Applied Microbiology and Biotechnology

, Volume 103, Issue 10, pp 4113–4124 | Cite as

Enhancement of substrate supply and ido expression to improve 4-hydroxyisoleucine production in recombinant Corynebacterium glutamicum ssp. lactofermentum

  • Feng ShiEmail author
  • Shuping Zhang
  • Yongfu Li
  • Zhengke Lu
Applied genetics and molecular biotechnology


4-Hydroxyisoleucine (4-HIL) has potential value in treating diabetes. L-isoleucine dioxygenase (IDO) catalyzes the hydroxylation of L-isoleucine (Ile) to form 4-HIL with the concomitant oxidation of α-ketoglutarate (α-KG) and oxygen consumption. In our previous study, by expressing the ido gene in the Ile producer Corynebacterium glutamicum ssp. lactofermentum SN01, 4-HIL was de novo-synthesized from glucose without adding either Ile or α-KG. In this study, synergistically improving the substrates supply and IDO activity was applied to enhance the de novo biosynthesis of 4-HIL. Deletion of aceA and blocking of the glyoxylate pathway effectively enhanced α-KG supply and Ile synthesis and thus improved 4-HIL production to 69.47 ± 2.18 mM, 18.9% higher than in the original strain. Coexpression of mqo with ido further improved Ile synthesis but decreased 4-HIL production, partially due to the inadequate activity of IDO. Coexpression of another gene, ido3, with mqo and ido efficiently promoted IDO activity, thus improving 4-HIL production to 91.54 ± 8.29 mM. Further expression of vgb and promotion of the oxygen uptake rate did not change the 4-HIL titer significantly but increased the 4-HIL production rate in the first 72 h of fermentation. After fermentation in the optimized medium, 4-HIL production by the final strains increased to 112–117 mM, indicating these strains are promising candidates for producing 4-HIL. These results demonstrate that synergistically promoting substrate supply and improving IDO activity are efficient approaches to enhance 4-HIL production in C. glutamicum.


4-Hydroxyisoleucine Corynebacterium glutamicum Substrate supply aceA deletion ido vgb 



This work was supported by the program of State Key Laboratory of Food Science and Technology (SKLF-ZZA-201904) and national first-class discipline program of the Light Industry Technology and Engineering (LITE2018-10).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Compliance with ethics requirements

