Optimisation of culture conditions with respect to biotin requirement for the production of recombinant avidin in Pichia pastoris

Due to its very high affinity to biotin, avidin is one of the most widely exploited proteins in modern biotechnological and biomedical applications. Since biotin is an essential vitamin for the growth of many microorganisms, we examined the effect of biotin deficiency on growth for a recombinant Pichia pastoris strain expressing and secreting a recombinant glycosylated avidin. The results showed that biotin deficiency lowers growth rate and biomass yield for P. pastoris. Substitution of biotin in the medium by the two structurally unrelated compounds, aspartic acid and oleic acid, which do not bind to recombinant avidin was analyzed quantitatively.These two compounds had a growth promoting effect in biotin-deficient medium, but did not replace biotin completely. Indeed, in chemostat culture, wash-out occured after about six liquid residence times and recombinant avidin productivity was lowered. However, addition of low amounts of biotin (20 μg L−1 of biotin for a cell density of 8 g L−1) resulted in stable chemostat cultures on methanol with the production of recombinant biotin-free avidin. The specific avidin production rate was 22 μg g−1 h−1 at a dilution rate of 0.06 h−1.