Over-expression of gluconic acid in Aspergillus oryzae RP-21 mutants generated by a random mutagenesis approach

Abstract

Random mutagenesis with N-methyl-N′ nitro-N-nitrosoguanidine (NTG) was used to mutate Aspergillus oryzae RP-21 to develop high gluconic acid-producing mutants. Forty mutant colonies (designated as A. oryzae strains RP-NTG-01 to RP-NTG-40) screened for gluconic acid, glucose dehydrogenase and glucose oxidase production using a 12-well plate method showed that 17 strains (positive mutants) produced high concentrations of these three products, whereas 12 strains (negative mutants) showed low concentrations and the remaining 11 strains (non-mutants) did not produce any of the three products. Detailed studies of A. oryzae RP-NTG-12, a positive mutant, produced gluconic acid of up to 72 g/L in batch fermentation, which was a 2.4-fold increase in yield to that of the strain and as expected it also possessed higher activities of cell-bound glucose dehydrogenase and glucose oxidase, key enzymes of the multi-functional gluconic acid synthesis pathway. We discuss changes in the cell-bound enzyme activities of the mutants and the wild type and speculate on a mechanism for this increase. The mutant strain, A. oryzae RP-NTG-12, and the random mutagenesis method used to increase bioproducts have a good potential for developing fermentation processes to an industrial scale as demonstrated by this study.