Applied Microbiology and Biotechnology

, Volume 101, Issue 10, pp 4041–4052 | Cite as

Engineering rTCA pathway and C4-dicarboxylate transporter for l-malic acid production

  • Xiulai Chen
  • Yuancai Wang
  • Xiaoxiang Dong
  • Guipeng Hu
  • Liming LiuEmail author
Biotechnological products and process engineering


l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L−1, 0.30 g g−1, and 0.32 g L−1 h−1 in the resulting strain W4209 with CaCO3 as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L−1, 0.31 g g−1, and 0.32 g L−1 h−1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.


l-Malic acid Saccharomyces cerevisiae Reductive TCA pathway C4-Dicarboxylate transporter Pathway optimization 



This work was financially supported by the National Natural Science Foundation of China (21676118, 21422602), the Provincial Natural Science Foundation of Jiangsu Province (BK20160163), the Fundamental Research Funds for the Central Universities (JUSRP51611A), the Special Foundation for State Key Research and Development Program of China (2016YFD0400801), and the National Science Foundation for Post-doctoral Scientists of China (2016M600362).

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_2017_8141_MOESM1_ESM.pdf (190 kb)
ESM 1 (PDF 189 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Xiulai Chen
    • 1
    • 2
    • 3
  • Yuancai Wang
    • 1
    • 2
    • 3
  • Xiaoxiang Dong
    • 1
    • 2
    • 3
  • Guipeng Hu
    • 1
    • 2
    • 3
  • Liming Liu
    • 1
    • 2
    • 3
    Email author
  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.Key Laboratory of Industrial Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina
  3. 3.Laboratory of Food Microbial-Manufacturing EngineeringJiangnan UniversityWuxiChina

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