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Applied Microbiology and Biotechnology

, Volume 63, Issue 3, pp 267–273 | Cite as

Optimization of recombinant aminolevulinate synthase production in Escherichia coli using factorial design

  • L. Xie
  • D. Hall
  • M. A. EitemanEmail author
  • E. Altman
Original Paper

Abstract

The production of recombinant Rhodobacter sphaeroides aminolevulinate (ALA) synthase was optimized in two strains of Escherichia coli: the wild-type strain MG1655, and a ptsG mutant AFP111. The effects of initial succinate, glucose and isopropyl-β-d-thiogalactopyranoside (IPTG) concentrations and the time of induction on enzyme activity were studied. One-way analysis was used to approximate the optimal ranges for these factors, followed by a full factorial design to quantify the effects of each factor and the interactions between the factors. Initial succinate, glucose, and IPTG concentration were observed to be the key factors affecting ALA synthase activity with the optimal levels determined to be above 6 g/l succinate, 0 g/l glucose, and 0.10 mM IPTG. ALA synthase activity was generally lower with AFP111 than with MG1655, and the effect of these three key factors was also lower with AFP111 than with MG1655. Based on the full factorial design results, a fermentation was completed that yielded 296 mU/mg protein with a final ALA concentration of 5.2 g/l (39 mM).

Keywords

Succinate IPTG Shake Flask Full Factorial Design IPTG Concentration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

The authors are grateful for the financial support from the Consortium for Plant Biotechnology Research, and for the technical assistance of Sarah Lee and Kris DeWitt. We thank Ellen Neidle for providing us with the pUI1015 plasmid.

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Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Center for Molecular BioEngineering, Driftmier EngineeringUniversity of GeorgiaAthensUSA
  2. 2.Department of StatisticsUniversity of GeorgiaAthensUSA

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