Fungi are ubiquitous organisms which have made a beneficial contribution to human development. This association dates back to 3000 B.C. when it is believed that for the first time, Egyptians used yeast for baking bread and brewing beer. Later with the discovery of penicillin from Penicillium chrysogenum, fungal antibiotics gained widespread use in treatment of infections. In the research field too, fungi serve as a model for genetics and cell biology. Filamentous fungi are known for secreting huge amounts of secondary metabolites like enzymes, organic acids, cyclosporins, and steroids. These valuable compounds have vast applications in several industries such as food, beverages, textiles, and pharmaceuticals. A well-known filamentous fungi is Aspergillus niger, an industrial workhorse for production of high titers of organic acids and enzymes.
Owing to its high genetic diversity, these fungi can be engineered to produce several organic acids as well as other building-block compounds. The production capacity of these versatile filamentous fungi is indeed promising. However, some bottlenecks such as slower growth rates, fewer genetic engineering tools, and longer strain development times make it lag behind other well established organisms like Escherichia coli or Saccharomyces cerevisiae. Therefore, it is essential to develop time-saving and more efficient tools for fungal engineering. In the recent past, several transformation and genetic manipulation strategies have been developed for filamentous fungi.
Recently, our group developed a genetic engineering method for DNA construction and its site-specific integration into the genome. Modern state-of-the-art techniques such as Golden-gate cloning along with CRISPR-Cas9 were employed in the method development. The golden-gate cloning approach, which involves assembly of several DNA fragments into a single construct, was adapted for application in multiple organisms. Based on the organism of your choice, you could design different constructs using this flexible DNA cloning toolbox. Recently, the bacterial and archaeal immune mechanism CRISPR-Cas9 has been engineered as a versatile DNA editing method. Any site in the genome can be targeted using the Streptococcus pyogenes Cas9 protein and a specific guide RNA. Double strand breaks created by the protein in the genome are then repaired by the host cell, which leads to mutations at this site. This gene targeting technique worked successfully in Aspergillus niger. Moreover, the excising ability of Cas9 was also used to linearize the DNA construct containing the desired set of genes. Site specific integration of this linearized DNA fragment in the genome of the organism then takes place by the homologous recombination ability of the host cell.
A.niger strains capable of secreting aconitic acid were generated by this method. Aconitic acid is used as a food additive as a flavouring agent and also as an intermediate for itaconic acid production. The method proved to be very efficient as all strains showed a correct integration event at the site. Besides, the side effects due to Cas9 mediated off-target changes are reduced by transient expression of this protein. This method enables a quicker and more efficient screening process and engineered strains can be generated in a shorter span of time compared to previously established methods. One can integrate several genes or even an entire metabolic pathway at a specific site and thus engineer strains for production of other bio-based chemicals. With the advent of sophisticated genetic engineering tools, it gets easier now to engineer fungi and harness the untapped potential of this ubiquitous organism.
This work is based on:
Sarkari P, Marx H, Blumhoff ML, Mattanovich D, Sauer M, Steiger MG: An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger. 2017, Bioresour Technol, DOI 10.1016/j.biortech.2017.05.004
Picture credits: acib