Team:Cornell/project/background/fungal toolkit

This year, we are working to develop a toolkit of genetic parts for engineering complex fungi, particularly plant-pathogenic basidiomycetes. We were inspired to do so by a local company, Ecovative Design, that uses lignin-degrading fungi and plant matter to produce a biodegradable Styrofoam substitute. Upon consulting the company on their production process, we found that their production efficiency suffered due to contamination from pathogenic molds, which inhibits the growth of the desired fungus and compromises entire batches of material. Our group recognized that this problem could be solved with a very rudimentary genetic circuit, which confers resistance to specific mold species, but that both industry and academia largely lack standardized fundamental tools for engineering fungi. We seek to empower Ecovative and other organizations to use fungi to develop sustainable technologies.

We are working with the fungus Ganoderma lucidum, a model organism for the class of wood-rot fungi that is used in Ecovative’s products. We have assembled a number of constructs for the production of pigmentation, fluorescent proteins, as well as simple antibiotic resistances that we are seeking to transform by both random insertion and homologous recombination into the organism’s genome. These proof-of-concept tools are necessary to optimize our process so that we can then obtain rigorous, reliable characterization data for the antifungal gene we are seeking to introduce, a protein that specifically targets Aspergillus niger. However, as Ganoderma does not have a well-standardized transformation protocol and takes several days to grow to an appropriate density, we are also conducting much of our basic fungal characterization work in a species of Cochliobolus, a simpler fungus. In addition, we are seeking to introduce a novel viral regulation system into fungi; similar systems have been used successfully in mammalian cells, and this system would greatly expand the accessibility of fungal genetic engineering beyond experienced mycologists. This system allows us to conduct preliminary characterization within E. coli, a much simpler system for acquiring quantitative data.

We are also implementing a number of biosafety measures within our toolkit, including a recombination system that has previously been demonstrated to work in simpler fungi, which will allow us to remove antibiotic resistance genes and targeted antifungal compounds before the end of the production process, in order to prevent them from spreading into the environment. Our products would also be subjected to heat treatment that is able to kill all cells and degrade much of its DNA in the final packaging product, so as to avoid transfer of genes from engineered to native strains. The strain we are seeking to develop would be huge boon to the economic viability of their product, as it would allow them to more easily maintain the high levels of quality control that are necessary in scaled-up production.

The product that we are seeking to improve, known as “mushroom packaging,” is a sustainable and necessary alternative to Styrofoam. Polystyrene can take hundreds of years to degrade in landfills and produces dozens of identified chemical toxins upon combustion, thus posing difficulties for disposal. Recycling of Styrofoam is incredibly inefficient at all levels; it suffers from low rates of recovery from consumers as most cities do not accept it for recycling, it is expensive to sort and transport to the appropriate specialized processing plant, and the actual process of recycling it involves high temperature and pressure conditions that consume large amounts of energy. Ecovative’s alternative requires only two feedstocks, fungal inoculum and sterilized plant matter, both of which readily degrade in the environment. The fungi used in their products are not typically used for food purposes and can be easily isolated and grown in controlled conditions. The feedstocks can include any form of plant matter, as cellulose is the key component of the plant matter that is needed in the final product, but the material properties vary with the feedstock. They have found that dense materials like stalks and seed husks, under-utilized components of agricultural waste, are optimal for their production. In this way, they are able to avoid using components that are in higher demand, such as food crops or even compost. For these and other considerations, their product has been granted the Cradle to Cradle Gold Certification.