Developing Chassis for LDPE Upcycling from Microbes Native to the Gut Microbiome of Yellow Mealworms
Ross Klauer1,2* (firstname.lastname@example.org), Lummy Maria Oliveira Monteiro1,2, Jyoti Singh1,2, Bill Alexander3, Adam Guss3, Carrie Eckert3, Kevin Solomon1,2, and Mark Blenner1,2
1Delaware Biotechnology Institute; 2University of Delaware; and 3Oak Ridge National Laboratory
This project aims to enable the efficient depolymerization of polyolefins and upcycling to itaconic acid, through novel genomic insights into nutrient enhanced polyolefin degradation by the yellow mealworm gut microbiome and genetic tool development for gut microbiome isolates and engineered microbial communities.
Waste plastics represent a significant untapped source of carbon. Annually more than 200 million tonnes of plastic waste in the form of high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polypropylene (PP) are generated and pollute soils, waterways, and bodies. No robust system exists to capture this carbon; however, in prior work in labs, researchers observed that yellow mealworm gut microbes are able to chemically modify these wastes suggesting an opportunity for biological upcycling. Microbial profiling of these communities reveals non-model taxa as the dominant microbes. Furthermore, the plastic degrading metabolic pathway remains unelucidated. A barrier to understanding plastic metabolism and engineering biological upcycling platforms is the lack of genetic tools to manipulate these species.
As a first step, the team previously enriched Tenebrio molitor guts for fast-degrading plastic microbial communities to identify optimal chasses for development for upcycling. Enrichment including growth on plastics and plastics with nutritional supplements such as oats, bran, and banana peels. Mealworm gut communities supplemented with oats were found to be optimal for plastics degradation rate. Researchers are further studying the effect of micronutrients and macronutrients in mealworm diets to enrich for an optimal polyolefin degrading community. The nutritional composition of the plastic and oats diet will be altered by supplementing with macronutrients such as protein and fats and with micronutrients such as nitrogen and potassium. From enriched communities, the team plans to cultivate key players in the degradation process and understand how the optimized community is structured, informing future creation of reduced complexity engineered microbial communities for plastic degradation and upcycling.
More than 300 microbial isolates were cultivated from microbial communities of LDPE, HDPE, and PP-fed mealworm guts, with and without oats supplementation through standard microbiological methods and isolating morphologically distinct colonies from each gut condition. By screening these isolates for growth with polyethylene powder as the primary carbon source, 30 taxonomically unique isolates were found to use LDPE powder as a carbon source for growth in a liquid mineral medium. Improved growth with LDPE particles as the primary carbon source relative to a mineral medium suggested that microbes participate in the degradation process. LDPE degradation is demonstrated by scanning electron microscopy (SEM) through visualized microbial colonization of plastic particles, biofilm formation, and surface modifications to the particles. Contrarily, no surface modifications were observed via SEM on LDPE films treated by the same microbial isolates, indicating that degradation efficiency varies depending on polymeric mechanical and chemical properties such as form factors, additives, and processing conditions. From this characterization survey, and microbial profile abundance, five taxa were identified belonging to genera Staphylococcus, Enterococcus, Corynebacterium, Brevibacterium, and Kocuria as robust chassis for development as upcycling platforms.
To understand degradation and upcycling potential of these isolates, researchers bioinformatically screened these isolates for polyolefin degrading enzymes. Polyolefin backbone cleavage is anticipated to be initiated by secreted enzymes via oxygen radical chemistry due to their high reactivity on carbon-carbon bonds. Protein families (pfam) that perform oxygen radical chemistry such as monooxygenases, dioxygenases, and peroxidases were identified in isolate genomes in order to identify PE-active enzymes. Genomes of isolates that grow best on LDPE were mined to find pfam that have a high number of genes that perform said chemistry relative to closely related microbial taxa. Select genes were heterologously expressed into Escherichia coli, purified, and tested on plastic substrates. Plastic substrate modification as a result of enzymatic activity is observed via (%) crystallinity increase after enzyme treatment on LDPE films.
As a first step towards the development of robust genetic engineering tools for these isolates, the team is collecting genome and methylome sequence data. The genome sequences are a prerequisite for targeted genetic engineering. The methylome data will help researchers understand which restriction modification systems are present in each isolate and design genetic parts to work in the presence of these systems.
In summary, mealworm microbiomes were enriched for optimum plastics degradation by supplementing with various co-feeds. New co-feed studies will identify key nutrients that further improve plastic degradation. From previously enriched communities, over 300 organisms have been isolated, 30 of which are able to grow with plastic as their primary carbon source. Five organisms hailing from non-model genera appear able to grow on LDPE powder and are somewhat abundant in enriched communities, indicating these microbes are suitable chassis for engineered polyolefin degradation and upcycling. Enzymes initiating the first degradation step of the upcycling process are being mined bioinformatically from microbial isolates and communities. Degradation capability of these enzymes is being analyzed chemically by Fourier-transform infrared spectroscopy and mechanically by differential scanning calorimetry. In parallel, downstream metabolic degradation processes are being evaluated using metagenomics and metatranscriptomics. Upon elucidation of polyolefin deconstruction processes, said processes will be enhanced in isolated strains and reassembled back into reduced complexity communities, as well as engineered into a heterologous host for the eventual production of itaconic acid from plastic substrates via metabolically engineering of the host.
This material is based upon work supported by the U.S. Department of Energy, Office of Science, Biological and Environmental Research (BER) Program, Genomic Science program under Award Number DE-SC0023085.