Applied Microbiology and Biotechnology

, Volume 94, Issue 4, pp 1107–1117 | Cite as

Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance

  • Hongfang Zhang
  • Huiqing Chong
  • Chi Bun Ching
  • Hao Song
  • Rongrong Jiang
Bioenergy and biofuels

Abstract

One major challenge in biofuel production, including biobutanol production, is the low tolerance of the microbial host towards increasing biofuel concentration during fermentation. Here, we have demonstrated that Escherichia coli 1-butanol tolerance can be greatly enhanced through random mutagenesis of global transcription factor cyclic AMP receptor protein (CRP). Four mutants (MT1–MT4) with elevated 1-butanol tolerance were isolated from error-prone PCR libraries through an enrichment screening. A DNA shuffling library was then constructed using MT1–MT4 as templates and one mutant (MT5) that exhibited the best tolerance ability among all variants was selected. In the presence of 0.8 % (v/v, 6.5 g/l) 1-butanol, the growth rate of MT5 was found to be 0.28 h−1 while that of wild type was 0.20 h−1. When 1-butanol concentration increased to 1.2 % (9.7 g/l), the growth rate of MT5 (0.18 h−1) became twice that of the wild type (0.09 h−1). Microbial adhesion to hydrocarbon test showed that cell surface of MT5 was less hydrophobic and its cell length became significantly longer in the presence of 1-butanol, as observed by scanning electron microscopy. Quantitative real-time reverse transcription PCR analysis revealed that several CRP regulated, 1-butanol stress response related genes (rpoH, ompF, sodA, manX, male, and marA) demonstrated differential expression in MT5 in the presence or absence of 1-butanol. In conclusion, direct manipulation of the transcript profile through engineering global transcription factor CRP can provide a useful tool in strain engineering.

Keywords

1-butanol tolerance cAMP receptor protein Error-prone PCR DNA shuffling Transcriptional engineering Global transcription factor 

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Hongfang Zhang
    • 1
  • Huiqing Chong
    • 1
  • Chi Bun Ching
    • 1
  • Hao Song
    • 1
  • Rongrong Jiang
    • 1
  1. 1.School of Chemical and Biomedical EngineeringNanyang Technological UniversitySingaporeSingapore

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