Applied Microbiology and Biotechnology

, Volume 102, Issue 9, pp 4117–4130 | Cite as

Promoter library-based module combination (PLMC) technology for optimization of threonine biosynthesis in Corynebacterium glutamicum

  • Liang Wei
  • Ning Xu
  • Yiran Wang
  • Wei Zhou
  • Guoqiang Han
  • Yanhe Ma
  • Jun LiuEmail author
Applied genetics and molecular biotechnology


Due to the lack of efficient control elements and tools, the fine-tuning of gene expression in the multi-gene metabolic pathways is still a great challenge for engineering microbial cell factories, especially for the important industrial microorganism Corynebacterium glutamicum. In this study, the promoter library-based module combination (PLMC) technology was developed to efficiently optimize the expression of genes in C. glutamicum. A random promoter library was designed to contain the putative − 10 (NNTANANT) and − 35 (NNGNCN) consensus motifs, and refined through a three-step screening procedure to achieve numerous genetic control elements with different strength levels, including fluorescence-activated cell sorting (FACS) screening, agar plate screening, and 96-well plate screening. Multiple conventional strategies were employed for further precise characterizations of the promoter library, such as real-time quantitative PCR, sodium dodecyl sulfate polyacrylamide gel electrophoresis, FACS analysis, and the lacZ reporter system. These results suggested that the established promoter elements effectively regulated gene expression and showed varying strengths over a wide range. Subsequently, a multi-module combination technology was created based on the efficient promoter elements for combination and optimization of modules in the multi-gene pathways. Using this technology, the threonine biosynthesis pathway was reconstructed and optimized by predictable tuning expression of five modules in C. glutamicum. The threonine titer of the optimized strain was significantly improved to 12.8 g/L, an approximate 6.1-fold higher than that of the control strain. Overall, the PLMC technology presented in this study provides a rapid and effective method for combination and optimization of multi-gene pathways in C. glutamicum.


Promoter library Multi-module combination technology Optimization of multi-gene pathway Threonine biosynthesis C. glutamicum 



We are grateful to Prof. Masayuki Inui (Research Institute of Innovative Technology for the Earth, Japan) for generously providing strains and plasmids. This study was supported by the National Natural Science Foundation of China (Nos. 31500044 and 31601460), the Natural Science Foundation of Tianjin (Nos. 17JCQNJC09600 and 17JCYBJC24000), and “Hundred Talents Program” of the Chinese Academy of Sciences.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2018_8911_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1220 kb)


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Copyright information

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

Authors and Affiliations

  • Liang Wei
    • 1
    • 2
    • 3
  • Ning Xu
    • 1
    • 2
  • Yiran Wang
    • 4
  • Wei Zhou
    • 1
    • 2
  • Guoqiang Han
    • 5
  • Yanhe Ma
    • 1
    • 2
  • Jun Liu
    • 1
    • 2
    Email author
  1. 1.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina
  2. 2.Key Laboratory of Systems Microbial BiotechnologyChinese Academy of SciencesTianjinChina
  3. 3.University of Chinese Academy of SciencesBeijingChina
  4. 4.Key Laboratory of Food Nutrition and Safety, Ministry of EducationTianjin University of Science and TechnologyTianjinChina
  5. 5.School of Life Science and BiotechnologyYangtze Normal UniversityChongqingChina

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