FunBiotics: Efficient production of antibiotics from fungi

FunBiotics has been a Xellia project performed in cooperation with SINTEF Materials and Chemistry with the aim to develop new technology and processes for production of Echinocandin antibiotics to be used for treatment of human fungal infections. The main focus has been to develop high yielding production strains suitable for large scale industrial production. Mutant strains with improved Echinocandin type 1 production and strongly (100 fold) reduced co-production of an unwanted toxic compound, has been developed. Despite these achievements, it was not possible to make a strain completely free from toxin formation. Such a strain would have been a great advantage during the development and up-scaling of a fermentation process to large scale manufacturing. A lot of work was done with four different mutant strains originally characterized not to produce toxin. DNA sequencing results showed that these strains have frameshift, deletion and point mutations within the genes needed for toxin production. Despite of this, the strains were still able to produce low levels of toxin. To make sure that this was not linked to mix of genomes, a flow-cytometric method for sorting of single fungal spores was developed. Furthermore, a lot of effort was put on developing better high throughput screening methods in micro-titer plates, a challenging task due to the extreme viscosity usually obtained in cultures of these types of fungi. The developed methods were applied throughout the project to screen for new mutants with reduced toxin and increased Echinocandin production. At the end of the project a library of mutants with improved production capacity was generated, but a verification regarding level of toxin production will be needed for any future use of the strains. Another activity in the project was to apply the new genetic technology, CrispR/Cas9 as an alternative way to remove the toxin producing abilities. This technology has not to our knowledge been tried on these type of fungal strains. Promising results with regards to transformation of production strain were obtained. Three different primer pairs, for potential introduction of frameshifts in the toxin genes, were made and introduced in to a selected vector. However, it turned out to be a challenge to find true transformants due to ineffective selection markers discriminating transformed cells from background cells. As a consequence the potential of CrispR/Cas9 in this setting was not clarified before the project was terminated. Two years after start, the project switched from Echinocandin type 1 to Echinocandin type 2 due to a change in market potential for Echinocandin 1. Based on knowledge from the first part of the project methods for spore-isolation and mutagenesis, as well as UPLC and rapid LC-MS analyses, were rapidly established for an Echinocandin 2 production strain. Additionally, fermentation and purification studies were initiated for this antibiotic and some milligrams of pure product of Echinocandin type 2 was made, and later deacylated. A deacylation of the compound is required before the production of the final semi-synthetic product. The deacylation enzyme was produced by a separate fermentation process developed in the project. Summer 2017, the leadership team in Xellia decided to reallocate resources from Echinocandins to other antibiotics. As a consequence, and in alignment with RCA and SINTEF, the project was terminated end of September. It is very likely that these products will be produced by Xellia in a near future where the results from this project will be of high value. All results and know-how have been compiled in several reports and all strains have been conserved for future use.

FunBiotics is a collaboration between Xellia Pharmaceuticals and SINTEF Materials and Chemistry. Its primary objective is to establish new methods and technology for faster development of fungal production processes with major focus on developing the production strains. The new methods will be used in development of strains suitable for industrial production of 1-2 selected antifungal echinocandins. The market for antifungal drugs is increasing. The echinocandins are the latest class of agents introduced to this market. The echinocanidins are produced by fungal species. Development of fungal production strains and fermentation processes for antibiotics are highly challenging. The main research challenges are coupled to the filamentous growth form of the fungal strains, complex regulation of the antibiotic biosynthesis and co-production of unwanted by-products. The basis for the project is the state of the art infrastructure and competence at Xellia and SINTEF. A broad range of novel methods will be developed using top level infrastructure to select, screen, characterize and cultivate new fungal production strains, from gene sequencing of mutants, classical mutagenesis, miniaturized cultivation with robotic material handling, ultra high throughput MS analyses for high capacity screening, and strain evaluation in parallel bioreactor systems. Evolution of the natural fungal strain will be done by chemical mutagenesis and high capacity screening and also by developing engineered strains for heterologous expression of echinocandin gene clusters (synthetic biology). The project will strengthen the position of Xellia's R&D situated in Norway. The socio-economic benefits of a successful project are substantial. New and Improved processes will reduce product costs and make life saving medicines more available to the worlds population. The project will contribute to further development generic important national competence related to pharmaceutical process development.