Applied Microbiology and Biotechnology

, Volume 102, Issue 20, pp 8739–8751 | Cite as

Enhancing l-malate production of Aspergillus oryzae FMME218-37 by improving inorganic nitrogen utilization

  • Qiang Ding
  • Qiuling Luo
  • Jie Zhou
  • Xiulai Chen
  • Liming LiuEmail author
Biotechnological products and process engineering


Microbial l-malate production from renewable feedstock is a promising alternative to petroleum-based chemical synthesis. However, high l-malate production of Aspergillus oryzae was achieved to date using organic nitrogen, with inorganic nitrogen still unable to meet industrial applications. In the current study, we constructed a screening system and nitrogen supply strategy to improve l-malate production with ammonium sulphate [(NH4)2SO4] as the sole nitrogen source. First, we generated and identified a high-producing mutant FMME218-37, which stably boosted l-malate production from 30.73 to 78.12 g/L, using a combined screening system with morphological characteristics. Then, by analyzing the fermentation parameters and physiological characteristics, we further speculated the key factor was the unbalance of carbon and nitrogen absorption. Finally, the titer and productivity of l-malate was increased to 95.2 g/L and 0.57 g/(L h) by regulating the nitrogen supply module to balance carbon and nitrogen absorption, which represented the highest level in A. oryzae with (NH4)2SO4 as nitrogen source achieved to date. Moreover, our findings using a low-cost substrate may lead to building an economical cell factory of A. oryzae for l-malate production.


l-Malate Aspergillus oryzae (NH4)2SO4 Screening system Nitrogen supply module Carbon and nitrogen absorption 



This work was funded by the National Natural Science Foundation of China (21676118, 21706095).

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_9272_MOESM1_ESM.pdf (99 kb)
Supplemental Table S1 (PDF 98 kb)


