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Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production

Abstract

Sucrose is one of the most promising carbon sources for industrial fermentation. To achieve sucrose catabolism, the sucrose utilization operons have been introduced into microorganisms that are not able to utilize sucrose. However, the rates of growth and sucrose uptake of these engineered strains were relatively low to be successfully employed for industrial applications. Here, we report a practical example of developing sucrose-utilizing microorganisms using Escherichia coli K-12 as a model system. The sucrose utilizing ability was acquired by introducing only β-fructofuranosidase from three different sucrose-utilizing organisms (Mannheimia succiniciproducens, E. coli W, and Bacillus subtilis). Among them, the M. succiniciproducens β-fructofuranosidase was found to be the most effective for sucrose utilization. Analyses of the underlying mechanism revealed that sucrose was hydrolyzed into glucose and fructose in the extracellular space and both liberated hexoses could be transported by their respective uptake systems in E. coli K-12. To prove that this system can also be applied for the production of useful metabolites, the M. succiniciproducens β-fructofuranosidase was introduced into the engineered l-threonine production strain of E. coli K-12. This recombinant strain was able to produce 51.1 g/L l-threonine by fed-batch culture, resulting in an overall yield of 0.284 g l-threonine per g sucrose. This simple approach to make E. coli K-12 to acquire sucrose-utilizing ability and its successful biotechnological application can be employed to develop sustainable bioprocesses using renewable biomass.

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Acknowledgements

This work was supported by the Korea–Australia Collaborative Research Project on the Development of Sucrose-Based Bioprocess Platform (10030795) from the Korean Ministry of Knowledge Economy. Further support by World Class University program (R322009000101420) by the Ministry of Education, Science and Technology through the National Research Foundation is appreciated.

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Correspondence to Jin Hwan Park or Sang Yup Lee.

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Lee, J.W., Choi, S., Park, J.H. et al. Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production. Appl Microbiol Biotechnol 88, 905–913 (2010). https://doi.org/10.1007/s00253-010-2825-7

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  • DOI: https://doi.org/10.1007/s00253-010-2825-7

Keywords

  • Sucrose
  • β-Fructofuranosidase
  • Sucrose 6-phosphate hydrolase
  • Sucrase
  • l-threonine