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Applied Microbiology and Biotechnology

, Volume 100, Issue 2, pp 637–647 | Cite as

Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production

  • Ying-Jia Tong
  • Xiao-Jun Ji
  • Meng-Qiu Shen
  • Lu-Gang Liu
  • Zhi-Kui Nie
  • He Huang
Biotechnological products and process engineering

Abstract

Many microorganisms could naturally produce (R, R)-2,3-butanediol ((R, R)-2,3-BD), which has unique applications due to its special chiral group and spatial configuration. But the low enantio-purity of the product hindered the development of large-scale production. In this work, a synthetic constitutive metabolic pathway for enantiomerically pure (R, R)-2,3-BD biosynthesis was constructed in Escherichia coli with vector pUC6S, which does not contain any lac sequences. The expression of this artificial constructed gene cluster was optimized by using two different strength of promoters (AlperPLTet01 (P01) and AlperBB (PBB)). The strength of P01 is twice stronger than PBB. The fermentation results suggested that the yield of (R, R)-2,3-BD was higher when using the stronger promoter. Compared with the wild type, the recombinant strain E. coli YJ2 produced a small amount of acetic acid and showed higher glucose consumption rate and higher cell density, which indicated a protection against acetic acid inhibition. In order to further increase the (R, R)-2,3-BD production by reducing the accumulation of its precursor acetoin, the synthetic operon was reconstructed by adding the strong promoter P01 in front of the gene ydjL coding for the enzyme of (R, R)-2,3-BD dehydrogenase which catalyzes the conversion of acetoin to (R, R)-2,3-BD. The engineered strain E. coli YJ3 showed a 20 % decrease in acetoin production compared with that of E. coli YJ2. After optimization the fermentation conditions, 30.5 g/L of (R, R)-2,3-BD and 3.2 g/L of acetoin were produced from 80 g/L of glucose within 18 h, with an enantio-purity over 99 %.

Keywords

(R, R)-2,3-butanediol Promoter Escherichia coli Metabolic engineering 

Notes

Acknowledgments

This work was financially supported by the National Science Foundation for Distinguished Young Scholars of China (No. 21225626), the National Natural Science Foundation of China (Nos. 21376002, 21476111), the Jiangsu Provincial Natural Science Foundation of China (No. BK20131405), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Conflict of interest

The authors declare that they have no competing interests.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical EngineeringNanjing Tech UniversityNanjingPeople’s Republic of China

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