An explosive growth in the requirement for recombinant protein drugs has been apparent since the turn of the millennium, and there has been much analysis and speculation on how drug development, production and marketing companies will address the predicted shortfall in manufacturing capacity at every stage of the product life cycle. Alongside the installation of additional capacity and process improvement in conventional fermenter-based technologies, transgenic whole organisms offer a more radical option to increase supply of heterologous proteins. The attraction lies in the potential to apply tried and tested agricultural husbandry and crop production techniques to new transgenic crops and breeds, and to operate at a scale unthinkable to fermenter technologies constrained by the capital costs of stainless steel, On the downside is the challenge of the molecular biology, fiendishly difficult in mammals and birds, and the whole genetically modified organisms debate on the protection of the food chain and the environment,
Mushrooms are, uniquely, a high-value horticultural crop grown around the world in purpose-built structures, independent of climate and season. The technologies for growing mushrooms on a commercial scale for culinary use have been developed and refined over the past 40 years by researchers at Warwick Horticulture Research International (HRI), part of the University of Warwick, UK, and by other groups in the US and the Netherlands. In best practice, the mushroom houses in which the crop is grown are closely controlled for environmental variables and the exclusion of microscopic vectors of disease–ideal, in fact, for the production of specialist crops requiring containment.
The mushroom crop has three important features which have particular advantages for industrial production and molecular pharming:
- The energy for mushroom growth is provided by the growing medium so light is not required. Consequently, production can continue all year round without expensive glasshouse structures or supplementary lighting,
- Mushrooms are propagated vegetatively. Each new crop is initiated by ‘spawn’, fragments of mycelium which grow to colonise the growing medium and generate the fruiting body, the mushroom, Production can be scaled up rapidly from master cultures without the delays of sexual reproduction, for example in bulking up seed supplies of plants, and every crop is genetically identical.
- Production is modular, Mushroom farms consist of a series of identical production units. If additional production is required the number of mushroom houses is increased but the process remains the same, This compares favourably with the process development and regulatory approvals required for scale-up in fermenter-based systems.
In short, mushrooms offer a model crop for molecular pharming, with contained continuous production, rapid scale-up and production facilities which can be constructed or converted at moderate cost. On top of this, mushrooms are highly productive and can be grown intensively on racking systems many layers high, Typical yields are more than 3200t/year of biomass for each hectare of growing facility, compared with 100t/hectare per crop quoted for tobacco, (1) for which one or two crops each year are possible in favoured production areas.
Although pharmaceutical proteins were first produced in plants in the 1980s, molecular pharming in mushrooms has been developed only recently, Although mushroom growing is global in scope and the horticultural crop is valued at 3bn [pounds sterling]/year ($5.3bn), (2) the industry is small compared with livestock and field crops, Accordingly, much more research, both publicly funded and commercial, has been devoted to molecular pharming in plants and animals than in mushrooms, In addition, the molecular biology of mushrooms is complex and specialised with few centres of excellence, However, since the late 1990s the range of transformation technologies available to introduce heterologous DNA into mushrooms has increased and this is now routine and efficient. A range of candidate proteins, including human growth hormone and other pharmaceuticals, an industrial enzyme and an insecticidal toxin, have successfully been incorporated into mushrooms and expression in the fruiting body has been demonstrated. (3,4)
The next stage will be to refine and develop the molecular tools which control expression to increase yields and restrict expression to specific tissues and developmental stages. Kerry Burton, Mike Challen and their coworkers at Warwick HRI have already identified promoters that switch the heterologous genes on as the fruiting body starts to develop, and are developing an expression cassette, the envelope which contains the gene and controls its expression, which will maximise the yield and concentrate the protein in the harvestable part of the crop.
It is still too early to be definitive about the commercial potential of this technology: relatively few candidates have been addressed and yields, while encouraging, are capable of significant improvement. Customers are particularly concerned about whether the protein product would function correctly. Glycosylation, the pattern of oligosaccharides attached to the protein as a result of post-translational modification, has often been seen as a key point of difference between native human proteins and proteins manufactured in bacteria, plants and other systems, potentially affecting efficacy and immunogenicity. However, this does not appear to present an insurmountable problem: glycosylation in higher fungi is closer to the mammalian model than in plants, (5) and there is also the option to develop non-glycosylated products where these are biologically active. (6)
While mushrooms offer the potential for cost savings and increased flexibility in the manufacture of conventionally formulated drugs, they have a further advantage worth consideration for new product formulations: they are safe for human consumption and have GRAS (generally recognised as safe) status, In conventional fermenter-based technologies, the investment required for the purification facility is about half the total capital cost, and the costs associated with purification are about half the total cost of goods. (7) Mushrooms could provide the basis for edible drug formulations that do not require extraction and purification of the active ingredient, and which would drive down costs still further. This would not only be of interest to drug companies in the developed world but might provide a route to affordable medical treatments in less developed countries,
There has been keen interest from the pharmaceuticals industry and agribusiness in this highly attractive yet relatively unfamiliar production system, and both commercial and technical aspects are being evaluated. Biomanufacturing in mushrooms is fast becoming a feasible proposition as molecular biology is unravelled and supporting resources, such as gene libraries, are put in place, The crippling disadvantages of other systems–enormous capital cost, safety concerns, containment–are now giving impetus to the development of mushrooms as a new challenger for biomanufacturing.
(1) J Ma, P Drake & P Christou, Nature Reviews of Genetics 2003, 4, 794
(2) T Elliott, K Burton & M Challen, ‘Farming fungi: the potential of edible mushrooms’, in ‘Proceedings of the OECD workshop’, J Toutant & E Balazs (eds), La Grande Motte, France, 3-6 September 2000
(3) A Velcko Jr, R Kerrigan, L MacDonald, M Wach, C Schlagnhaufer & C Romaine, in ‘Science and cultivation of edible and medicinal fungi’, Romaine, Keil, Pinker & Royse (eds) University Park Pennsylvania, Penn State Press 2004, 591-597
(4) C Zhang, V Odon, H Kim, M Challen, K Burton, D Hartley & T Elliott, in ‘Science and cultivation of edible and medicinal fungi’, (as above), 611-617
(5) Q Zeng, A Morales & G Cottarel, Trends in Genetics 200I, 17, 682-684
(6) M Ward, Genetic Engineering News 2002.22 (21), 48
(7) U Steiner, ‘The business case for plant-factories’, paper presented at the conference on Plant-made pharmaceuticals, Quebec, Canada, 16-19 March 2003
David Hartley is chief executive of Agarico, Horticulture Research International, Warwick, UK.