Team:DTU-Denmark/Ganoderma-protoplastation
Ganoderma Protoplastation
Introduction
The generation of protoplasts involves the removal of the cell wall of the fungal cells by enzymatic digestion. Thereafter, the addition of calcium will permeabilize the protoplasts, facilitating the intake of foreign DNA (1), see also fig. 1. When generating protoplasts, the enzymatic mix used for the cell wall digestion is critical, as the components in the cell wall can vary significantly between species. Besides the percentage of glucan, mannan, and chitin, there can be variations in the hyphal branching, as well as differences when fungi are sporulating (2).
Fig. 1: Schematic representation of the generation of protoplasts. After the spores or hyphae from mycelium has been collected, the cells are incubated with an enzymatic mix. This disrupts the cell wall, liberating the protoplasts. The lysate is filtered, allowing the protoplasts to go through the filter. To transform the protoplasts, DNA of interest is added together with calcium ions and PEG, facilitating the fusion of exogenous DNA into the cells. Eventually, the transformants will be inoculated in selective media, being able to distinguish transformants from non-transformant cells. 
In our project, we were interested in generating protoplasts for the species Ganoderma resinaceum. This species has been employed by Ecovative Design to use mycelium as sustainable biomaterials (3). In addition, besides being a source of inspiration for our project, we had the chance to collaborate with them. Our objective in this collaboration was to improve their protoplast generation protocol for Ganoderma resinaceum. Therefore, we received a sample of the strain they were using so we could experiment with it.
Within the Ganoderma protoplast protocol, we decided to investigate if we could perform the protoplastation avoiding the use of Driselase. This is a costly enzyme targeting the cell wall, that causes the release of carbohydrates. It contains a mix of enzymes (fungal carbohydrate hydrolases). Instead, we proposed the use of only one cell lysis component, Glucanex. This is a cost-effective enzymatic mix containing β-glucanase, cellulase, protease, and chitinase activities (4).
Results
During the tissue generation, tissue was obtained from Ganoderma resinaceum mycelium and incubated for 24 hours at 30ºC and either 85 rpm or 150 rpm. Thereafter, the samples were digested with a lysis solution containing different concentrations of Glucanex: 10, 20, 30 and 40 mg/mL. Then, all samples were incubated for cell wall digestion either for 2-3 hours or 19-24 hours. Finally, the protoplasts were filtered, centrifuged and re-suspended in sorbitol solution.
After having digested the cell wall with several Glucanex® concentrations, the digest was filtered through Mira cloth (22-25 µm). None of the Glucanex concentrations (10, 20, 30 and 40 mg/mL) seemed to have lysed the cell wall successfully, as no protoplasts were observed after centrifugation. This indicates that no protoplasts were generated from the mycelium. This might be due to the components of the cell wall, not being fully degraded by the enzymatic mix. These results suggest that other enzymes targeting the cell wall would be necessary as well to successfully digest the cell wall.
Future work towards making the protoplast generation more effective would be using Driselase at lower concentrations. In addition, we would try to improve the yield of protoplasts generated, by increasing the number of initial mycelia.
(1) Turgeon BG, Condon B, Liu J, Zhang N. 2010. Protoplast Transformation of Filamentous Fungi. Methods Mol Biol 638:3-19.
(2) Li D, Tang Y, Lin J. 2017. Methods for Genetic Transformation of Filamentous Fungi. Microb Cell Fact 16:168.
(3) Ecovative Design. 2018. Beyond the Basics.
(4) Petit J, Boisseau P, Arveiler B. 1994. Glucanex: A cost-effective yeast lytic enzyme. Trends Genet 10:4-5.