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


  1. Ehira S, Teramoto H, Inui M, Yukawa H (2009) Regulation of Corynebacterium glutamicum heat shock response by the extracytoplasmic-function sigma factor SigH and transcriptional regulators HspR and HrcA. J Bacteriol 191(9):2964–2972. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Eikmanns BJ, Rittmann D, Sahm H (1995) Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J Bacteriol 177(3):774–782. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Haeri MR, Limaki HK, White CJ, White KN (2012) Non-insulin dependent anti-diabetic activity of (2S, 3R, 4S) 4-hydroxyisoleucine of fenugreek (Trigonella foenum graecum) in streptozotocin-induced type I diabetic rats. Phytomedicine 19(7):571–574. CrossRefPubMedGoogle Scholar
  4. Hirasawa T, Kim J, Shirai T, Furusawa C, Shimizu H (2012) Molecular mechanisms and metabolic engineering of glutamate overproduction in Corynebacterium glutamicum. Subcell Biochem 64:261–281. CrossRefPubMedGoogle Scholar
  5. Hoffmann J, Altenbuchner J (2014) Hyaluronic acid production with Corynebacterium glutamicum: effect of media composition on yield and molecular weight. J Appl Microbiol 117(3):663–678. CrossRefPubMedGoogle Scholar
  6. Hu J, Tan Y, Li Y, Hu X, Xu D, Wang X (2013) Construction and application of an efficient multiple-gene-deletion system in Corynebacterium glutamicum. Plasmid 70(3):303–313. CrossRefPubMedGoogle Scholar
  7. Hu J, Li Y, Zhang H, Tan Y, Wang X (2014) Construction of a novel expression system for use in Corynebacterium glutamicum. Plasmid 75:18–26. CrossRefPubMedGoogle Scholar
  8. Huang S, Shi F (2018) Directed evolution and site-specific mutagenesis of L-isoleucine dioxygenase derived from Bacillus weihenstephanensis. Biotechnol Lett 40(8):1227–1235. CrossRefPubMedGoogle Scholar
  9. Jetté L, Harvey L, Eugeni K, Levens N (2009) 4-Hydroxyisoleucine: a plant-derived treatment for metabolic syndrome. Curr Opin Investig Drugs 10(4):353–358PubMedGoogle Scholar
  10. Jo JH, Seol HY, Lee YB, Kim MH, Hyun HH, Lee HH (2012) Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum. Can J Microbiol 58(3):278–286. CrossRefPubMedGoogle Scholar
  11. Jojima T, Fujii M, Mori E, Inui M, Yukawa H (2010) Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid L-alanine under oxygen deprivation. Appl Microbiol Biotechnol 87(1):159–165. CrossRefPubMedGoogle Scholar
  12. Kennerknecht N, Sahm H, Yen MR, Pátek M, Saier Jr MH Jr, Eggeling L (2002) Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family. J Bacteriol 184(14):3947–3956. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Kimura E (2003) Metabolic engineering of glutamate production. Adv Biochem Eng Biotechnol 79:37–57PubMedGoogle Scholar
  14. Kivero AD, Novikova AE, Smirnov SV (2012) Modification of E. coli central metabolism to optimize the biotransformation of L-isoleucine into 4-hydroxyisoleucine by enzymatic hydroxylation. Appl Biochem Microbiol 48:639–644CrossRefGoogle Scholar
  15. Kodera T, Smirnov SV, Samsonova NN, Kozlov YI, Koyama R, Hibi M, Ogawa J, Yokozeki K, Shimizu S (2009) A novel l-isoleucine hydroxylating enzyme, l-isoleucine dioxygenase from Bacillus thuringiensis, produces (2S,3R,4S)-4-hydroxyisoleucine. Biochem Biophys Res Commun 390(3):506–510. CrossRefPubMedGoogle Scholar
  16. Liu Q, Zhang J, Wei XX, Ouyang SP, Wu Q, Chen GQ (2008) Microbial production of L-glutamate and L-glutamine by recombination Corynebacterium glutamicum harboring Vitreoscilla hemoglobin gene vgb. Appl Microbiol Biotechnol 77:1297–1304. CrossRefPubMedGoogle Scholar
  17. Mao Y, Li G, Chang Z, Tao R, Cui Z, Wang Z, Tang YJ, Chen T, Zhao X (2018) Metabolic engineering of Corynebacterium glutamicum for efficient production of succinate from lignocellulosic hydrolysate. Biotechnol Biofuels 11:95. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Mindt M, Risse JM, Gruss H, Sewald N, Eikmanns BJ, Wendisch VF (2018) One-step process for production of N-methylated amino acids from sugars and methylamine using recombinant Corynebacterium glutamicum as biocatalyst. Sci Rep 8(1):12895. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Molenaar D, van der Rest ME, Petrović S (1998) Biochemical and genetic characterization of the membrane-associated malate dehydrogenase (acceptor) from Corynebacterium glutamicum. Eur J Biochem 254(2):395–403. CrossRefPubMedGoogle Scholar
  20. Molenaar D, van der Rest ME, Drysch A, Yucel R (2000) Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Corynebacterium glutamicum. J Bacteriol 182(24):6884–6891. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Neelakantan N, Narayanan M, de Souza RJ, van Dam RM (2014) Effect of fenugreek (Trigonella foenum-graecum L.) intake on glycemia: a meta-analysis of clinical trials. Nutr J 13:7. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Shi F, Jiang J, Li Y, Li Y, Xie Y (2013) Enhancement of gamma-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis. J Ind Microbiol Biotechnol 40(11):1285–1296. CrossRefPubMedGoogle Scholar
