CoReCo – New metabolic modelling tool for the production strain development

by Mikko Arvas

 
Metabolic engineering is required to make a microbe to produce a new chemical or to improve the production of an existing product. But how to select the right genes and pathways to be engineered?

 
Stoichiometric metabolic modelling encompasses numerous techniques to make these selections using state-of-the-art computational tools and databases of chemical reactions and compounds. At the heart of stoichiometric metabolic modelling are the metabolic models of organisms. In order to model the metabolism of an organism and hence to select the required genetic modifications a metabolic model for that organism is required.

 
Our task at VTT is to develop products, production strains and production processes for the biotechnology industry using microbial production systems. Our focus is on the production of bulk chemicals (for example polymer precursors or biofuels) and proteins (for example biomass degrading enzymes such as cellulases).

 
In collaboration with Aalto University and University of Helsinki, we have developed a novel tool, CoReCo (Comparative ReConstruction), to reconstruct genome wide metabolic models from genome sequence alone (Figure). Unlike previous tools it takes into account information from related species through a phylogenetic approach and verifies the correctness of reactions by atom-maps.

FigureFigure. CoReCo (Comparative ReConstruction) process. Sequence homology searches (InterProScan, Blast and GTG) are carried out for a set of genomes i.e. a genome of interest and some related genomes. A probabilistic model is built for the presence of each enzyme in the set of species and their ancestors. After that atom mapped, electron and element balanced reactions are taken from a reaction database (for example KEGG) to reconstruct a metabolic network of the reactions that the enzymes can carry out. The end product of the process is a Systems Biology Markup Language – model which contains the stoichiometric matrix required for stoichiometric modelling.

 
We have demonstrated the functionality and usability of the reconstructed models with computational steady-state biomass production experiments (Pitkänen et al. (2014) PLoS Computational Biology, 10(2), e1003465). For example, we show that functional models can be built for species that are very distant from major model organisms such as baker’s yeast and for incomplete genome sequences. After the publication we have carried out extensive development of bacterial and fungal reaction databases and also made algorithmic improvements.

 
With novel long read sequencing techniques such as PacBIO, purchasing a high quality genome for a micro-organism starts to be very cost efficient. For example, a yeast genome costs around 5 – 10 000 €. This opens up efficient genetic modifications techniques and now also, with CoReCo, metabolic modelling for any cultivable micro-organism.

 
Well-established microbial production organisms, such as baker’s yeast, are heavily patented and only represent a tiny fraction of natural variability of metabolism. Therefore, exploration of novel production organisms utilizing CoReCo represents considerable opportunities for the industrial biotechnology sector to create new production strains and IPR.

Mikko photoAsk more information about CoReCo and metabolic modelling from the author Senior Scientist Dr Mikko Arvas. He has background in genetics, but for the last ten years he has concentrated on computational genome analysis of fungi for the needs of industrial biotechnology.

Great future ahead with the help of Plant Molecular Farming!

by Anneli Ritala and Suvi T. Häkkinen

Green

Roughly a year ago, we wrote a commentary “Molecular pharming in plants and plant cell cultures – a great future ahead?” to Pharmaceutical Bioprocessing [1]. And now a huge breakthrough has been made with the help of Plant Molecular Pharming: Two Ebola patients were saved with a plant-made antibody that is still in the experimental phase.

Pharming or Farming?

The term Molecular Pharming is used to highlight the production of protein-based biopharmaceuticals, which contribute to the sustainable production of drugs that promote human and animal wellbeing. It also applies to the production of valuable secondary metabolites such as the analgesic drug morphine and the anticancer drugs paclitaxel, vincristine and vinblastine which are far too complex molecules to be synthetized in an economically feasible way.

In broader perspective, term Molecular Farming can be used in the context of utilization of the versatility of plants and plant cells to produce diverse valuable proteins and other compounds for any applications. Thus Molecular Farming covers also other fields than pharmaceuticals and describes better the approach taken at VTT Ltd.

Biopharmaceuticals

Biopharmaceuticals are on the commercial forefront of the pharmaceutical sector and roughly 30% of the new drugs under development belong to this class. The biopharmaceutical market has been steadily rising and reached total cumulative sales of US$ 140 billion in 2013 [2].

