Team:DTU-Denmark/Results-choosing-organism
Choice of organism
Choice of organism
Growing conditions between fungal species can vary, depending on how they grow in nature. Factors could be temperature, amount of light and the substrate, among other things. With the goal of growing fungal biobricks, a selection of the species used for our research needed to be done.
Pleurotus ostreatus, Aspergillus oryzae and Schizophyllum commune were initially investigated. First, S. commune has been investigated and used for the generation of bricks by the company Ecovative Design (1), which we collaborated with. In addition, S. commune also allowed us to study genes affecting the structure of mycelium, by using mutants as Δsc3. This strain is characterized by a lack of sc3 genes, responsible for the production of hydrophobins. Hydrophobins are small proteins present in filamentous fungi that have the ability to repel water and create a coating in hydrophobic: hydrophilic surfaces (2).
Secondly, P. ostreatus is a common edible mushroom also known as the oyster mushroom that has been widely cultivated. In addition to having a strong mycelium, it is used in industrial processes and mycoremediation. (3).
As for A. oryzae, it has been used for fermentation processes for at least 2000 years (4). In addition, its genome has been sequenced (5), is an optimal choice for the generation of a genetic toolbox.
Results
In the first experiment, the species were seeded at 3 different concentrations of MEA (47,5 g, 50 g and 52,5), PDA (19,5 g, 39 g and 58,5 g) and YPD. Thereafter, growth pictures were taken every 24 hours and the size of the fungal colonies was compared. In addition, different growth conditions (temperature and light) were tested for each of the species. S. commune was incubated 30ºC at light and 27ºC at light, P. ostreatus at 28ºC at dark and 25ºC at light, and A. oryzae at 28ºC at dark and 30ºC at dark.
In addition, we also investigated the growth of a mutated S. commune strain ,Δsc3.
Fig. 1: - Growth of A. oryzae on PDA 58.5g (A), S. commune on PDA 39g (B) and P. ostreatus on PDA 19.5g (C) plates after 24, 48 and 72 hours respectively after inoculation.
S. commune, P. ostreatus and A. oryzae grew at a similar rate on all media. However, the mutant strain of S. commune, Δsc3, was able to grow only at PDA 39 g (Figure 2).
Fig. 2: - Growth of S. commune Δsc3 on PDA 39g plates after 24, 48 and 72 hours respectively after inoculation.
Furthermore, to successfully transform the species we tested the sporulation of the fungi under the conditions we researched in the literature. The presence of spores facilitates the generation of protoplasts from fungi cells, that can then be transformed.
After incubation at the following conditions: S. commune at 30ºC at light, P. ostreatus at 28ºC at dark and A. oryzae at 28ºC at dark, only A. oryzae showed sporulation.
This preliminary experiment showed that several species were interesting for our research, as they were able to grow in widely used media, and they produced mycelium that could form fungal bricks. In addition, only A. oryzae was able to sporulate. This was possible when cultured at 28ºC at dark for 5-7 days. Moreover, it is a well-studied species that are used in fermentation processes and has been engineered previously. Therefore, we decided to focus the research on transformation with genes of interest on A. oryzae.
(1) Flores, R. (2015). Plastic Alternatives: Exploring Mycelium as a Medium. Parsons The New School for Design
(2) van Wetter, MA., Wösten, HA., Sietsma, JH., Wessels, JG. (2000). Hydrophobin gene expression affects hyphal wall composition in Schizophyllum commune. Fungal Genet Biol. 2000 Nov;31(2):99-104.
(3) Sanchez, C. (2010). Cultivation of Pleurotus ostreatus and other edible mushrooms. Appl Microbiol Biotechnol. 2010 Feb;85(5):1321-37.
(4) Rokas, A. (2009). The effect of domestication on the fungal proteome. Trends in Genetics Vol.25 No.2.
(5) Machida, M., Asai, K., Kikuchi, H. (2005). Genome sequencing and analysis of Aspergillus oryzae. Nature volume 438, pages 1157–1161