Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Effect of dissolved oxygen on l-methionine production from glycerol by Escherichia coli W3110BL using metabolic flux analysis method


l-Methionine is an essential amino acid in humans, which plays an important role in the synthesis of some important amino acids and proteins. In this work, metabolic flux of batch fermentation of l-methionine with recombinant Escherichia coli W3110BL was analyzed using the flux balance analysis method, which estimated the intracellular flux distributions under different dissolved oxygen conditions. The results revealed the producing l-methionine flux of 4.8 mmol/(g cell·h) [based on the glycerol uptake flux of 100 mmol/(g cell·h)] was obtained at 30% dissolved oxygen level which was higher than that of other dissolved oxygen levels. The carbon fluxes for synthesizing l-methionine were mainly obtained from the pathway of phosphoenolpyruvate to oxaloacetic acid [15.6 mmol/(g cell·h)] but not from the TCA cycle. Hence, increasing the flow from phosphoenolpyruvate to oxaloacetic acid by enhancing the enzyme activity of phosphoenolpyruvate carboxylase might be conducive to the production of l-methionine. Additionally, pentose phosphate pathway could provide a large amount of reducing power NADPH for the synthesis of amino acids and the flux could increase from 41 mmol/(g cell·h) to 51 mmol/(g cell·h) when changing the dissolved oxygen levels, thus meeting the requirement of NADPH for l-methionine production and biomass synthesis. Therefore, the following modification of the strains should based on the improvement of the key pathway and the NAD(P)/NAD(P)H metabolism.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3


  1. 1.

    Bai Y, Zhou PP, Fan P, Zhu YM, Tong Y, Wang HB, Yu LJ (2015) Four-stage dissolved oxygen strategy based on multi-scale analysis for improving spinosad yield by Saccharopolyspora spinosa ATCC49460. Microb Biotechnol 8:561–568.

  2. 2.

    Bellou S, Makri A, Triantaphyllidou IE, Papanikolaou S, Aggelis G (2014) Morphological and metabolic shifts of Yarrowia lipolytica induced by alteration of the dissolved oxygen concentration in the growth environment. Microbiology 160:807–817.

  3. 3.

    Chua PS, Salleh AHM, Mohamad MS, Deris S, Omatu S, Yoshioka M (2015) Identifying a gene knockout strategy using a hybrid of the bat algorithm and flux balance analysis to enhance the production of succinate and lactate in Escherichia coli. Biotechnol Bioprocess E 20:349–357.

  4. 4.

    Dubitzky W, Wolkenhauer O, Cho KH, Yokota H (2013) Flux balance. Analysis.

  5. 5.

    Garcia A, Ferrer P, Albiol J, Castillo T, Segura D, Pena C (2018) Metabolic flux analysis and the NAD(P)H/NAD(P)(+) ratios in chemostat cultures of Azotobacter vinelandii. Microb Cell Fact 17:10.

  6. 6.

    Georgi T, Rittmann D, Wendisch VF (2005) Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase. Metab Eng 7:291–301.

  7. 7.

    He L, Xiao Y, Gebreselassie N, Zhang F, Antoniewiez MR, Tang YJ, Peng L (2014) Central metabolic responses to the overproduction of fatty acids in Escherichia coli based on 13C-metabolic flux analysis. Biotechnol Bioeng 111:575–585.

  8. 8.

    Hendry JI, Prasannan CB, Joshi A, Dasgupta S, Wangikar PP (2016) Metabolic model of Synechococcus sp. PCC 7002: prediction of flux distribution and network modification for enhanced biofuel production. Bioresour Technol 213:190–197.

  9. 9.

    Hua Q, Yang C, Baba T, Mori H, Shimizu K (2003) Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts. J Bacteriol 185:7053–7067.

  10. 10.

    Huang JF, Liu ZQ, Jin LQ, Tang XL, Shen ZY, Yin HH, Zheng YG (2017) Metabolic engineering of Escherichia coli for microbial production of l-methionine. Biotechnol Bioeng 114:843–851.

  11. 11.

    Huang JF, Shen ZY, Mao QL, Zhang XM, Zhang B, Wu JS, Liu ZQ, Zheng YG (2018) Systematic analysis of bottlenecks in a multibranched and multilevel regulated pathway: the molecular fundamentals of l-methionine biosynthesis in Escherichia coli. ACS Synth Biol 7:2577–2589.

  12. 12.

    Huang JF, Zhang B, Shen ZY, Liu ZQ, Zheng YG (2018) Metabolic engineering of E. coli for the production of O-succinyl-l-homoserine with high yield. 3 Biotech 8:310.

  13. 13.