  1. Anglov T, Petersen IM, Kristiansen J (1999) Uncertainty of nitrogen determination by the Kjeldahl method. Accred Qual Assur 4(12):504–510CrossRefGoogle Scholar
  2. Battat E, Peleg Y, Bercovitz A, Rokem JS, Goldberg I (2010) Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor. Biotechnol Bioeng 37(11):1108–1116CrossRefGoogle Scholar
  3. Bork P, Sander C, Valencia A (1993) Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases. Protein Sci 2(1):31–40CrossRefGoogle Scholar
  4. 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 Biotech 97(20):8903–8912CrossRefGoogle Scholar
  5. Chen XL, Wang Y, Dong X, Hu G, Liu L (2017) Engineering rTCA pathway and C4-dicarboxylate transporter for L-malic acid production. Appl Microbiol Biotech 101(10):4041–4052CrossRefGoogle Scholar
  6. Chen XL, Xu GQ, Xu N, Zou W, Zhu P, Liu LM, Chen J (2013) Metabolic engineering of Torulopsis glabrata for malate production. Metab Eng 19(5):10–16CrossRefGoogle Scholar
  7. Deng Y, Li S, Xu Q, Gao M, Huang H (2012) Production of fumaric acid by simultaneous saccharification and fermentation of starchy materials with 2-deoxyglucose-resistant mutant strains of Rhizopus oryzae. Bioresour Technol 107(3):363–367CrossRefGoogle Scholar
  8. Dong X, Chen X, Qian Y, Wang Y, Wang L, Qiao W, Liu L (2017) Metabolic engineering of Escherichia coli W3110 to produce L-malate. Biotechnol Bioeng 114(3):656–664CrossRefGoogle Scholar
  9. Dörsam S, Fesseler J, Gorte O, Hahn T, Zibek S, Syldatk C, Ochsenreither K (2017) Sustainable carbon sources for microbial organic acid production with filamentous fungi. Biotechnol Biofuels 10(1):242–254CrossRefGoogle Scholar
  10. Downes DJ, Davis MA, Kreutzberger SD, Taig BL, Todd RB (2013) Regulation of the NADP-glutamate dehydrogenase gene gdhA in Aspergillus nidulans by the Zn(II)2Cys6 transcription factor LeuB. Microbiology 159(12):2467–2480CrossRefGoogle Scholar
  11. Driouch H, Hänsch R, Wucherpfennig T, Krull R, Wittmann C (2012) Improved enzyme production by bio-pellets of Aspergillus niger: targeted morphology engineering using titanate microparticles. Biotechnol Bioeng 109(2):462–471CrossRefGoogle Scholar
  12. Driouch H, Sommer B, Wittmann C (2010) Morphology engineering of Aspergillus niger for improved enzyme production. Biotechnol Bioeng 105(6):1058–1068PubMedGoogle Scholar
  13. Du ZQ, Zhang Y, Qian ZG, Xiao H, Zhong JJ (2017) Combination of traditional mutation and metabolic engineering to enhance ansamitocin P-3 production in Actinosynnema pretiosum. Biotechnol Bioeng 114(12):2794–2806CrossRefGoogle Scholar
  14. Engel CAR, Straathof AJJ, Zijlmans TW, Gulik WMV, Wielen LAMVD (2008) Fumaric acid production by fermentation. Appl Microbiol and Biotech 78(3):379–389CrossRefGoogle Scholar
  15. Gao C, Wang S, Hu G, Guo L, Chen X, Xu P, Liu L (2017) Engineering Escherichia coli for malate production by integrating modular pathway characterization with CRISPRi-guided multiplexed metabolic tuning. Biotechnol Bioeng 115(2–3):661–672PubMedGoogle Scholar
  16. Gao D (2014) Microbial lipid production by oleaginous fungus Mortierella isabellina through morphology engineering. Biotechnol Bioeng 111(9):15–20CrossRefGoogle Scholar
  17. Goldberg I, Rokem JS, Pines O (2010) Organic acids: old metabolites, new themes. J Chem Technol Biot 81(10):1601–1611CrossRefGoogle Scholar
  18. Gu C, Wang G, Mai S, Wu P, Wu J, Wang G, Liu H, Zhang J (2017) ARTP mutation and genome shuffling of ABE fermentation symbiotic system for improvement of butanol production. Appl Microbiol Biotech 101(5):2189–2199CrossRefGoogle Scholar
  19. Khan I, Nazir K, Wang ZP, Liu GL, Chi ZM (2014) Calcium malate overproduction by Penicillium viticola 152 using the medium containing corn steep liquor. Appl Microbiol Biotech 98(4):1539–1546CrossRefGoogle Scholar
  20. Knuf C, Nookaew I, Brown SH, McCulloch M, Berry A, Nielsen J (2013) Investigation of malic acid production in Aspergillus oryzae under nitrogen starvation conditions. Appl Environ Microb 79(19):6050–6058CrossRefGoogle Scholar
  21. Knuf C, Nookaew I, Remmers I, Khoomrung S, Brown S, Berry A, Nielsen J (2014) Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68. Appl Microbiol Biotech 98(8):3517–3527CrossRefGoogle Scholar
  22. Kusnan MB, Berger MG, Fock HP (1987) The involvement of glutamine synthetase/glutamate synthase in ammonia assimilation by Aspergillus nidulans. J Gen Appl Microbiol 133(5):1235–1242Google Scholar
  23. Li X, Liu R, Li J, Chang M, Liu Y, Jin Q, Wang X (2015) Enhanced arachidonic acid production from Mortierella alpina combining atmospheric and room temperature plasma (ARTP) and diethyl sulfate treatments. Bioresour Technol 177(177C):134–140CrossRefGoogle Scholar
  24. Lin SY, Wang LY, Jones G, Trang H, Yin YG, Liu JB (2012) Optimized extraction of calcium malate from eggshell treated by PEF and an absorption assessment in vitro. Int J Biol Macromol 50(5):1327–1333CrossRefGoogle Scholar
  25. Liu J, Li J, Shin HD, Du G, Chen J, Liu L (2017a) Metabolic engineering of Aspergillus oryzae for efficient production of l-malate directly from corn starch. J Biotechnol 262(5):40–46CrossRefGoogle Scholar
