Biotechnology Letters

, Volume 28, Issue 20, pp 1695–1700 | Cite as

Fed-batch two-phase production of alanine by a metabolically engineered Escherichia coli

  • Geoffrey M. Smith
  • Sarah A. Lee
  • Kevin C. Reilly
  • Mark A. Eiteman
  • Elliot Altman
Original Paper

Abstract

dl-Alanine was produced from glucose in an Escherichia coli pfl pps poxB ldhA aceEF pTrc99A-alaD strain which lacked pyruvate-formate lyase, phosphoenolpyruvate (PEP) synthase, pyruvate oxidase, lactate dehydogenase, components of the pyruvate dehydogenase complex and over-produced alanine dehydrogenase (ALD). A two-phase process was developed with cell growth under aerobic conditions followed by alanine production under anaerobic conditions. Using the batch mode, cells grew to 5.3 g/l in 9 h with the accumulation of 6–10 g acetate/l, and under subsequent anaerobic conditions achieved 34 g alanine/l in 13 h with a yield of 0.86 g/g glucose. Using the fed-batch mode at μ = 0.15 h−1, only about 1 g acetate/l formed in the 25 h required for the cells to reach 5.6 g/l, and 88 g alanine/l accumulated during the subsequent 23 h. This fed-batch process attained an alanine volumetric productivity of 4 g/lh during the production phase, and a yield that was essentially 1 g/g.

Keywords

Acetate Alanine Alanine dehydrogenase Fed-batch process Overflow metabolism 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Notes

Acknowledgements

Financial support from the U.S. Dept. of Energy Biobased Products Industry Education Program (DE-FG36-01ID14007), the USDA-NRI Program (2003-35504-13666) and the Georgia Experiment Station is gratefully acknowledged. We also acknowledge J. E. Cronan, Jr. for providing us with strain YYC202.

References

  1. Åkesson M, Hagander P, Axelsson JP (2001) Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding. Biotechnol Bioeng 73(3):223–230PubMedCrossRefGoogle Scholar
  2. Bunch PK, Mat-Jan F, Lee N, Clark DP (1997) The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiol 142:187–195Google Scholar
  3. Chang YY, Cronan Jr JE (1983) Genetic and biochemical analyses of Escherichia coli strains having a mutation in the structural gene (poxB) for pyruvate oxidase. J Bacteriol 154(2):756–762PubMedGoogle Scholar
  4. Chibata TI, Kakimoto T, Kato J (1969) Process for producing l-alanine. US Patent 3,458,400Google Scholar
  5. Eiteman MA, Chastain MJ (1997) Optimization of the ion-exchange analysis of organic acids from fermentation. Anal Chim Acta 338:69–75CrossRefGoogle Scholar
  6. Hashimoto S, Katsumata R (1998) l-alanine fermentation by an alanine racemase-deficient mutant of the dl-alanine hyperproducing bacterium Arthrobacter oxydans HAP-1. J Ferment Bioeng 86:385–390CrossRefGoogle Scholar
  7. Katsumata R, Hashimoto S (1996) Process for producing alanine. US Patent 5,559,016 Google Scholar
  8. Kleman GL, Strohl WR (1994) Acetate metabolism in Escherichia coli in high-cell-density fermentation. Appl Environ Microbiol 60(11):3952–3958PubMedGoogle Scholar
  9. Kleman GL, Chalmers JJ, Luli GW, Strohl WR (1991) A predictive and feedback control algorithm maintains a constant glucose concentration in fed-batch fermentations. Appl Environ Microbiol 57(4):910–917PubMedGoogle Scholar
  10. Lee M, Smith GM, Eiteman MA, Altman E (2004) Aerobic production of alanine by Escherichia coli aceF ldhA mutants expressing the Bacillus sphaericus alaD gene. Appl Microbiol Biotechnol 65:56–60PubMedGoogle Scholar
  11. Luli GW, Strohl WR (1990) Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl Environ Microbiol 56(4):1004–1011PubMedGoogle Scholar
  12. Mat-Jan F, Alam KY, Clark DP (1989) Mutants of Escherichia coli deficient in the fermentative lactate deydrogenase. J Bacteriol 171(1):342–348PubMedGoogle Scholar
  13. Ohashima T, Soda K (1979) Purification and properties of alanine dehydrogenase from Bacillus sphaericus. Eur J Biochem 100:29–39PubMedCrossRefGoogle Scholar
  14. Uhlenbusch I, Sahm H, Sprenger GA (1991) Expression of an l-alanine dehydrogenase gene in Zymomonas mobilis and excretion of l-alanine. Appl Environ Microbiol 57:1360–1366PubMedGoogle Scholar
  15. Vemuri GN, Altman E, Sangudekar DP, Khodursky AB, Eiteman MA (2006) Overflow metabolism in Escherichia coli during steady-state growth: transcriptional regulation and effect of the redox ratio. Appl Environ Microbiol 72(5):3653–3661PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Geoffrey M. Smith
    • 1
  • Sarah A. Lee
    • 1
  • Kevin C. Reilly
    • 1
  • Mark A. Eiteman
    • 1
  • Elliot Altman
    • 1
  1. 1.Center for Molecular BioEngineering, Driftmier EngineeringUniversity of GeorgiaAthensUSA

Personalised recommendations