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Metabolic engineering of Propionibacterium freudenreichii: effect of expressing phosphoenolpyruvate carboxylase on propionic acid production

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Abstract

Propionic acid is currently produced mainly via petrochemicals, but there is increasing interest in its fermentative production from renewable biomass. However, the current propionic acid fermentation process suffers from low product yield and productivity. In this work, the gene encoding phosphoenolpyruvate carboxylase (PPC) was cloned from Escherichia coli and expressed in Propionibacterium freudenreichii. PPC catalyzes the conversion of phosphoenolpyruvate to oxaloacetate with the fixation of one CO2. Its expression in P. freudenreichii showed profound effects on propionic acid fermentation. Compared to the wild type, the mutant expressing the ppc gene grew significantly faster, consumed more glycerol, and produced propionate to a higher final titer at a faster rate. The mutant also produced significantly more propionate from glucose under elevated CO2 partial pressure. These effects could be attributed to increased CO2 fixation and resulting changes in the flux distributions in the dicarboxylic acid pathway.

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References

  • Ammar EM, Wang Z, Yang ST (2013) Metabolic engineering of Propionibacterium freudenreichii for n-propanol production. Appl Microbiol Biotechnol 97:4677–4690

    Article  CAS  PubMed  Google Scholar 

  • Boyaval P, Corre C (1995) Production of propionic acid. Lait 75:453–461

    Article  CAS  Google Scholar 

  • Cheong DE, Lee HI, So JS (2008) Optimization of electrotransformation conditions for Propionibacterium acnes. J Microbiol Methods 72(1):38–41

    Article  CAS  PubMed  Google Scholar 

  • Coral J, Karp SG, Porto de Souza Vandenberghe L, Parada JL, Pandey A, Soccol CR (2008) Batch fermentation model of propionic acid production by Propionibacterium acidipropionici in different carbon sources. Appl Biochem Biotechnol 151:333–341

    Article  CAS  PubMed  Google Scholar 

  • Dishisha T, Ståhl K, Lundmark S, Hatti-Kaul R (2013) An economical biorefinery process for propionic acid production from glycerol and potato juice using high cell density fermentation. Bioresour Technol 135:504–512

    Article  CAS  PubMed  Google Scholar 

  • Falentin H, Deutsch SM, Jan G, Loux V, Thierry A, Parayre S, Maillard MB, Dherbécourt J, Cousin FJ, Jardin J, Siguier P, Couloux A, Barbe V, Vacherie B, Wincker P, Gibrat JF, Gaillardin C, Lortal S (2010) The complete genome of Propionibacterium freudenreichii CIRM-BIA1, a hardy actinobacterium with food and probiotic applications. PLoS One 5(7):e11748

    Article  PubMed Central  PubMed  Google Scholar 

  • Faye T, Asebø A, Salehian Z, Langsrud T, Nes IF, Brede DA (2008) Construction of a reporter vector system for in vivo analysis of promoter activity in Propionibacterium freudenreichii. Appl Environ Microbiol 74:3615–3617

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Genevieve P (1988) Presence of plasmids in propionic acid bacteria. Lait 68:103–107

    Article  Google Scholar 

  • Gu Z, Glatz BA, Glatz CE (1998) Effects of propionic acid on propionibacteria fermentation. Enzym Microb Technol 22:13–18

    Article  CAS  Google Scholar 

  • Himmi EH, Bories A, Boussaid A, Hassani L (2000) Propionic acid fermentation of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. Appl Microbiol Biotechnol 53:435–440

    Article  CAS  PubMed  Google Scholar 

  • Horváth B, Hunyadkürti J, Vörös A, Fekete C, Urbán E, Kemény L, Nagy I (2012) Genome sequence of Propionibacterium acnes type II strain ATCC 11828. J Bacteriol 194:202–203

    Article  PubMed Central  PubMed  Google Scholar 

  • Hsu ST, Yang ST (1991) Propionic acid fermentation of lactose by Propionibacterium acidipropionici: effects of pH. Biotechnol Bioeng 38:571–578

    Article  CAS  PubMed  Google Scholar 

  • Jore JP, van Luijk N, Luiten RG, van der Werf MJ, Pouwels PH (2001) Efficient transformation system for Propionibacterium freudenreichii based on a novel vector. Appl Environ Microbiol 67(2):499–503

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kagliwal LD, Survase SA, Singhal RS, Granström T (2013) Wheat flour based propionic acid fermentation: an economic approach. Bioresour Technol 129:694–699

    Article  CAS  PubMed  Google Scholar 

  • Kiatpapan P, Murooka Y (2001) Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii. Appl Microbiol Biotechnol 56:144–149

    Article  CAS  PubMed  Google Scholar 

  • Kiatpapan P, Murooka Y (2002) Genetic manipulation system in propionibacteria. J Biosci Bioeng 93:1–8

    Article  CAS  PubMed  Google Scholar 

  • Kiatpapan P, Hashimoto Y, Nakamura H, Piao YZ, Ono H, Yamashita M, Murooka Y (2000) Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria. Appl Environ Microbiol 66:4688–4695

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Liu Y, Zhang YG, Zhang RB, Zhang F, Zhu J (2011) Glycerol/glucose co-fermentation: one more proficient process to produce propionic acid by Propionibacterium acidipropionici. Curr Microbiol 62:152–158

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Liu Z, Ma C, Gao C, Xu P (2012) Efficient utilization of hemicellulose hydrolysate for propionic acid production using Propionibacterium acidipropionici. Bioresour Technol 114:711–714

