AshByofactory

Project Title
Ashbya gossypii: a systems metabolic engineered cell factory
Project Type
Nacional / Public
Funding Body
Funding Program
PTDC
Reference
EBB-EBI/101985/2008
Funding
  • CEB: 164 000,00
  • Total: 164 000,00
Start
01-04-2010
End
31-03-2013
Partnership
Universidade do Minho
External link

Principal Investigator

Team Members - CEB

Abstract

Although Ashbya gossypii has been known in the scientific community for 80 years (mainly as a plant pathogen) and in the industrial community for near 20 years (as a riboflavin producer), interest in this microorganism increased as a result of the publication of its genome sequence in 2004. This fungus is a very promising experimental system because it has a small genome (the smallest eukaryotic genome known to date) and haploid nuclei. Moreover, efficient gene targeting, propagation of plasmids and growth on defined liquid and solid media are possible. A. gossypii also grows well in waste vegetable oils and thus a cheap, environmental-friendly culture medium can be designed for growth and metabolite production. Ashbya is a GRAS organism and is used industrially for riboflavin production by e.g. BASF, Germany, and thus large-scale industrial fermentations are well developed for this host. Taking advantage of these unique features, the main purpose of this project is the development of A. gossypii as a tailor-made cell factory organism. In the first stage of this project, using information obtained from genome sequencing and annotation, the genome-scale metabolic model (GSMM) of this organism will be constructed. Results obtained using Metabolic Flux Analysis for growth on defined carbon sources will be used to validate and complement the metabolic model, based on pseudo-steady state assumptions. The stoichiometric model obtained will then be used to simulate in silico the phenotypic behaviour of A. gossypii under different environmental and genetic conditions, thus creating an important tool for metabolic engineering design. In a second stage, and using the information provided by the in silico simulations, optimization of riboflavin and recombinant proteins production will be envisaged, or even the identification of other potentially interesting chemicals for which high yields are predicted in silico. Several systems-wide analyses will be performed in the rationally constructed strains, namely analysis of the transcriptome, proteome and fluxome, with the corresponding results being used, in conjunction with the metabolic model, to consider improvements in the production of riboflavin and/or recombinant proteins. This project proposes to focus on the whole metabolism of Ashbya and follow a truly systems biology approach in order to be able to take advantage of all potentialities of this organism and develop an environmental-friendly cell factory organism. In this proposal, to develop a tailor-made cell factory system based on the fungus A. gossypii, three main objectives are pursued: (1) to develop a GSMM that can be used to predict the behaviour of the organism when subject to environmental and genetic modifications and, consequently, help the metabolic engineering target identification and systems-wide data analysis. (2) to rationally engineer A. gossypii’s recombinant strains, in order to obtain improved riboflavin and/or recombinant protein producing strains.\n(3) to characterize the rationally engineered strains and use that information to validate the model and improve the metabolic engineering strategies. The team experience in both yeast and filamentous fungal metabolism and recombinant protein production is crucial to the success of the project, since A. gossypii is very close to Saccharomyces cerevisiae but is nevertheless, a filamentous fungus. For this project proposal, collaboration with VTT Technical Research Centre of Finland with Prof. Merja Penttilä’s group has been settled which will strengthen the knowledge of systems biology at CEB. The access to the systems biology platform implemented at VTT, together with the exchange of results and methodologies, will facilitate rapid results. BASF (industrial riboflavin producer with A. gossypii) Germany, is willing to collaborate with this project, sharing tools and methodologies. This collaboration will help to direct the research towards industrial needs and can be strengthened as the project progresses. It is worth noting that two PhD grants have been awarded by FCT to Orquídea Ribeiro (SFRH/BD/30229/2006) and Tatiana Aguiar (SFRH/BD/39112/2007), on this topic. This project will finance that work and reinforce this research area.