  26. Liu J, Xie Z, Shin HD, Li J, Du G, Chen J, Liu L (2017b) Rewiring the reductive tricarboxylic acid pathway and L-malate transport pathway of Aspergillus oryzae for overproduction of L-malate. J Biotechnol 253(2):1–9CrossRefGoogle Scholar
  27. Margelis S, D’Souza C, Small AJ, Hynes MJ, Adams TH, Davis MA (2001) Role of glutamine synthetase in nitrogen metabolite repression in Aspergillus nidulans. J Bacteriol 183(20):5826–5833CrossRefGoogle Scholar
  28. Marzluf GA (1997) Genetic regulation of nitrogen metabolism in the fungi. Mol Microbiol 61(1):17–32Google Scholar
  29. Muller MM, Kugler JH, Henkel M, Gerlitzki M, Hormann B, Pohnlein M, Syldatk C, Hausmann R (2012) Rhamnolipids--next generation surfactants? J Biotechnol 162(4):366–380CrossRefGoogle Scholar
  30. Nakayama S, Tabata K, Oba T, Kusumoto K, Mitsuiki S, Kadokura T, Nakazato A (2012) Characteristics of the high malic acid production mechanism in Saccharomyces cerevisiae sake yeast strain No. 28. J Biosci Bioeng 114(3):281–285CrossRefGoogle Scholar
  31. Ochsenreither K, Fischer C, Neumann A, Syldatk C (2014) Process characterization and influence of alternative carbon sources and carbon-to-nitrogen ratio on organic acid production by Aspergillus oryzae DSM1863. Appl Microbiol Biotech 98(12):5449–5460CrossRefGoogle Scholar
  32. Papagianni M, Mattey M (2006) Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Microb Cell Factories 5(1):3–14CrossRefGoogle Scholar
  33. Peleg Y, Stieglitz B, Goldberg I (1989) Malic acid accumulation by Aspergillus flavus. Appl Microbiol Biotech 30(2):176–183CrossRefGoogle Scholar
  34. Schwartz H, Radler F (1988) Formation of l (−)malate by Saccharomyces cerevisiae during fermentation. Appl Microbiol Biotech 27(5–6):553–560CrossRefGoogle Scholar
  35. Stojkovič G, Plazl I, Žnidaršič-Plazl P (2011) L-Malic acid production within a microreactor with surface immobilised fumarase. Microfluid Nanofluid 10(3):627–635CrossRefGoogle Scholar
  36. Thakker C, Martínez 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 Biot 42(3):403–422CrossRefGoogle Scholar
  37. Veiter L, Rajamanickam V, Herwig C (2018) The filamentous fungal pellet—relationship between morphology and productivity. Appl Microbiol Biotech 102(7):1–10CrossRefGoogle Scholar
  38. Wang B, Han X, Bai Y, Lin Z, Qiu M, Nie X, Wang S, Zhang F, Zhuang Z, Yuan J (2017) Effects of nitrogen metabolism on growth and aflatoxin biosynthesis in Aspergillus flavus. J Hazard Mater 324(Pt B):691–702CrossRefGoogle Scholar
  39. Werpy TA, Holladay JE, White JF (2004) Top value added chemicals from biomass: I. Results of Screening for Potential Candidates from Sugars and Synthesis Gas. Synthetic FuelsGoogle Scholar
  40. West TP (2011) Malic acid production from thin stillage by Aspergillus species. Biotechnol Lett 33(12):2463–2467CrossRefGoogle Scholar
  41. Yasuhara T, Nokihara K (2001) High-throughput analysis of total nitrogen content that replaces the classic Kjeldahl method. J Agr Food Chem 49(10):4581–4590CrossRefGoogle Scholar
  42. Ye X, Honda K, Morimoto Y, Okano K, Ohtake H (2013) Direct conversion of glucose to malate by synthetic metabolic engineering. J Biotechnol 164(1):34–40CrossRefGoogle Scholar
  43. Zambanini T, Kleineberg W, Sarikaya E, Buescher JM, Meurer G, Wierckx N, Blank LM (2016a) Enhanced malic acid production from glycerol with high-cell density Ustilago trichophora TZ1 cultivations. Biotechnol Biofuels 9(1):135–144CrossRefGoogle Scholar
  44. Zambanini T, Sarikaya E, Kleineberg W, Buescher JM, Meurer G, Wierckx N, Blank LM (2016b) Efficient malic acid production from glycerol with Ustilago trichophora TZ1. Biotechnol Biofuels 9(1):67–75CrossRefGoogle Scholar
  45. Zambanini T, Tehrani HH, Geiser E, Sonntag CK, Buescher JM, Meurer G, Wierckx N, Blank LM (2017) Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production. Metab Eng Commun 4(C):12–21CrossRefGoogle Scholar
  46. Zelle RM, de Hulster E, van Winden WA, de Waard P, Dijkema C, Winkler AA, Geertman JMA, van Dijken JP, Pronk JT, van Maris AJA (2008) Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export. Appl Environ Microb 74(9):2766–2777CrossRefGoogle Scholar
  47. Zhang X, Wang X, Shanmugam KT, Ingram LO (2011) L-malate production by metabolically engineered Escherichia coli. Appl Environ Microb 77(2):427–434CrossRefGoogle Scholar
  48. Zhao B, Li Y, Li C, Yang H, Wang W (2018) Enhancement of Schizochytrium DHA synthesis by plasma mutagenesis aided with malonic acid and zeocin screening. Appl Microbiol Biotech 102(5):2351–2361CrossRefGoogle Scholar
  49. Zhe C, Wang ZP, Wang GY, Khan I, Chi ZM (2014) Microbial biosynthesis and secretion of l-malic acid and its applications. Crit Rev Biotechnol 36(1):99–107Google Scholar
  50. Zhu X, Zhang W, Chen X, Wu H, Duan Y, Xu Z (2010) Generation of high rapamycin producing strain via rational metabolic pathway-based mutagenesis and further titer improvement with fed-batch bioprocess optimization. Biotechnol Bioeng 107(3):506–510CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Qiang Ding
    • 1
    • 2
    • 3
  • Qiuling Luo
    • 1
    • 2
    • 3
  • Jie Zhou
    • 1
    • 2
    • 3
  • Xiulai Chen
    • 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.National Engineering Laboratory for Cereal Fermentation TechnologyJiangnan UniversityWuxiChina

Personalised recommendations