Metabolic engineering and process development for enhanced propionic acid production by Propionibacterium acidipropionici.

摘要

Propionic acid is an important mold inhibitor. The goal of this project was to develop an economical fermentation process for propionic acid production from glucose and processing wastes via integrated metabolic and process engineering approaches.;Propionibacterium acidipropionici has been extensively studied for propionic acid production, with acetic acid as the main byproduct. Compared to the wild type strain, the mutant (ACK-Tet) produced more propionate and less acetate, but the specific growth rate of the mutant was also decreased due to less ATP can be generated from the impaired acetic acid synthesis pathway. In fed-batch fibrous bed bioreactor (FBB) fermentation, the final propionic acid concentration reached ∼104 g/l, which was 43% higher than the highest concentration (∼72 g/l) previously reported. Clearly, the bacteria in the FBB had adapted and acquired a higher tolerance to propionic acid. A growth kinetics experiment showed that the adapted mutant from the FBB was ∼10 times less sensitive to propionic acid inhibition. The increased acid tolerance was partially attributed to increased expression of H+-ATPase, which plays a key role in proton pumping and maintaining the intracellular pH. Furthermore, after adaptation in the FBB, the ACK-Tet mutant recovered its specific growth rate to the same level as that of its parent wild-type strain.;Besides the final concentration of propionic acid in the fermentation broth, the P/A ratio (propionic acid vs. acetic acid) is another key factor affecting downstream purification and the overall production cost of propionic acid. In general, the lower the oxidation level of the carbon source is, the higher the P/A ratio can be obtained due to the intracellular NADH/NAD + balance. Glycerol is thus an attractive substrate for the production of reductive chemicals (e.g., H2, ethanol, and propionic acid) because of its low oxidation state. There are also abundant supplies of low-cost glycerol as a waste product from the biodiesel industry. P. acidipropionici could use glycerol for growth and propionic acid production, with a high yield of 0.71 g/g glycerol, which was ∼30% higher than that from glucose (0.55 g/g glucose). In addition, almost no acetic acid was produced from glycerol; the acetate yield was only 0.03 g/g glycerol (vs. 0.1 g/g glucose). Thus, glycerol fermentation produced a high-purity propionic acid with the propionic acid to acetic acid ratio of ∼24 (vs. ∼5 from glucose fermentation), facilitating the recovery and purification of propionic acid from the fermentation broth by simple solvent extraction. The highest propionic acid concentration obtained from glycerol fermentation was ∼106 g/l, which was 2.5 times of the maximum concentration of ∼42 g/l reported in the literature. Moreover, a stoichiometric metabolic model was set up based on the NADH/NAD+ balance and maximum ATP production. The trend predicted by the model fitted the experimental data very well.;The effects of CO2 (HCO3-) on cell growth and acids production from glycerol were studied. The productivity of propionic acid in glycerol fermentation with CO2 (HCO3 -) reached 2.94 g/l/day, which was markedly higher than that without CO2 (HCO3-) (1.56 g/l/day). However, the propionic acid yield was decreased slightly from 0.77 to 0.67 g/g glycerol due to the higher biomass production when CO2 (HCO3 -) was supplemented in the media. Meanwhile, the yield and productivity of succinate increased 81% and 280%, respectively, suggesting a significant increase in the Wood-Werkman cycle rate that could be attributed to the increased activities of key enzymes (e.g. phosphoenolpyruvate carboxylase and propionyl CoA transferase) stimulated by CO2 (HCO3 -).;Propionyl-CoA:succinate CoA transferase (CoA T, EC