    Kaushal M, Chary KVN, Ahlawat S, Palabhanvi B, Goswami G, Das D (2018) Understanding regulation in substrate dependent modulation of growth and production of alcohols in Clostridium sporogenes NCIM 2918 through metabolic network reconstruction and flux balance analysis. Bioresour Technol 249:767–776.

  14. 14.

    Kim S, Moon DB, Lee CH, Nam SW, Kim P (2009) Comparison of the effects of NADH- and NADPH-perturbation stresses on the growth of Escherichia coli. Curr Microbiol 58:159–163.

  15. 15.

    Kromer JO, Wittmann C, Schroder H, Heinzle E (2006) Metabolic pathway analysis for rational design of l-methionine production by Escherichia coli and Corynebacterium glutamicum. Metab Eng 8:353–369.

  16. 16.

    Kumar D, Gomes J (2005) Methionine production by fermentation. Biotechnol Adv 23:41–61.

  17. 17.

    Li H, Wang BS, Li YR, Zhang L, Ding ZY, Gu ZH, Shi GY (2017) Metabolic engineering of Escherichia coli W3110 for the production of L-methionine. J Ind Microbiol Biotechnol 44:75–88.

  18. 18.

    Lin H, Bennett GN, San KY (2005) Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield. Metab Eng 7:116–127.

  19. 19.

    Litsanov B, Brocker M, Bott M (2012) Toward homosuccinate fermentation: metabolic engineering of Corynebacterium glutamicum for anaerobic production of succinate from glucose and formate. Appl Environ Microbiol 78:3325–3337.

  20. 20.

    Liu ZQ, Dong SC, Yin HH, Xue YP, Tang XL, Zhang XJ, He JY, Zheng YG (2017) Enzymatic synthesis of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase in an aqueous-organic solvent system. Bioresour Technol 229:26–32.

  21. 21.

    Liu ZQ, Lu MM, Zhang XH, Cheng F, Xu JM, Xue YP, Jin LQ, Wang YS, Zheng YG (2018) Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid. Int J Biol Macromol 116:563–571.

  22. 22.

    Liu ZQ, Wu L, Zhang XJ, Xue YP, Zheng YG (2017) Directed evolution of carbonyl reductase from Rhodosporidium toruloides and its application in stereoselective synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. J Agril Food Chem 65:3721–3729.

  23. 23.

    Liu ZQ, Wu L, Zheng L, Wang WZ, Zhang XJ, Jin LQ, Zheng YG (2018) Biosynthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by carbonyl reductase from Rhodosporidium toruloides in mono and biphasic media. Bioresour Technol 249:161–167.

  24. 24.

    Lou F, Li N, Zhao Y, Guo S, Wang Z, Chen T (2016) Effects of overexpression of carboxylation pathway genes and inactivation of malic enzymes on malic acid production in Escherichia coli. Chin J Biotechnol 32:1539–1548.

  25. 25.

    Manish S, Venkatesh K, Banerjee R (2007) Metabolic flux analysis of biological hydrogen production by Escherichia coli. Int J Hydrogen Energy 32:3820–3830.

  26. 26.

    Matsuoka Y, Kurata H (2017) Modeling and simulation of the redox regulation of the metabolism in Escherichia coli at different oxygen concentrations. Biotechnol Biofuels 10:183.

  27. 27.

    Mitsuhashi S (2014) Current topics in the biotechnological production of essential amino acids, functional amino acids, and dipeptides. Curr Opin Biotechnol 26:38–44.

  28. 28.

    Mohandas SP, Balan L, Jayanath G, Anoop BS, Philip R, Cubelio SS, Bright Singh IS (2018) Biosynthesis and characterization of polyhydroxyalkanoate from marine Bacillus cereus MCCB 281 utilizing glycerol as carbon source. Int J Biol Macromol 119:380–392.

  29. 29.

    Muthuraj M, Palabhanvi B, Misra S, Kumar V, Sivalingavasu K, Das D (2013) Flux balance analysis of Chlorella sp. FC2 IITG under photoautotrophic and heterotrophic growth conditions. Photosynth Res 118:167–179.

  30. 30.

    Niu K, Zhang X, Tan WS, Zhu ML (2011) Effect of culture conditions on producing and uptake hydrogen flux of biohydrogen fermentation by metabolic flux analysis method. Bioresour Technol 102:7294–7300.

  31. 31.

    Park JH, Lee SY (2010) Metabolic pathways and fermentative production of L-aspartate family amino acids. Biotechnol J 5:560–577.

  32. 32.

