Dm/Tt.Lcc1 in water did not appear to decolorize indigo carmine compared to controls (Fig. 4b). When the Cu2+ co-factor was included, supernatant from centrifuged lyophilized powder in water decolorized nearly 100% of indigo carmine in Dm/Tt.Lcc1 and 25% from WT flies after 90 hours (Fig. 4c-d).
We also evaluated Dm/Tt.Lcc1 lyophilized powder activity against BPA. Incubating 12.5 µg/mL BPA with soluble enzyme from the lyophilized powder in the presence of Cu2+ co-factor resulted in a 96% reduction of BPA from Dm/Tt.Lcc1 fly powder after 90 hours. Incubation with WT fly powder resulted in a 3% reduction of BPA (Fig. 4e-f).
Discussion
Engineering animals to heterologously express fungal/microbial enzymes may improve sustainable waste management, bioremediation, and facilitate the use of low-value waste streams as inputs for the production of high-value products such as industrial enzymes. Here, we engineer D. melanogaster to express a functional laccase from the fungus T. trogii. Lysates of this engineered fly (Dm/Tt.Lcc1) oxidized the endocrine disruptor BPA by 95% in vitro compared to the WT flies. Engineered flies also degraded BPA by 56% in vivo when added to their diet. Further, a powder prepared from lyophilized engineered flies also degraded BPA by 96% and the textile dye indigo carmine by almost 100% in vitro.
Mediator compounds are often used to improve the substrate range and activity of laccases56. Although Dm/Tt.Lcc1 lyophilized powder did not require the addition of ABTS as a mediator for degrading BPA, in vivo BPA degradation did. White rot fungi such as Phanerochaete chrysosporium and Pycnoporus cinnabarinus synthesize and secrete endogenous redox mediators57,58 and it may be possible to engineer animals to secrete redox mediators to also enhance their bioremediation activities. Alternatively, colloidal lignin or derivatives of lignin also show potential to act as redox mediators59,60, and including them may be feasible to assist with in vivo activity.
Using transgenic insects as platforms to convert waste to high-value products, such as industrial enzymes, will require high production titres. Dm/Tt.Lcc1 lyophilized powder made from whole flies had 1.4 U/g of laccase activity without any attempt at optimization compared to 75.3 U/g of activity from a commercially available purified T. versicolor laccase. This is a very promising start and developing tools to genetically manipulate more relevant hosts and strain engineering is needed for insects to reach their potential as bioproduction platforms.
In summary, our findings demonstrate for the first time that insects can be stably engineered to express a functional fungal laccase, which demonstrated in vivo and in vitro laccase activity. Transgenic flies were capable of oxidizing laccase substrates in their environment and they can be used as a lyophilized powder to oxidize substrates in aqueous solutions. These results provide strong support for further research in using transgenic animals as platforms for bioremediation, waste stream management, and recombinant protein production.
Methods
Construct design and assembly
NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) was used to assemble all constructs. pMBO274415 was used as a backbone to generate the enzyme expression plasmids. A gBLOCK (IDT®) was synthesized that encodes a short alpha-tubulin promoter, a downstream SV40 polyadenylation sequence, and a NotI restriction site in between. This gBLOCK was installed into NotI linearized pMB02744 by HiFi assembly to produce pMC-1-1-1 (Supplementary Table 4). This plasmid was then linearized with NotI, and gBLOCKs that encode for T. trogii laccase or P. brumalis laccase enzyme were installed with HiFi assembly to generate pMC-1-2-3 and pMC-1-2-6, respectively (Supplementary Table 4). The DNA sequences were codon optimized for expression in D. melanogaster using the IDT® codon optimization tool. The native fungal signal peptides were replaced with the larval cuticle protein 9 signal peptide. The Kozak sequence, AATCTTACAAA, was upstream of the start codon. See Supplementary Table 2 for plasmid descriptions and Supplementary Table 3 for primers used in this study.
Insect rearing and generation of transgenic fly strains
Canton S wild-type flies were from the Bloomington Stock Centre (64349, RRID: BDSC 64349). Enzyme expression plasmids were purified using ZymoPURE™ II Plasmid Midiprep Kit (Zymo Research #D4200) and sent to BestGene Inc (Chino Hills, Ca) for C31 mediated integration. All strains were maintained on a cornmeal diet based on the Bloomington standard Nutri-Fly formulation (catalog number 66-113; Genesee Scientific). In preparation for the in vitro or in vivo laccase enzyme activity assay, the diet for both the transgenic and wild-type strains was supplemented with CuSO4•5H2O (1 mM, Merck, CAS-no: 7758-99-9). Flies were reared in a controlled environment room at 25.0 °C, 75% humidity, and a 12 hour light/dark cycle with a 30 minute transition period. See Supplementary Table 1 for all of the fly strains used in this study. Biosafety approval was granted by the Institutional Biosafety Committee of Macquarie University (#5049).
