Pichia pastoris has been used to produce various recombinant proteins under high oxygen demand conditions. To improve the heterologous production of beta-galactosidase, the vgb gene encoding Vitreoscilla hemoglobin (VHb) was co-expressed in the P. pastoris cytoplasm under the control of the methanol-inducible promoter. Co-expression of VHb under different aeration conditions improved cell performance in terms of growth, viability, respiratory rate, and p-galactosidase production. Under limiting aeration conditions, the VHb(+) strain produced 28.2% more biomass but 31.2% less total beta-galactosidase activity than the VHb(-) strain. Under non-limiting aeration conditions, the VHb(+) strain showed 20.3% higher cell growth and 9.9% more total beta-galactosidase activity than the VHb(-) strain. Moreover, under these conditions, the VHb(+) strain was 7.7% more viable and had a 28.2% higher oxygen uptake rate (OUR) than the VHb(-) strain. Evidently, VHb can enhance the OUR and promote methanol metabolism, thereby improving cell performance and beta-galactosidase production. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.