    Shi F, Li K, Huan X, Wang X (2013) Expression of NAD(H) kinase and glucose-6-phosphate dehydrogenase improve NADPH supply and l-isoleucine biosynthesis in Corynebacterium glutamicum ssp. lactofermentum. Appl Biochem Biotechnol 171:504–521.

  33. 33.

    Sun Y, Guo W, Wang F, Peng F, Yang Y, Dai X, Liu X, Bai Z (2016) Transcriptome and multivariable data analysis of Corynebacterium glutamicum under different dissolved oxygen conditions in bioreactors. PLoS One 11:e0167156.

  34. 34.

    Usuda Y, Kurahashi O (2005) Effects of deregulation of methionine biosynthesis on methionine excretion in Escherichia coli. Appl Environ Microbiol 71:3228–3234.

  35. 35.

    Wang XX, Lin CP, Zhang XJ, Liu ZQ, Zheng YG (2018) Improvement of a newly cloned carbonyl reductase and its application to biosynthesize chiral intermediate of duloxetine. Process Biochem 70:124–128.

  36. 36.

    Weiner M, Trondle J, Albermann C, Sprenger GA, Weuster BD (2014) Improvement of constraint-based flux estimation during l-phenylalanine production with Escherichia coli using targeted knock-out mutants. Biotechnol Bioeng 111:1406–1416.

  37. 37.

    Willke T (2014) Methionine production—a critical review. Appl Microbiol Biotechnol 98:9893–9914.

  38. 38.

    Xu JZ, Yang HK, Zhang WG (2018) NADPH metabolism: a survey of its theoretical characteristics and manipulation strategies in amino acid biosynthesis. Crit Rev Biotechnol 38:1061–1076.

  39. 39.

    Xue LL, Chen HH, Jiang JG (2017) Implications of glycerol metabolism for lipid production. Prog Lipid Res 68:12–25.

  40. 40.

    Yamamoto S, Sakai M, Inui M, Yukawa H (2011) Diversity of metabolic shift in response to oxygen deprivation in Corynebacterium glutamicum and its close relatives. Appl Microbiol Biotechnol 90:1051–1061.

  41. 41.

    Yang JK, Xiong W, Xu L, Li J, Zhao XJ (2015) Constitutive expression of Campylobacter jejuni truncated hemoglobin CtrHb improves the growth of Escherichia coli cell under aerobic and anaerobic conditions. Enzyme Microb Technol 75–76:64–70.

  42. 42.

    Yin L, Zhao J, Chen C, Hu X, Wang X (2014) Enhancing the carbon flux and NADPH supply to increase l-isoleucine production in Corynebacterium glutamicum. Biotechnol Bioprocess E 19:132–142.

  43. 43.

    Zamboni N, Fendt SM, Ruhl M, Sauer U (2009) 13C-based metabolic flux analysis. Nat Protoc 4:878–892.

  44. 44.

    Zhang X, Shanmugam KT, Ingram LO (2010) Fermentation of glycerol to succinate by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol 76:2397–2401.

  45. 45.

    Zhang X, Zhang D, Zhu J, Liu W, Xu G, Zhang X, Shi J, Xu Z (2019) High-yield production of l-serine from glycerol by engineered Escherichia coli. J Ind Microbiol Biotechnol 46:221–230.

  46. 46.

    Zheng YG, Yin HH, Yu DF, Chen X, Tang XL, Zhang XJ, Xue YP, Wang YJ, Liu ZQ (2017) Recent advances in biotechnological applications of alcohol dehydrogenases. Appl Microbiol Biotechnol 101:987–1001.

  47. 47.

    Zheng Y, Chang Y, Zhang R, Song J, Xu Y, Liu J, Wang M (2018) Two-stage oxygen supply strategy based on energy metabolism analysis for improving acetic acid production by Acetobacter pasteurianus. J Ind Microbiol Biotechnol.

  48. 48.

    Zhou HY, Wu WJ, Niu K, Xu YY, Liu ZQ, Zheng YG (2019) Enhanced l-methionine production by genetically engineered Escherichia coli through fermentation optimization. 3 Biotech.

Download references


This research is supported by the National Natural Science Foundation of China (Nos. 31971342 and 31700095).

Author information

Correspondence to Zhi-Qiang Liu.

Ethics declarations

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 by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 111 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Niu, K., Xu, Y., Wu, W. et al. Effect of dissolved oxygen on l-methionine production from glycerol by Escherichia coli W3110BL using metabolic flux analysis method. J Ind Microbiol Biotechnol (2020).

Download citation


  • Escherichia coli
  • l-Methionine
  • Dissolved oxygen
  • Metabolic flux
  • NAD(P)/NAD(P)H metabolism