The FDA (The US Food and Drug Administration) and EMA (The European Medicines Agency) are already familiar with the two major biopharmaceutical production systems:

  • Microbes – mainly Escherichia coli and different yeast hosts
  • Mammalian cells such as the Chinese hamster ovary (CHO) platform,

and standard protocols can be followed to ensure the approval of new products.

Currently, the equivalent protocols are emerging for plant-based production systems, and there is one plant-derived biopharmaceutical protein on the market: Elelyso™ (taliglucerase alfa). It is produced in carrot cells by the Israeli company Protalix Biotherapeutics and licensed to Pfizer Inc., and is used for the treatment of the life-threatening lysosomal storage disorder, Gaucher’s disease. The recombinant product gained FDA approval in 2012 and the product is currently for sale in USA and Israel.

Advantages of plant-based production systems

Picture2The plant-based systems are starting to compete with the above mentioned established biopharmaceutical production systems, and on a technological basis plant-based systems have the advantage in following areas:

 

  • Speed
  • Scalability
  • Improved product quality

In need-for-speed situations, like in case of epidemic diseases as Ebola and bioterrorist threats, the transient plant expression systems benefit from the rapid onset of recombinant protein production. The plant material is propagated before the introduction of foreign DNA, allowing plants to be grown in the open or in greenhouse conditions after which the plant material is moved into contained, GMP-compliant facilities for protein production.

The greatest advantage of intact plants that are stably transformed to produce a target protein is their unparalleled scalability. For biopharmaceutical products, manufacturing will probably be restricted to greenhouses and other closed environments to ensure product safety and batch-to-batch consistency when production is carried out under controlled conditions. The Canadian company SemBioSys developed a safflower-based production system for insulin before filing for bankruptcy in 2012. The SemBioSys platform was so outstandingly efficient that theoretically 16 mid-sized Canadian farms could have produced enough insulin to meet the entire exponentially growing global demand. At VTT we have taken an initiative in producing food allergen specific antibodies for diagnostic and safety verification purposes. The barley-produced antibody can recognize and precipitate beta-lactoglobulin, which is the major allergen in cow´s milk. The established platform has potential in development of hypoallergenic products for milk allergic patients [3].

The high product quality is gained with the use of plant cell suspension cultures for Molecular Pharming as well as Farming purposes. At VTT we have harnessed the traditional microbial bioreactors to cultivate plant cells at the 600-litre scale [3]. We are currently working on a pharmaceutical target, Transferrin, in a project getting financial support from the Academy of Finland. We also only recently got funding from ERA-Anihwa, and we are entering with our plant cell culture expression system on fish vaccine production which is a very relevant target for Plant Molecular Pharming . The annual loss in aquaculture caused by viral diseases is remarkable and in order to be able to keep the fast-growing aquaculture industry ecologically, environmentally and ethically sustainable, good health for farmed aquaculture organisms is essential.

Plant Molecular Farming provides a safe and sustainable platform for the production of valuable proteins and other compounds – the great future is here and we are happy to be part of it!

References

  1. Ritala A, Häkkinen ST, Schillberg S. 2014. Molecular pharming in plants and plant cell cultures: a great future ahead? Pharm. Bioprocess. 2:223-226.
  2. Walsh G. 2014. Biopharmaceutical benchmarks 2014. Nat. Biotechnol. 32:992-1000.
  3. Ritala A, Leelavathi S, Oksman-Caldentey KM, Reddy VS, Laukkanen ML. 2014. Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, ß-lactoglobulin. Transgen. Res. 23:477-487.
  4. Reuter LJ, Bailey MJ, Joensuu JJ, Ritala A. 2014. Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells. Plant Biotech. J. 12:402-410.

AnneliThe author Dr. Anneli Ritala, Principal Scientist (PhD Pharm., Docent in Pharmaceutical Biology) has special expertise in production of recombinant proteins and small molecules in plant cell cultures. She has over 20 years´ experience on plant biotechnology, especially genetic and metabolic engineering of plants and plant cell cultures including barley, oats, tobacco and other medicinal plants. anneli.ritala@vtt.fi

SuviThe author Dr. Suvi T. Häkkinen, Senior Scientist (D.Sc.(Tech)) has special expertise in medicinal plants and natural compound research. She obtained her doctoral degree for her work related to alkaloid biosynthesis and she has over 15 years´ experience on plant biotechnology including metabolic engineering, recombinant protein production and plant cell culture technology. suvi.hakkinen@vtt.fi