(507c) Metabolic Engineering of Rhizopus Oryzae: Effects of Overexpressing Fumr Genes On Fumaric Acid Biosynthesis From Glucose

Fumaric acid, a dicarboxylic acid extensively used as a food acidulant and in the manufacturing of synthetic resins, can be produced from glucose in aerobic fermentation by the filamentous fungus Rhizopus oryzae. However, the fumaric acid yield is limited by the co-production of ethanol and other fermentation byproducts. Also, the fumaric acid accumulation mechanism in R. oryzae has not been fully resolved. Fumarase, which converts malate to fumarate in cytosol and the reverse reaction in the TCA cycle, is the key enzyme regulating fumaric acid biosynthesis. In order to understand the mechanism for fumaric acid biosynthesis and optimize the fermentation, effects of overexpressing the endogenous fumarase (fumR) gene were investigated. R. oryzae M16, a uracil auxotrophic mutant of R. oryzae 99880, was used as the parental strain. The uracil auxotroph has a mutation in pyrF gene encoding OMP pyrophosphorylase (pyrF). Only transformants carrying plasmids containing the pyrF gene could grow on the selective medium. The fumR gene was amplified from R. oryzae genomic DNA and cloned into the expression plasmids containing pyrF gene. Then, the expression plasmids were transformed into R. oryzae M16 spores using microprojectile particle bombardment. After culturing for 5-7 days, several fumR tranformants were obtained from the resulting colonies on the agar plates. These transformants were stable in maintaining the recombinant plasmids even after culturing for over 10 days on PDA plates without the selective pressure. Enzyme activity assay showed that the specific fumarase activity in the fumR mutants increased 5- to 10-fold compared to the wild type. However, in shake-flask fermentations, fumaric acid production by fumR mutants produced about the same amount of fumaric acid, but twice more malic acid (0.28-0.38 g/g glucose vs. 0.14 g/g for the wild type). The result suggested that the cloned fumarase catalyzed the reversible hydration of fumaric acid to malic acid. This is the first metabolic engineering study of fumaric acid biosynthesis in R. oryzae. Although overexpressing fumR did not increase fumaric acid production, the mutants produced much more malic acid and are promising for industrial production of malic acid.