Filamentous Fungi

Fungi are a diverse group of organisms, whose biological activities affect our daily life in many ways. The filamentous fungi particularly are of great importance in production of medicine, in the industry, in agriculture, and in basic biological research. Some of the filamentous fungal species are pathogenic to humans, whereas others have great value in the production of antibiotics such as penicillin. The fungi are therefore importance for industrial as well as medical production.
Filamentous fungi produce a diverse array of secondary metabolites, which serve the pharmaceutical sciences as prolific source of chemical compounds for the development of new drugs.

Growth of filamentous fungi

The vegetative growth of filamentous fungi starts with the germination of a spore when the conditions are right (Adams et al., 1998). The spore germination leads to formation of hyphae (Adams et al., 1998). A fungal hypha is a long tubular modular structure composed of individual cells (Harris, 1997). Hyphae extend only at their tips and are typically divided into individual cellular compartments by the formation of septa (Harris et al., 2009).

Filamentous fungi grow by the polar extension of hyphae and multiply by branching (Timberlake, 1990). The branched hyphae forms a network of interconnected cells called a mycelium (Adams et al., 1998). The mycelium forms a radially symmetric colony that expands over large area until growth is limited by for example lack of nutrients (Adams et al., 1998; Nielsen, 2008). The fungal mycelium appears to be a formless collection of corresponding vegetative cells. However, the various cells within the mycelium interact to form an ordered network with different hyphae or cells playing distinct roles in the acquirement of nutrients from the environment and development (Adams et al., 1998).

Aspergillus nidulans

The filamentous fungus Aspergillus nidulans is a widely recognised model organism (Harris et al., 2009). A. nidulans possesses today, in contrast to most other aspergilla, a well characterized sexual cycle and a well-developed genetics system (Galagan et al., 2005). Furthermore, in A.nidulans the parasexual cycle has been extensively utilized. Parasexual genetic involves examination of recombination in the absence of sexual reproduction.

The genetic analysis has produced a deep understanding of both the physiology of Aspergillus and the organisation of the genome (Doonan, 1992). This research has advanced the study of eukaryotic cellular physiology and contributed to our understanding of metabolic regulation, development, DNA repair, morphogenesis, and human genetics diseases (Galagan et al., 2005). Furthermore, the recent sequencing of the complete genome of A.nidulans has created a tremendous potential to obtain insight into important aspects of fungal biology such as transcriptional regulation, secondary metabolite production and pathogenicity (Nielsen et al., 2006).

Gene targeting

Accurate manipulation of genes is a key approach in fungal molecular biology (Krappmann, 2007). The method of gene targeting facilitates precise genome manipulations which are called site directed alterations. They are performed by use of deletions, replacements and insertion of the target locus. Gene targeting is achieved by transforming an organism with a suitable linear DNA fragment that contains sequences that are identical to the target site in the genome, see figure. Different DNA fragment are constructed depending on deletion, replacement or insertion. The fungi can integrates linear DNA fragment in its genome by using its repair system of DNA double stranded breaks.

Two mechanisms of DNA double strand break repair ensure that the introduced piece of DNA is pasted into the fungal genome, to be replicated stably: Homologous Recombination and Non Homologous End Joining, also called illegitimate recombination (Krappmann, 2007). Homologous recombination involves interaction between homologous sequences, whereas NHEJ involves ligation of the strand ends independently of DNA homology (Ninomiya et al., 2004). Precise genome manipulation can often be tedious and time-consuming, because fungi appear to favour Non Homologous End Joining over Homologous recombination resulting in low gene targeting efficiencies (Krappmann, 2007).

Proof of concept

Several separate devices were conducted with Plug ‘n’ Play assembly to verify if the system will work in fungi as well as in mammalian cells. The idea was to target the fungi with a fluorescence protein to specific compartments and in general to express the fluorescence proteins. All the devices have the same promoter, terminator and marker cassette, though with different gene of interest and were all transformed in laboratory Aspergillus nidulans stain: argB2, pyrG89, veA1 by random non-homologues-end-joining integration.

We succeed in proving that Plug ‘n’ Play assembly easily transformed and expressed in fungi as we succeed in tagging A. nidulans with two fluorescence proteins. The transformants with integration of our 4 devices change morphology compared with the wild type control strain since the DNA were randomly intergraded and have inflicted some pathways in the fungi.

Device BBa_K678060

Aspergillus nidulans with device BBa_K678060 - DIC light.	Aspergillus nidulans with device BBa_K678060 - GFP light.

Aspergillus nidulans with device BBa_K678060 - Front	Aspergillus nidulans with device BBa_K678060 - Back

Device BBa_K678061

Aspergillus nidulans with device BBa_K678061 - DIC light.	Aspergillus nidulans with device BBa_K678061- GFP light.

Aspergillus nidulans with device BBa_K678061 - Front	Aspergillus nidulans with device BBa_K678061 - Back

Device BBa_K678062

Aspergillus nidulans with device BBa_K678062 - DIC light.	Aspergillus nidulans with device BBa_K678062 - RFP light.

Aspergillus nidulans with device - Front	Aspergillus nidulans with device - Back

Device BBa_K678063

Aspergillus nidulans with device BBa_K678063 - DIC light.	Aspergillus nidulans with device BBa_K678063 - RFP light.

Aspergillus nidulans with device BBa_K678063 - Front	Aspergillus nidulans with device BBa_K678063 - Back

Control strain

Wild type Aspergillus nidulans - DIC light.	Wild type Aspergillus nidulans - RFP light.	Wild type Aspergillus nidulans - YFP light.

Wild type Aspergillus nidulans - Front	Wild type Aspergillus nidulans - Back