In vitro enzyme activity assay
To obtain a fly lysate, ten frozen adult flies were homogenized with a motorized pestle in the stated buffer supplemented with 10 mM CuSO4•5H2O and acid-washed glass beads. The samples were then agitated along a tube rack 10 times. Each tube was incubated for 15 minutes at 25 °C and centrifuged twice to remove insoluble debris.
For the ABTS oxidation assay, fly lysates were prepared in citrate-phosphate buffer (pH 5) and added to 1 mM ABTS. The tubes were incubated at 25 °C for 2.5 hours. The reaction was quenched by adding 5% trichloroacetic acid (TCA, Sigma Aldrich, catalogue #T0699, CAS-no:76-03-9). A UV-Vis spectrophotometer (Jasco V-760 UV-Vis spectrophotometer) was used to measure the absorbance at 420 nm. The measurements were blanked to an equivalent sample that did not contain ABTS.
For the BPA degradation assay, fly lysates or controls were prepared in sodium acetate buffer (pH 5) and added to 25 µg/mL BPA. The assay mixtures were incubated at room temperature for 21 hours. The reaction was quenched by adding 5% (v/v) trichloroacetic acid (Sigma Aldrich, catalog #T0699, CAS-no:76-03-9). BPA concentration was analyzed by LC-MS as described below.
In vivo laccase activity assays
Minimal media
The minimal media components were for 100 mL: 1.58 g yeast, 0.66 g agar, 5 g sugar, 4 mL 1 M propionic acid (pH 4.4), 12.4 mg CuSO4•5H2O (1 mM final concentration). A buffer of 20 mM citrate-phosphate (pH 5) was added for the initial ABTS assays, 25 mM sodium acetate buffer (pH 5) was used for all BPA experiments.
ABTS only in vivo experiments
To minimal media 100 mg ABTS (1 mg/mL final concentration) was added. Three female and three male adult flies were incubated on the media for 48 hours, after which they were removed. For the all-male ABTS assay, the adult flies remained in the vial for 11 days. The appearance of the dark green radical cation was monitored and recorded with photographs.
For the ABTS + BPA experiments
To minimal media, BPA was added (25 µg/mL final concentration). ABTS (1 mg/mL final concentration) was added for the +BPA, + ABTS condition.
For each condition, the liquid media was pipetted into a 14 mL polypropylene round-bottom culture tube. 5 male and 5 female adult flies were incubated on the media for 3 days and then removed. For the lysate condition, 50 µL of lysate from ∼100 flies was added to the tube. After a total of 10 days the enzymatic reaction was stopped with TCA (5% v/v final concentration). The media was melted in a water bath, after which methanol (making ∼1:1 methanol: aqueous TCA for a two-fold dilution of BPA) was added, followed by vortexing.
The tubes were then incubated at 300 rpm and 37 °C for 30 minutes. After incubation, the tubes were centrifuged twice with the final supernatant reserved for BPA quantification by UHPLC-FLD as described below.
Lyophilized Dm/Tt.Lcc1 enzyme activity assays
Frozen Dm/Tt.Lcc1 or wild-type flies were lyophilized (Alpha 1-4 LDplus, Christ) at −45 °C at mBar. The freeze-dried flies were homogenized in a porcelain mortar and pestle and stored at 4 °C.
For the activity assays, lyophilized Dm/Tt.Lcc1 and commercial T. versicolor laccase (Sigma-Aldrich, Catalogue 38429) were resuspended in 25 mM sodium acetate buffer (pH 5) supplemented with 10 mM CuSO4•5H2O. Lyophilized Dm/Tt.Lcc1 did not dissolve, and the soluble fraction was extracted at 25 °C for 15 minutes at 800 rpm and centrifuged twice to remove the insoluble debris. Enzyme serial dilutions were added in duplicate to 1 mM ABTS and ABTS oxidation was monitored on a PHERAStar FS plate reader (BMG Labtech) at 420 nm at 25 °C. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the oxidation of 1 μmol ABTS min-1 using the molar extinction coefficient 36 mM-1 cm-1 61.
For the indigo carmine decolorization assay, 5 mg/mL of lyophilized powder or controls in Millipore water were added to 100 mg/L indigo carmine (Sigma-Aldrich catalog 131164) and incubated at RT. At 0 and 48 hours, indigo carmine decolorization was monitored on a PHERAStar FS plate reader (BMG Labtech) at 610 nm.