  23. Shi F, Niu T, Fang H (2015) 4-Hydroxyisoleucine production of recombinant Corynebacterium glutamicum ssp. lactofermentum under optimal corn steep liquor limitation. Appl Microbiol Biotechnol 99(9):3851–3863. CrossRefPubMedGoogle Scholar
  24. Shi F, Fang H, Niu T, Lu Z (2016) Overexpression of ppc and lysC to improve the production of 4-hydroxyisoleucine and its precursor L-isoleucine in recombinant Corynebacterium glutamicum ssp. lactofermentum. Enzym Microb Technol 87(88):79–85. CrossRefGoogle Scholar
  25. Shi F, Luan M, Li Y (2018a) Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing γ-aminobutyrate in Corynebacterium glutamicum. AMB Express 8(1):61. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Shi F, Zhang M, Li Y, Fang H (2018b) Sufficient NADPH supply and pknG deletion improve 4-hydroxyisoleucine production in recombinant Corynebacterium glutamicum. Enzym Microb Technol 115:1–8. CrossRefGoogle Scholar
  27. Šilar R, Holátko J, Rucká L, Rapoport A, Dostálová H, Kadeřábková P, Nešvera J, Pátek M (2016) Use of in vitro transcription system for analysis of Corynebacterium glutamicum promoters recognized by two sigma factors. Curr Microbiol 73(3):401–408. CrossRefPubMedGoogle Scholar
  28. Smirnov SV, Kodera T, Samsonova NN, Kotlyarova VA, Rushkevich NY, Kivero AD, Sokolov PM, Hibi M, Ogawa J, Shimizu S (2010) Metabolic engineering of Escherichia coli to produce (2S, 3R, 4S)-4-hydroxyisoleucine. Appl Microbiol Biotechnol 88(3):719–726. CrossRefPubMedGoogle Scholar
  29. Smirnov SV, Sokolov PM, Kodera T, Sugiyama M, Hibi M, Shimizu S, Yokozeki K, Ogawa J (2012) A novel family of bacterial dioxygenases that catalyse the hydroxylation of free L-amino acids. FEMS Microbiol Lett 331(2):97–104. CrossRefPubMedGoogle Scholar
  30. Stark BC, Pagilla KR, Dikshit KL (2015) Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation. Appl Microbiol Biotechnol 99(4):1627–1636. CrossRefPubMedGoogle Scholar
  31. Theodosiou E, Breisch M, Julsing MK, Falcioni F, Bühler B, Schmid A (2017) An artificial TCA cycle selects for efficient α-ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli. Biotechnol Bioeng 114(7):1511–1520. CrossRefPubMedGoogle Scholar
  32. Tsai PS, Rao G, Bailey JE (1995) Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: new insights from NAD(P)H fluorescence and culture redox potential. Biotechnol Bioeng 47(3):347–354. CrossRefPubMedGoogle Scholar
  33. Wei L, Wang H, Xu N, Zhou W, Ju J, Liu J, Ma Y (2019) Metabolic engineering of Corynebacterium glutamicum for L-cysteine production. Appl Microbiol Biotechnol 103(3):1325–1338. CrossRefPubMedGoogle Scholar
  34. Xu D, Tan Y, Huan X, Hu X, Wang X (2010) Construction of a novel shuttle vector for use in Brevibacterium flavum, an industrial amino acid producer. J Microbiol Methods 80(1):86–92. CrossRefPubMedGoogle Scholar
  35. Xu M, Rao Z, Xu H, Lan C, Dou W, Zhang X, Xu H, Jin J, Xu Z (2011) Enhanced production of L-arginine by expression of Vitreoscilla hemoglobin using a novel expression system in Corynebacterium crenatum. Appl Biochem Biotechnol 163(6):707–719. CrossRefPubMedGoogle Scholar
  36. Yamamoto S, Gunji W, Suzuki H, Toda H, Suda M, Jojima T, Inui M, Yukawa H (2012) Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions. Appl Environ Microbiol 78(12):4447–4457. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Yokota A, Sawada K, Wada M (2017) Boosting anaplerotic reactions by pyruvate kinase gene deletion and phosphoenolpyruvate carboxylase desensitization for glutamic acid and lysine production in Corynebacterium glutamicum. Adv Biochem Eng Biotechnol 159:181–198. CrossRefPubMedGoogle Scholar
  38. Zafar MI, Gao F (2016) 4-Hydroxyisoleucine: a potential new treatment for type 2 diabetes mellitus. BioDrugs 30(4):255–262. CrossRefPubMedGoogle Scholar
  39. Zhang C, Li Y, Ma J, Liu Y, He J, Li Y, Zhu F, Meng J, Zhan J, Li Z, Zhao L, Ma Q, Fan X, Xu Q, Xie X, Chen N (2018a) High production of 4-hydroxyisoleucine in Corynebacterium glutamicum by multistep metabolic engineering. Metab Eng 49:287–298. CrossRefPubMedGoogle Scholar
  40. Zhang C, Ma J, Li Z, Liang Y, Xu Q, Xie X, Chen N (2018b) A strategy for L-isoleucine dioxygenase screening and 4-hydroxyisoleucine production by resting cells. Bioengineered 9(1):72–79. CrossRefPubMedGoogle Scholar
  41. Zhang HL, Zhang C, Pei CH, Han MN, Xu ZD, Li CH, Li W (2018c) Efficient production of trans-4-Hydroxy-l-proline from glucose by metabolic engineering of recombinant Escherichia coli. Lett Appl Microbiol 66(5):400–408. CrossRefPubMedGoogle Scholar
  42. Zhao TX, Li M, Zheng X, Wang CH, Zhao HX, Zhang C, Xing XH (2017) Improved production of trans-4-hydroxy-L-proline by chromosomal intergration of the Vitreoscilla hemoglobin gene into recombinant Escherichia coli with expression of proline-4-hydroxylase. J Biosci Bioeng 123(1):109–115. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina
  3. 3.International Joint Laboratory on Food SafetyJiangnan UniversityWuxiChina
  4. 4.National Engineering Laboratory for Cereal Fermentation TechnologyJiangnan UniversityWuxiChina

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