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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 Liu
Biotechnological products and process engineering

Abstract

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.

Keywords

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

Notes

Acknowledgements

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)

References

  1. Ajikumar PK, Xiao WH, Tyo KE, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G (2010) Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330(6000):70–74CrossRefPubMedPubMedCentralGoogle Scholar
  2. Battat E, Peleg Y, Bercovitz A, Rokem JS, Goldberg I (1991) Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor. Biotechnol Bioeng 37(11):1108–1116CrossRefPubMedGoogle Scholar
  3. Brown SH, Bashkirova L, Berka R, Chandler T, Doty T, McCall K, McCulloch M, McFarland S, Thompson S, Yaver D, Berry A (2013) Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of L-malic acid. Appl Microbiol Biotechnol 97(20):8903–8912CrossRefPubMedGoogle Scholar
  4. Canelas AB, ten Pierick A, Ras C, Seifar RM, van Dam JC, van Gulik WM, Heijnen JJ (2009) Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics. Anal Chem 81(17):7379–7389CrossRefPubMedGoogle Scholar
  5. Chen X, Xu G, Xu N, Zou W, Zhu P, Liu L, Chen J (2013) Metabolic engineering of Torulopsis glabrata for malate production. Metab Eng 19:10–16CrossRefPubMedGoogle Scholar
  6. Chen J, Zhu X, Tan Z, Xu H, Tang J, Xiao D, Zhang X (2014) Activating C4-dicarboxylate transporters DcuB and DcuC for improving succinate production. Appl Microbiol Biotechnol 98(5):2197–2205CrossRefPubMedGoogle Scholar
  7. Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH (2002) A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res 30(6):e23CrossRefPubMedPubMedCentralGoogle Scholar
  8. Juminaga D, Baidoo EE, Redding-Johanson AM, Batth TS, Burd H, Mukhopadhyay A, Petzold CJ, Keasling JD (2012) Modular engineering of L-tyrosine production in Escherichia coli. Appl Environ Microbiol 78(1):89–98CrossRefPubMedPubMedCentralGoogle Scholar
  9. Kenealy W, Zaady E, du Preez JC, Stieglitz B, Goldberg I (1986) Biochemical aspects of fumaric acid accumulation by Rhizopus arrhizus. Appl Environ Microbiol 52(1):128–133PubMedPubMedCentralGoogle Scholar
  10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275PubMedGoogle Scholar
  11. Lv Y, Zhao X, Liu L, Du G, Zhou J, Chen J (2013) A simple procedure for protein ubiquitination detection in Saccharomyces cerevisiae: Gap1p as an example. J Microbiol Methods 94(1):25–29CrossRefPubMedGoogle Scholar
  12. MacGurn JA, Hsu PC, Emr SD (2012) Ubiquitin and membrane protein turnover: from cradle to grave. Annu Rev Biochem 81:231–259CrossRefPubMedGoogle Scholar
  13. Peleg Y, Rahamim E, Kessel M, Goldberg I (1988) Malic acid accumulation by Aspergillus flavus—II. Crystals and hair-like processes formed by A. flavus in a l-malic acid production medium. Appl Microbiol BiotechnolGoogle Scholar
  14. Radivojac P, Vacic V, Haynes C, Cocklin RR, Mohan A, Heyen JW, Goebl MG, Iakoucheva LM (2010) Identification, analysis, and prediction of protein ubiquitination sites. Proteins 78(2):365–380CrossRefPubMedPubMedCentralGoogle Scholar
  15. Taing O, Taing K (2007) Production of malic and succinic acids by sugar-tolerant yeast Zygosaccharomyces rouxii. Eur Food Res Technol 224(3):343–347CrossRefGoogle Scholar
  16. Thakker C, Martinez I, Li W, San KY, Bennett GN (2015) Metabolic engineering of carbon and redox flow in the production of small organic acids. J Ind Microbiol Biotechnol 42(3):403–422CrossRefPubMedGoogle Scholar
  17. van Maris AJ, Geertman JM, Vermeulen A, Groothuizen MK, Winkler AA, Piper MD, van Dijken JP, Pronk JT (2004) Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast. Appl Environ Microbiol 70(1):159–166CrossRefPubMedPubMedCentralGoogle Scholar
  18. West TP (2011) Malic acid production from thin stillage by Aspergillus species. Biotechnol Lett 33(12):2463–2467CrossRefPubMedGoogle Scholar
  19. Xu G, Liu L, Chen J (2012) Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae. Microb Cell Factories 11:24CrossRefGoogle Scholar
  20. Xu P, Gu Q, Wang W, Wong L, Bower AG, Collins CH, Koffas MA (2013) Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat Commun 4:1409CrossRefPubMedGoogle Scholar
  21. Yan D, Wang C, Zhou J, Liu Y, Yang M, Xing J (2014) Construction of reductive pathway in Saccharomyces cerevisiae for effective succinic acid fermentation at low pH value. Bioresour Technol 156:232–239CrossRefPubMedGoogle Scholar
  22. Zelle RM, de Hulster E, van Winden WA, de Waard P, Dijkema C, Winkler AA, Geertman JM, van Dijken JP, Pronk JT, van Maris AJ (2008) Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export. Appl Environ Microbiol 74(9):2766–2777CrossRefPubMedPubMedCentralGoogle Scholar
  23. Zhang X, Wang X, Shanmugam KT, Ingram LO (2011) L-malate production by metabolically engineered Escherichia coli. Appl Environ Microbiol 77(2):427–434CrossRefPubMedGoogle Scholar

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
  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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