To optimize the secretory expression of recombinant human serum albumin (HSA) under the control of methanol oxidase (MOX promoter in the methylotrophic yeast Hansenula polymorpha DL-1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5 ' -untranslated region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational level. With the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus, a single copy of the expression vector integrated into the MOX locus became sufficient for the maximal expression of HSA. Northern blot analysis revealed that the levels of HSA transcript did not increase any further upon increasing copy number. The mox-disrupted (mox Delta) transformant was constructed, in which the genomic MOX gene was transplaced with the HSA expression cassette, to examine the effect of the methanol oxidase-deficient phenotype of the host on HSA expression. The mox Delta transformant showed higher levels of HSA production in shake-flask cultures than the MOX wild-type transformant, especially at low concentrations of methanol and a twofold higher specific HSA production rate in fed-batch fermentation with an abrupt induction mode. The native prepro signal sequence of HSA secreted in H. polymorpha was correctly processed and the mature recombinant protein had a pl value identical to that of the authentic HSA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large-scale production of recombinant albumin, (C) John Wiley & Sons, Inc.