Biotechnology Journal DOI 10.1002/biot.200600036 Biotechnol. J. 2006, 1, 822–827 822 © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 Introduction Oxygen is essential for maximal energy yield and optimal utilization of substrate in every aerobic organism [1]. Dur- ing growth of Aspergillus oryzae on solid substrates, the aerial hyphae account for 70% of the oxygen uptake [2]. It is shown that diffusion of oxygen is limited in the fila- mentous fungal layer that covers the solid substrate and that the substrate penetrative hyphae are limited in oxy- gen consumption and growth [2, 3]. Therefore, oxygen supply to filamentous fungal cells that are in close contact with the substrate is considered as a bottleneck in solid- state fermentation [2, 3]. Hemoglobins bind oxygen reversibly and have been discovered in a wide range of organisms including verte- brates, invertebrates, higher plants, fungi and bacteria [4]. Despite the fact that all known hemoglobins have a highly variable primary amino acid sequence, they all show a six to eight α-helical arrangement that facilitates binding of heme in the hydrophobic core of the protein [1]. Hemoglobin bridges a wide variation in oxygen tensions at the sites of oxygen loading and unloading, and plays therefore a major role in oxygen transport, although spe- cific hemoglobins may be specialized for other particular functions [4]. Several hemoglobin domain (HBD) genes have been recently identified in various fungi, including Aspergillus species (Te Biesebeke et al., An Aspergillus oryzae gene expressed during hyphal growth is part of an unusual family of fungal proteins, submitted). These HBD Research Article Expression of Aspergillus hemoglobin domain activities in Aspergillus oryzae grown on solid substrates improves growth rate and enzyme production Rob te Biesebeke 1,2 , Amandine Boussier 1,2 , Nick van Biezen 2 , Machtelt Braaksma 2 , Cees A. M. J. J. van den Hondel 3 , Willem M. de Vos 1,4 and Peter J. Punt 1,2 1 Wageningen Center for Food Sciences (WCFS), Wageningen, The Netherlands 2 TNO Quality of Life, Zeist, The Netherlands 3 Clusius Laboratory, Institute of Biology, Leiden University, Leiden, The Netherlands 4 Wageningen University and Research Center, Microbiology, Hesselink van Suchtelenweg, Wageningen, The Netherlands DNA fragments coding for hemoglobin domains (HBD) were isolated from Aspergillus oryzae and Aspergillus niger. The HBD activities were expressed in A. oryzae by introduction of HBD gene frag- ments under the control of the promoter of the constitutively expressed gpdA gene. In the trans- formants, oxygen uptake was significantly higher, and during growth on solid substrates the de- veloped biomass was at least 1.3 times higher than that of the untransformed wild-type strain. Growth rate of the HBD-activity-producing strains was also significantly higher compared to the wild type. During growth on solid cereal substrates, the amylase and protease activities in the ex- tracts of the HBD-activity-producing strains were 30–150% higher and glucoamylase activities were at least 9 times higher compared to the wild-type strain. These results suggest that the As- pergillus HBD-encoding gene can be used in a self-cloning strategy to improve biomass yield and protein production of Aspergillus species. Keywords: Aspergillus · Hemoglobin · Growth rate · Protein production · Solid-state fermentation Correspondence: Dr.Rob te Biesebeke, Friesland Foods, P.O. Box 226, 8901 MA Leeuwarden, The Netherlands E-mail: rob.tebiesebeke@frieslandfoods.com Fax: +31-582992286 Abbreviations: CM, complete medium; DO, dissolved oxygen; HBD, hemo- globin domains; PDA, Potato dextrose agar; WK, wheat kernels; WSM, wheat-based solid medium Received 15 March 2006 Revised 23 May 2006 Accepted 25 May 2006