As indigo carmine appeared to adsorb to the lyophilized powder, the soluble fraction was tested for enzymatic activity. 5 mg/mL lyophilized powder or controls in Millipore water supplemented with and without 10 mM CuSO4•5H2O were extracted at 25 °C for 15 minutes at 800 rpm. The samples were centrifuged twice to remove the insoluble debris. The supernatant of the lyophilized powder and controls were added to 100 mg/L indigo carmine and incubated at room temperature. Indigo carmine decolorization was monitored on a PHERAStar FS plate reader (BMG Labtech) at 610 nm at 0, 21, 48, 72, and 90 hours.
For the BPA degradation assay, the soluble fraction of the lyophilized powder was extracted as described in the indigo carmine assay. The supernatant of the lyophilized powder and controls were added to 12.5 µg/mL BPA and incubated at room temperature. At 0, 48, and 96 hours, 100 μL aliquots were removed and the reaction was stopped using a 3 KDa nominal molecular weight limit (NMWL) centrifuge filter (Merck Amicon Ultra 0.5mL Centrifugal Filters, UFC500396). BPA concentration was analyzed by UHPLC-FLD as described below.
BPA quantification
BPA in samples was either quantified by UHPLC-FLD analysis or by LC-MS. UHPLC-FLD analysis was performed using an Agilent RRHD ZORBAX Eclipse Plus C18 column (3.0 × 150 mm 1.8 μm, p/n 959759-302) equipped with a UHPLC Eclipse Plus C18 Guard Column, (2.1 mm 1.8 µm, p/n 821725-901). The isocratic mobile phase consisted of acetonitrile (ACN):10 mM KH2PO4 (50:50 v/v) at a flow rate of 0.5 mL/min. A gradient wash step was introduced after each run with eluent B 100% ACN and eluent A 10mM KH2PO4, starting with 45% B1 from 0 - 1 minute, 70% B from 1 – 6 minutes, 5% B from 6 – 11 minutes, and finally 50% B from 11 - 16 minutes at 0.3 mL/min. BPA was detected by setting FLD excitation wavelength at 229 nm, and emission wavelength at 316 nm and a gain setting of 12. Linear regression of 7-point standards from 12.5 ng/mL to 12,500 ng/mL BPA was performed to calibrate BPA concentration. All samples and standards were either filtered using syringe filters (Filtropur S µm, Sarstedt, 83.1826.001) or centrifuge filters (Merck Amicon Ultra 0.5mL Centrifugal Filters, UFC500396) prior to injection into the UHPLC.
LC-MS validation and quantification of BPA was performed externally at the Mass Spectrometry Facility at the University of Sydney. LC separation was performed using a Waters Sunfire C18 column (5 μm, 2.1 mm ID x 15 mm) coupled to a Bruker amaZon SL mass spectrometer. Gradient elution was performed with a mobile phase consisting of (A) methanol and (B) water with 0.1% acetic acid at a flow rate of 0.3 mL/min. The gradient consisted of 20% A from 0 - 9.3 minutes, 76% A from 9.3 - 17.3 minutes, 92% A from 17.3 – 26 minutes, and finally 20% A from 26 - 35 minutes. MS analysis was performed using Atmospheric Pressure Chemical Ionisation (ACPI) under positive and negative ionization mode over a range of 200-1400 m/z. The operation parameters were: Nebulizer 27.3 psi, Dry Gas 4.0 L/min, Dry temperature 180 °C, APCI Vaporisation temperature: 400 °C, APCI Corona needle current: 6000 nA, Mass mode: enhanced resolution, SPS (Smart parameter setting) Target mass 150 m/z, compound stability 100%. Linear regression of 6-point BPA standards from 20 ng/mL to 10 µg/mL BPA was performed to calibrate BPA concentration. An internal deuterated BPA-D16 standard was spiked into the samples prior to injection to calibrate ionization in the sample matrix compared to the standard curve matrix prepared in methanol. All samples were centrifuge filtered (Merck Amicon Ultra 0.5mL Centrifugal Filters, UFC500396) prior to injection into the LC-MS.
Statistical analysis
Figures were generated in Prism 9 by GraphPad. For statistical analysis, a one-way ANOVA using Dunnet’s method was conducted. The significance level for the analysis was 0.05, and all error bars represent the standard error of the mean. The exact P values, statistical tests used, and sample numbers (n) can be found in the figure legend.
Funding
This material is based upon work supported by the International Technology Center Pacific (ITC-PAC) under Contract No. FA520920P0100, a Macquarie University seeding grant, and the CSIRO Synthetic Biology Future Science Platform.
Author Contributions
MC, KT, and MM conceived of this study. MC, KT, KP, AS, and MM designed experiments and analyzed data. MC, KT, KP, and SK performed the experiments. All authors contributed to writing the manuscript.
Competing Interests
The authors have filed a patent application for aspects of this work.
Data and materials availability
All data is available upon request.
Supplementary information
Acknowledgements
We are grateful Dr. Nick Proschogo from the Mass Spectrometry Facility at the University of Sydney.
Footnotes
Text/figure captions improved for clarity