    Article  CAS  PubMed  Google Scholar 

  • Luijk NV, Stierli MP, Schwenninger SM, Herve C, Dasen G, Jore JPM, Pouwels PH, van der Werf MJ, Teuber M, Meile L (2002) Genetics and molecular biology of propionibacteria. Lait 82:45–57

    Article  Google Scholar 

  • Meurice G, Jacoba D, Debordea C, Chailloub S, Rouaulta A, Leverriera P, Jana G, Thierrya A, Maillarda MB, Ameta P, Lalandec M, Zagorecb M, Boyavala P, Dimovaa D (2004) Whole genome sequencing project of a dairy Propionibacterium freudenreichii subsp. shermanii genome: progress and first bioinformatic analysis. Lait 84:15–24

    Article  CAS  Google Scholar 

  • Millard CS, Chao YP, Liao JC, Donnelly MI (1996) Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli. Appl Environ Microbiol 62:1808–1810

    CAS  PubMed Central  PubMed  Google Scholar 

  • Parizzi LP, Grassi MC, Llerena LA, Carazzolle MF, Queiroz VL, Lunardi I, Zeidler AF, Teixeira PJ, Mieczkowski P, Rincones J, Pereira GA (2012) The genome sequence of Propionibacterium acidipropionici provides insights into its biotechnological and industrial potential. BMC Genomics 13:562

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Piao Y, Kawaraichi N, Asegawa R, Kiatpapan P, Ono H, Yamashita M, Murooka Y (2004a) Molecular analysis of promoter elements from Propionibacterium freudenreichii. J Biosci Bioeng 97:310–316

    Article  CAS  PubMed  Google Scholar 

  • Piao Y, Yamashita M, Kawaraichi N, Asegawa R, Ono H, Murooka Y (2004b) Production of vitamin B12 in genetically engineered Propionibacterium freudenreichii. J Biosci Bioeng 98:167–173

    Article  CAS  PubMed  Google Scholar 

  • Playne MJ (1985) Propionic and butyric acids. In: Moo-Young M (ed) Comprehensive biotechnology, vol 3. Pergamon Press, New York, pp 731–759

    Google Scholar 

  • Sawada K, Zen-in S, Wada M, Yokota A (2010) Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032. Metab Eng 12:401–407

    Article  CAS  PubMed  Google Scholar 

  • Song CW, Kim DI, Choi S, Jang JW, Lee SY (2013) Metabolic engineering of Escherichia coli for the production of fumaric acid. Biotechnol Bioeng 110:2025–2034

    Article  CAS  PubMed  Google Scholar 

  • Suwannakham S, Yang ST (2005) Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor. Biotechnol Bioeng 91:325–337

    Article  CAS  PubMed  Google Scholar 

  • Suwannakham S, Huang Y, Yang ST (2006) Construction and characterization of ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation. Biotechnol Bioeng 94:383–395

    Article  CAS  PubMed  Google Scholar 

  • Vörös A, Horváth B, Hunyadkürti J, McDowell A, Barnard E, Patrick S, Nagy I (2012) Complete genome sequences of three Propionibacterium acnes isolates from the type IA(2) cluster. J Bacteriol 194:1621–1622

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang Z, Yang ST (2013) Propionic acid production in glycerol/glucose co-fermentation by Propionibacterium freudenreichii subsp. shermanii. Bioresour Technol 137:116–123

    Article  CAS  PubMed  Google Scholar 

  • Zhang A, Yang ST (2009a) Engineering Propionibacterium acidipropionici for enhanced propionic acid tolerance and fermentation. Biotechnol Bioeng 104:766–773

    CAS  PubMed  Google Scholar 

  • Zhang A, Yang ST (2009b) Propionic acid production from glycerol by metabolically engineered Propionibacterium acidipropionici. Process Biochem 44:1346–1351

    Article  CAS  Google Scholar 

  • Zhang B, Skory CD, Yang ST (2012) Metabolic engineering of Rhizopus oryzae: effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose. Metab Eng 14:512–520

    Article  CAS  PubMed  Google Scholar 

  • Zhu L, Wei P, Cai J, Zhu X, Wang Z, Huang L, Xu Z (2012) Improving the productivity of propionic acid with FBB-immobilized cells of an adapted acid-tolerant Propionibacterium acidipropionici. Bioresour Technol 112:248–253

    Article  CAS  PubMed  Google Scholar 

  • Zhuge X, Liu L, Shin H, Chen RR, Li J, Du G, Chen J (2013) Development of a Propionibacterium-Escherichia coli shuttle vector for metabolic engineering of Propionibacterium jensenii, an efficient producer of propionic acid. Appl Environ Microbiol 79:4595–4602

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

This work was supported in part by The Dow Chemical Company. We thank Prof. Murooka of Osaka University, Japan, for providing the plasmid pKHEM04 used in this work. The Egyptian government’s scholarship (General Mission) to Ammar is also acknowledged.

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Correspondence to Shang-Tian Yang.

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Ammar, E.M., Jin, Y., Wang, Z. et al. Metabolic engineering of Propionibacterium freudenreichii: effect of expressing phosphoenolpyruvate carboxylase on propionic acid production. Appl Microbiol Biotechnol 98, 7761–7772 (2014). https://doi.org/10.1007/s00253-014-5836-y

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  • DOI: https://doi.org/10.1007/s00253-014-5836-y

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