Applied Microbiology and Biotechnology

, Volume 102, Issue 5, pp 2129–2141 | Cite as

Efficient biosynthesis of l-phenylglycine by an engineered Escherichia coli with a tunable multi-enzyme-coordinate expression system

  • Qiaoli Liu
  • Junping Zhou
  • Taowei Yang
  • Xian Zhang
  • Meijuan Xu
  • Zhiming Rao
Biotechnological products and process engineering


Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of l-phenylglycine (l-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD+-dependent mutant formate dehydrogenase from Candida boidinii (CbFDHA10C) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDHA10C and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was 28.4 mg L−1 min−1 g−1 dry cell weight for l-Phg production using whole-cell transformation, it’s was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 °C), 60 g L−1 benzoylformic acid was completely converted to pure chiral l-Phg in 4.5 h with 10 g L−1 dry cells and 50.4 g L−1 ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved l-Phg productivity and demonstrated a novel low-cost method for enantiopure l-Phg production.


l-Phenylglycine Leucine dehydrogenase Formate dehydrogenase Tunable multi-enzyme-coordinate expression Whole-cell catalysis 


Funding information

This work was supported by the National Natural Science Foundation of China (863 Program) (2015AA021004), Jiangsu Province Science Fund for Distinguished Young Scholars (BK20150002), National Natural Science Foundation of China (31300028, 21778024), Natural Science Foundation of Jiangsu Province (BK20161292), 111 Project (111-2-06), and Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8741_MOESM1_ESM.pdf (193 kb)
ESM 1 (PDF 193 kb).


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.The Key Laboratory of Industrial Biotechnology, Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of BiotechnologyJiangnan UniversityWuxiChina

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