(635d) Combining Gene Inactivation and Gene Amplification to Increase Propionic Acid Production

Propionibacterium freudenreichii subsp. shermanii has been used to produce propionic acid via fermentation processes. This organism forms propionic acid, acetic acid and succinic acid as main fermentation products. In order to decrease the production of acetic acid and divert more carbon flux toward propionic acid forming steps, phosphotransacetylase gene (pta) and acetate kinase gene (ack) were inactivated using two strategies. First, pta was disrupted by insertion of an integrational nonreplicative plasmid containing chloramphenicol resistance gene (cmlA/cmxA) through single crossover recombination. Since pta is the upstream gene in the pta-ack operon, acetate kinase activity was also attenuated. Second, pta-ack was completely substituted on the chromosome by cmlA/cmxA through double crossover recombination. The resulting two mutants, named cm-PTA and cm-PTAK, produced less acetic acid compared with the wildtype. However, due to the loss of the ATP-forming acetate kinase activity, the specific growth rates of both mutants were lower. The insertion of antibiotics resistance gene was confirmed by PCR and the enzyme inactivation was tested by enzyme assay and SDS-PAGE. Propionyl-CoA:succinate-CoA transferase (CoA T) is the last and limiting enzyme in the propionic acid pathway. In order to enhance propionic acid production while reducing acetic acid level, an additional copy of CoA T gene was introduced into chromosome together with cmlA/cmxA via double crossover recombination, replacing pta-ack operon. The resulting mutant cm-COAT was characterized on molecular level. All the three mutants were used in batch fermentation and fed-batch fermentation in 5-L bioreactor to evaluate their acid production performance.