INCREASE IN COPY NUMBER OF AN INTEGRATED VECTOR DURING CONTINUOUS-CULTURE OF HANSENULA-POLYMORPHA EXPRESSING FUNCTIONAL HUMAN HEMOGLOBIN

Recombinant human haemoglobin A (rHbA) was produced by a leucine-requi ring strain of Hansenula polymorpha which had been transformed with an integration vector containing the Saccharomyces cerevisiae LEU2 gene and cDNAs for the expression of a and beta globin each driven by the H . polymorpha MOX promoter. After 40 generations in a chemostat it was found that the integrated vector had become amplified in the host stra in. In some cases this led to an increase in LEU2 gene dosage, but a l oss of globin expression cassettes. In other cases the globin gene dos age also increased. These changes coincided with an increase in rHbA p roduction in the culture, which was reversed when the dilution rate wa s increased. Isolates from a chemostat culture producing elevated leve ls of rHbA were grown in fed-batch fermentations, resulting in higher productivities than when inoculated with the parent strain. The rHbA p roduced was purified and characterized. Oxygen binding studies and ele ctrospray mass spectrometry showed that the rHbA had been processed an d assembled correctly, and behaved as a fully functional co-operative tetramer.