The development of a solid-phase fermentation system for the production of pharmaceutically useful secondary metabolites in fungi

摘要

A Phoma species, producing a squalestatin (Si) was grown on agar media derivedfrom wheat, oats, oil seed rape and malt extract over a range of water availabilityvalues corresponding to water activity (aa) levels of. 0.998,0.995,0.990,0.980 and0.960. Growth of the organism was not significantly affected by aw, except at thelowest value, but production of SI was significantly enhanced at intermediate aW inthe range 0.990-0.980. For example, at 25°C and 0.98 aw wheat extract produced al Ox increase in titre compared to 0.998 aw at the same temperature. Wheat extract wasshown to be the best substrate for SI production. For example, at 25°C and 0.98 aW,this substrate produced 2x, 5x and 8x increases in titres compared to oat, malt and oilseed rape extracts respectively.A range of raw and processed agricultural products, including those used as extractswith the Phoma species, as well as maize, rice, soya, wheat flakes, bulgar wheat,couscous and "shredded wheat", were selected as candidate materials for solidsubstrate fermentation (SSF). Moisture sorption isotherms were prepared for each ofthese so that aW could be accurately set in experimental work. Small scalefermentations (40 cm3 wet substrate volume) were carried out with these materials andthe fungi Epicoccum nigrum, Sarophorum palmicola, Drechslera dematioidea andCorynespora cassiicola over the aw range 0.998-0.970. Studies with E. nigrum inparticular produced a range of unique metabolites at low aw, and other metaboliteswhere titres were increased by as much as 20x compared to high aW conditions. Theoptimum aw level for metabolite production in this fungus appeared to be in the range0.990-0.980. Ultimately, E. nigrum was chosen as the model fungus and bulgar wheatas the model substrate, with 3 key target metabolites being followed (metabolites 1,2and 3).A series of scale-up studies (40 cm3-3 litres wet volume) were carried out utilising themodel system. These studies typically produced reasonable levels of metabolites, butwere subject to problematic water and heat accumulation, and bacterial contamination.These were identified as critical parameters. A system was ultimately developedaround a Bioengineering AG submerged liquid fermenter, modified for use with solid substrates, and incorporating forced aeration and mechanical agitation. This apparatusgave encouraging levels of metabolites, producing most of these rapidly anduniformly, and showed good critical parameter control.The overall scale-up achieved in the final fermenter studies was 75x, in terms of wetsubstrate volume. Increased titres were achieved for all three target metabolitescompared to small-scale studies with the same substrate. These increases wereapprox. 17x for metabolite 1, approx. 3x for metabolite 3, while metabolite 2 wasabsent from small scale studies at the relevant aW level.