ABSTRACT
Isocitrate lyase (ICL) was assayed during batch cultivations of Ashbya gossypii on soybean oil or glucose as carbon source. On soybean oil, a correlation between enzyme activity and riboflavin synthesis was observed. On glucose as carbon source, riboflavin overproduction started in the late growth phase when glucose was exhausted. ICL activity appeared in parallel and reached a maximum of 0.41 U (mg protein)-1. This suggested synthesis of vitamin B2 from the intracellular reserve fat. ICL specific activity correlated with the enzyme concentration detected by specific antibodies. Itaconate, an efficient inhibitor of ICL, was used as an antimetabolite to screen mutants with enhanced ICL activity. Cultivations of an itaconate-resistant mutant on soybean oil revealed a 15% increase in enzyme specific activity and a 25-fold increase in riboflavin yield compared to the wild-type. On the other hand, growth experiments on glucose resulted in an eightfold increase in riboflavin yield but showed a 33% reduction in ICL specific activity compared to the wild-type grown on the same medium. These results support the idea of an ICL bottleneck in the riboflavin overproducer A. gossypii when plant oil is used as the substrate.
