Final Report Summary - QUANTFUNG (Quantitative Biology for Fungal Secondary Metabolite Producers) Filamentous fungi are fascinating microorganisms. One of the reasons why it is so worthwhile to take a closer look at them is their capacity to produce secondary metabolites. Some of these substances have the potential to be of great use for mankind, such as it was the case with penicillin and its discovery in 1928. Almost a century later, the situation in healthcare could possibly turn back to the state before the development of the first antibiotics. Due to an overuse of antibiotics we are facing a surge of multiresistant bacteria that are not inhibited by any of the currently known drugs. That was part of the background why a European research project was launched in October 2013, titled “Quantitative Biology for Fungal Secondary Metabolite Producers”, or “QuantFung”. Fifteen young scientists embarked on a new phase in their career, moving to new work environments within Europe and dedicating their work lives intensively to the quest for useful secondary metabolites. After four years, the QuantFung project concluded in October 2017. The focus of the “QuantFung” project was to identify and isolate novel bioactive molecules from filamentous fungi. One of the triggers for launching this 4-year project was the crisis of antibiotic resistance. However, the quest for novel bioactive fungal products goes far beyond the problem of the antibiotics crisis, as it includes the need for novel drugs for multiple human health problems ranging from different cancers to increasing neurodegenerative diseases, which are especially prominent in aging societies.The driving force of QuantFung was the collaboration of 8 academic and 5 industrial partners to expedite the application of new secondary metabolites in areas such as health care, nutrition or agriculture. The main objectives of the consortium were to find novel bioactive molecules by exploiting the wealth of fungal biodiversity and to translate these into useful products. This required the (re-)design and engineering of fungal organisms with new characteristics, using highly sophisticated synthetic biology tools. The educational idea of QuantFung was to use this research context to train 11 PhD students and 4 Post-Docs as new problem-solving, creative European scientists in interdisciplinary and intersectorial biotechnological research. The fields varied from modelling and network analysis to systems biology (e.g. genomics, transcriptomics, proteomics and metabolomics), from molecular biology, like fungal genetics and biochemistry, to synthetic biology methods. Different work packages were designed which focused on discovery of secondary metabolite gene clusters, targeted activation of gene clusters, quantification of secondary metabolites in industrial hosts, and bioactivity testing to identify their mode of action. In order to bridge the gap between academia and industry, the industrial partners offered secondments and training modules to share their experience with the trainees. As these multidisciplinary projects required physical and intellectual flexibility, the training program for the young fellows included defined work periods in different QuantFung laboratories, as well as local and networked training events for researchers. The vision of the QuantFung partners was that such an approach would promote the development of a new generation of fungal biotechnologist with experience in both the academic and the industrial work culture, comprising significant translational and entrepreneurial skills. What has been achieved? The overall impact will be most likely revealed in one or two years’ time from now as much of QuantFung’s output cannot yet be taken into account (many manuscripts are still in preparation, have just been submitted, are currently under review). However, the fellows’ accomplishments are already very impressive at this early stage: On average, each fellow presented his/her work at four (inter)national conferences and has been co-author of 1.9 publications. Three of these publications have receive particular attention by the research community: (i) the establishment of a CRISPR/Cas9 based genome editing tool for Penicillium chrysogenum [Pohl et al., 2016, ACS Synth Biol 5:754-64], (ii) the sequencing of nine different Penicillium genomes and the identification of 1,317 putative secondary metabolite gene clusters hidden there [Nielsen et al., 2017, Nat Microbiol 2:17044], (iii) the establishment of polycistronic gene expression in the cell factory Aspergillus niger as a tool for high level production of secondary metabolites [Schütze and Meyer 2017, Microb Cell Fact 16:162]. As a team, the QuantFung participants jointly organised one Mini-symposium in 2014 during the annual meeting of the German Society of General and Applied Microbiology (VAAM) and a session during the highly prestigious Conference on the Physiology of Yeast and Filamentous Fungi 2016 in Lisbon (PYFF6). The fellows used the opportunity to publicize QuantFung to the broader audience of biotechnologists and the interested public, and collectively co-wrote a commentary piece in 2015 [Büttel et al., 2015, Fungal Biol Biotechnol 2:6], which resulted in over 2,590 downloads during the first two years after its publication. Further results which have been achieved in the QuantFung consortium are (amongst others):- A gene-coexpression network was established for A. niger collecting transcriptomics data for more than 150 growth conditions. This allowed the in silico prediction of global transcription factors which regulate a large set of SM core genes. Their functions were verified in vivo using deletion and targeted overexpression approaches.- A comparative proteomic approach uncovered a monodictyphenone cluster producing xanthones to be specifically active in Hülle cells of A. nidulans.- A comparative transcriptomics analysis in two industrial P. chrysogenum strains uncovered four secondary metabolite genes to be highly expressed under penicillin producing conditions. These genes were selected for targeted deletion in order to improve penicillin production.- Comparative genomics, transcriptomics and metabolome analysis of industrial P. chrysogenum strains uncovered point mutations in developmental genes as cause for low/high penicillin production. This was verified by targeted CRISPR/Cas9 gene editing in wild type isolates.- The genomes of ten environmental Penicillia strains have been sequenced and a comparative analysis of these strains cultivated under different controlled bioreactor conditions identified novel secondary metabolites. A anticancer polyketide was isolated from P. decumbens cultures and the respective gene cluster identified using a comparative genomic analysis.- 38 fungi were isolated from soil and marine environments and the genomes from five selected fungi sequenced. Their genomes are predicted to harbor 138 secondary metabolite gene clusters, among which compounds with antibacterial and antifungal activities were isolated.- P. chrysogenum strains were deleted in several (up to 3) SM gene clusters using CRISPR/Cas9 technology to study the impact of these genome modifications on penicillin production.- A SM gene cluster from Talaromyces marneffei was successfully integrated in the genome of P. chrysogenum and expressed using novel synthetic transcriptional control systems established in the consortium.- An optimized fermentation protocol for small-scale bioreactors was developed to overproduce the secondary metabolite YWA1 in A. nidulans.- 24 metabolic networks for Penicillium species were established and improved resulting in models which are functional. - An optimized fermentation protocol was developed for heterologous overproduction of enniatin in Aspergillus niger.- Bioactivity assays identified fractions with antibacterial and/or antifungal activity in fungal samples isolated from the environment.The QuantFung network provided an ideal framework for international exchange and for building up a vital and innovative research environment where knowledge, state-of-the-art technologies and the most modern equipment were shared. All of which is what is needed to train our next generation of high-calibre scientists, to foster their creativity and to jointly come to new scientific breakthroughs. How beneficial it is to have such a network was strikingly proven by the success that QuantFung had on many levels. There are the measurable results and there is the fact that 15 young researchers are now exquisitely prepared for a future career in biotechnology. The fellows are well qualified with a broad portfolio of skills and practical experience of cross sector working; this will make them strong candidates for future employers and underpin their career progression. Importantly, taking responsibility for their own development has instilled in them at an early stage the importance of continuous professional development. Such high quality and highly competitive candidates are needed in both the public and private sectors.The QuantFung website presents all fellows and their projects to the public (http://intern.mikrobiologie.tu-berlin.de/).
