Metabolic Engineering and Process Development for Enhanced Propionic acid Production by Propionibacterium acidipropionici
Abstract (Summary)Propionibacterium acidipropionici has been extensively studied for propionic acid production, with acetic acid as the main byproduct. 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. To eliminate or reduce acetate formation and increase propionic acid production, gene knock-out through homologous recombination was performed to inactivate acetate kinase gene (Ack) in the acetate formation pathway. 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. The mutant was used in a fibrous bed bioreactor to further improve propionic acid production from glucose. The results showed that the maximum theoretical propionic acid yield of ~0.54 g/g glucose could be achieved. In fed-batch 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. The increased acid tolerance was partially attributed to increased expression of H+-ATPase. Glycerol is an attractive substrate for the production of reductive chemicals because of its low oxidation state. P. acidipropionici could have a higher propionic acid yield of 0.71 g/g glycerol. In addition, the acetate yield was only 0.03 g/g glycerol. Thus, glycerol fermentation produced a high-purity propionic acid facilitating the recovery and purification of propionic acid. The highest propionic acid concentration obtained from glycerol fermentation was ~106 g/l. 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). 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 stimulated by CO2 (HCO3-). Propionyl-CoA:succinate CoA transferase (CoA T, EC# 2.8.3.- ) catalyzes the rate-limiting step in the propionic acid formation pathway. The genome of Propionibacterium acidipropionici ATCC 4875 was sequenced by 454 sequencing and annotated. The CoA transferase gene was identified in the 454 genome sequence database, and was obtained by PCR amplification and then inserted into an expression vector (pET-CoA). The CoA transferase gene was expressed in Escherichia coli BL21 (DE3) under both aerobic and anaerobic conditions. However, P. acidipropionici CoA transferase activity was only observed in the crude extract of BL21 (DE3) harboring pET-CoA grown anaerobically. In addition, a consensus Shine-Dalgarno sequence was found four bases upstream of the AUG codon and two inverted repeat regions were located at the downstream of the TGA stop codon. The amino acid alignment of the P. acidipropionici propionyl-CoA:succinate CoA transferase with other reported CoA transferases illustrated the presence of conserved sites in the amino acid sequence for CoA binding.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Date of Publication:01/01/2009