Aspergillus oryzae has drawn much attention as one of the most excellent hosts for protein production due to its ability to secrete vast amounts of proteins and the safety guaranteed by the use in fermentative food industry. While the production level of homologous (fungal) proteins by A. oryzae can reach gram-per-liter scale, the production of heterologous (mammalian and plant) proteins yields only milligram-per-liter scale. Although hyper-producing strains are required for higher-level production of heterologous proteins, only a few attempts have been reported for breeding of an excellent production host in A. oryzae. We previously reported that the double disruption of the proteinase genes (tppA and pepE) increased the production of human lysozyme (HLY) by 1.63-fold (15.6 mg/l to 25.4 mg/l).1) Moreover, by using the double proteinase disruptant, the production yield of bovine chymosin (CHY) was 65.1 mg/l, which was 1.93-fold increment from the control strain (33.8 mg/l). High productivity of the two heterologous proteins by the double disruptant raised the possibility that the strain showing a higher-level production of HLY might also produce other heterologous proteins in larger amounts. In order to test this hypothesis, hyper-producing mutants of HLY were screened from the double disruptant. For this purpose we developed an efficient screening system by employing halo assay with bacterial cells, the HLY substrate. Among 22 hyper-producing mutants obtained from ~80,000 colonies, the highest production level of HLY was 50.8 mg/l, which was 2.00-fold increment compared to that of parental strain (the double disruptant). Subsequently, the niaD-based plasmid for HLY production was cured from the mutants by positive selection using chlorate, which is metabolized into a cytotoxic compound by the nitrate assimilation pathway. The 8 strains obtained by the curing were named as AUT1~8 (A. oryzae hyper-producing strains bred in The University of Tokyo). The maximum production yield of CHY by the AUT strains was 107.9 mg/l, which was 1.66-fold increase from the double disruptant. In conclusion, we successfully increased heterologous protein production (3.26- and 3.19-folds for HLY and CHY, respectively) by double proteinase disruption and mutagenesis in A. oryzae. The AUT strains are expected as powerful hosts for higher-level production for many heterologous proteins.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)