Metabolic Profiling Of A Rhizopus Oryzae Fumaric Acid Production Mutant Generated By Femtosecond Laser Irradiation

Fumaric acid serves as a typical platform chemical (a carbon-carbon double bondand two carboxylic acid groups) and has been identified one of the top twelvebuilding block chemicals in the21stcentury by the US Department of Energy.Fumaric acid is produced chemically from maleic anhydride by isomerization andoxidation of furfural at present, but the process need severe reactive condition,poisonous catalyst, serious environmental pollution. While considering theenvironmental and energy crisis, it is of great urgency to develop a biologicalfermentation process for fumaric acid from renewable resources.Nowdays, rhizopus species were the best-producing ones for fumaric acidproduction, but the species are limited by genetic engineering due to low efficiency ofrecombinant DNA and site-specific recombination frequency. In this work,femtosecond laser irradiation technology was first employed in Rhizopus oryzae, apositive mutant FM19was obtained, which led to1.56-and1.37-fold increase to49.4g/L and0.56g fumaric acid/g glucose, respectively, compared with the parental strainWild1.22. Mutant FM19produced less ethanol (6.7g/L compared to13.2g/L) and anincrease in malic acid was observed from2.3to3.8g/L.Further, the metabolic profiling was adopted to reveal the intracellular metabolitedifferences for fumaric acid production caused by Gas chromatography-massspectrometry. The metabolites of high-yielding FM19and parental stain Wild1.22were analyzed during Phase B and Phase C, a total of61intracellular metaboliteswere identified and quantified and principal component analysis (PCA) indicated thatthe high-yielding strain exhibited different metabolic characteristics compared withwild type. The schematic representation of the metabolic network revealed that higherlevels of carbon (tricarboxylic acid cycle and Embden-Meyerhof-Parnas) and aminoacid metabolism were operating in the high-yielding strain. Particularly,4-aminobutyric acid and5-aminolevulinic acid were increased by10.33-and7.22-fold, respectively, compared with parental strain during the stationary phase. Thephysiological features helped FM19maintain cell viability and promotedaccumulation of fumaric acid. In addition, the ratio of unsaturated fatty acids tosaturated fatty acids was much higher in mutant FM19than in Wild1.22. Alternationsin the membrane fatty acid and phospholipid composition could enhanced the activity of transporters in fumaric acid transport. These findings provided new insights intometabolic characterization of high-yielding fumaric acid R. oryzae mutant FM19.