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Periodic Report Summary 1 - QUANTFUNG (Quantitative Biology for Fungal Secondary Metabolite Producers)

1. Summary QuantFung

Filamentous fungi are of great economic importance as a source of high-value products in the biotechnology industry. European companies are global leaders in using fungi for bulk manufacturing. The newly emerged fungal ‘omics’ era and the advent of systems and synthetic biology provide innovative concepts and ideas for the production of novel bioactive compounds from fungi. The QuantFung ITN is a collaboration of leading European academic and industrial groups active in the field of filamentous fungal research and development and trains eleven early-stage researchers and four experienced researchers. The highly multidisciplinary training includes gaining expertise in cultivation of fungal organisms, collecting data sets of fungal genomics, transcriptomics, proteomics and metabolomics approaches and processing enormous datasets using bioinformatics and systems biology tools. The final goal is the design and engineering of fungal organisms with new characteristics using highly sophisticated synthetic biology tools to produce novel bioactive products. Overall, four work packages (WP) encompass ten different tasks: (i) discovery of secondary metabolite clusters (SM), (ii) activation of secondary metabolite clusters which are not expressed under standard laboratory conditions or which derive from metagenomics approaches and (iii) quantification of SMs with the goal to identify and produce them in industrially established hosts including Penicillium chrysogenum, Aspergillus niger and Saccharomyces cerevisiae. These three work packages merge into WP 4 which focuses on the (iv) bioactivity testing of identified SMs as putative products.
The major goal of WP1 is the identification of interesting secondary metabolites and gene clusters from established organisms (Aspergillus nidulans), industrial fungal strains (A. niger, Penicillium chrysogenum) and environmental isolates including newly isolated marine strains. Additionally, co-cultivations of A. nidulans with bacteria such as Streptomycetes shall be performed to unlock fungal secondary metabolite clusters. Fungal genomic sequences from the environment and from collections were assembled based on 454 and Illumina sequencing and biosynthetic gene clusters predicted using web-based tools such as SMURF and AntiSmash. To integrate genomic, transcriptomic, proteomic and metabolomic data, a database for SM clusters for the different filamentous fungi was established.
The main results of WP1 are:
- A gene-coexpression network was established for A. niger collecting transcriptomics data for more than 150 growth conditions. It was found that 57 out of the 78 predicted gene clusters are expressed in at least one condition
- 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.
- A comparative analysis of seven environmental Penicillia strains cultivated under controlled bioreactor conditions identified several secondary metabolites
- 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.
The major goal of WP2 is to activate secondary metabolite clusters. As SM expression varies considerably with the host, different generic hosts will be established as chassis to produce SMs. These host platforms will be gained by employing synthetic biology methodologies to produce SMs derived from natural or non-natural SM variants. As a novel platform, a SM-deficient strain of P. chrysogenum of an industrial lineage will be constructed. A second platform, A. niger, will be used which is an established production host in biotechnology and for which genetic tools are already available to express any gene of interest under controlled conditions. The third approach is based on non-filamentous single cells of perfect Aspergilli, the Hülle cells.
Putative interesting gene clusters encoding novel SMs and identified in WP1 will be introduced into these host platforms using synthetic gene circuits allowing fine-tuning of the expression levels of different genes.
The main results of WP2 are:
- A polycistronic gene expression cassette was established for A. niger
- A modular synthetic biology toolbox was established for P. chrysogenum containing a large number of promoters, terminators, regulators, selection markers and fluorescent proteins
- An inducible expression system based on the Q-system from Neurospora crassa was established for P. chrysogenum
- A CRISPR/Cas9 based genome engineering tool was established for P. chrysogenum
- A preliminary pathway for the biosynthesis of the chrysogenine was proposed
- An in vivo imaging method was established to follow Hülle cell development
The major goal of WP3 is to quantify secondary metabolites. The metabolism of the host platforms developed in WP2 will be (re)directed towards optimum secondary metabolite production. To this end, metabolic network modelling will be performed to identify the timing of secondary metabolite precursor formation and possible metabolic bottlenecks. Based on this data, optimized fermentation protocols for bench-scale fermentation (up to 5 litres) and semi-pilot scale fermentation (up to 150 litres) will be developed to yield high titres of secondary metabolites.
The main results of WP3 are:
- An optimized fermentation protocol for small-scale bioreactors was developed to overproduce the secondary metabolite YWA1 in A. nidulans
- A global pipeline was developed that will enable the reconstruction of 30 Penicillium metabolic draft networks
- An optimized bench- and semi-pilot scale fermentation protocol was achieved for the high yield production of itaconic acid by Aspergillus terreus
The major goal of WP4 is to test bioactivity of natural and non-natural secondary metabolites. For this purpose, a panel of multi-drug resistant bacteria and fungal pathogens in humans is available.
The main results of WP4 are:
- Bioactivity assays identified fractions with antibacterial and/or antifungal activity in fungal samples isolated from the environment in WP1
Core to the training programme of QuantFung is training through research by means of individual personalised multidisciplinary projects (see above). Complementary, QuantFung Training Schools delivered network training through a structured onsite programme. The training schools and workshops were strongly multidisciplinary and included the following topics:
- Summer school “Quantitative biology: Current concepts and tools for Microbial Strain and Process Development”
- Seminar on Professional Responsibilities in Collaborative Projects
- Master class “Synthetic biology of antibiotics production”
- Workshop “Bioinformatics for secondary metabolite discovery”
- Workshop “Metabolic Engineering and Systems Biology”
- Summer School “Proteomics and Mass Spectrometry”
- Workshop “Advanced techniques for the cultivation of filamentous fungi”
- Fellow retreat
The scientific vision of QuantFung is to establish new engineered production strains which can be used as chassis for the production of new secondary metabolites used as pharmaceuticals, food and feed ingredients, or even fuel feedstocks. The gene(s) or gene cluster(s) of interest can be of fungal or even (non)fungal origin and respective expression constructs will be plugged into selected genomic loci allowing highest control over gene expression. The gene(s) of interest will be expressed under the control of synthetic promoters and the product repertoire, the timing of product formation and productivity has been optimised using metabolic engineering strategies.
The immediate benefit of QuantFung is to ensure 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. Several opportunities were taken including scientific conferences and publications as well as press releases and public days to discuss the vision and progress of QuantFung with the scientific community and general public. The QuantFung website presents all fellows and their projects to the public (
The coordinated research programs proposed within the QuantFung ITN brings together groups working in different disciplines. Although some of the teams have been working together in the past, most of the collaborations in the project are new and cover a much wider range of research questions and applications in fungal biotechnology. The integration efforts intended by all the teams will result in a more focused and coherent approach to the research objectives, promoting complementary work, sharing resources, and will catalyse research activities and drive new collaborations. The network will act as a vehicle of communication and exchange between research groups, benefiting the whole research area of filamentous fungi.

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