Skip to main content
SpringerLink
Log in

Advanced Whole-cell Conversion for D-allulose Production Using an Engineered Corynebacterium glutamicum

  • Research Paper
  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

D-allulose has received considerable attention as an alternative functional sugar for its zero caloric value with 70% relative sweetness compared to D-sucrose. Despite its strong potential as an alternative sweetener, recent industrial productions rely on a high-cost enzymatic method. Here, we advanced whole-cell conversion at high temperatures using Corynebacterium glutamicum expressing D-allulose 3-epimerase (DAE). By varying the reaction temperature from 25°C to 70°C, D-allulose conversion could reach the reaction equilibrium at high temperatures. The C. glutamicum showed superior reusability of cells at 60°C compared to Escherichia coli. We simplified the cell growth media and whole-cell conversion reaction solution. Clostridium hylemonae DAE (ChDAE) showed the highest thermostability and reusability among various DAE candidates. Finally, the ChDAE expression under the synthetic 2X-cT-T5 promoter could reduce the reaction time by 25%. Our result showed that 120 g/L of D-allulose can be produced from 400 g/L of D-fructose by reusable whole-cell conversion at 55°C in 1.5 h. This study can be highly applicable in industrial economic production.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Binkley, W. W. (1963) The fate of cane juice simple sugars during molasses formation. IV. Probable conversion of D-fructose to D-psicose. Int. Sugar J. 65: 105–106.

    CAS  Google Scholar 

  2. Miller, B. S. and T. Swain (1960) Chromatographic analyses of the free amino-acids, organic acids and sugars in wheat plant extracts. J. Sci. Food Agric. 11: 344–348.

    Article  CAS  Google Scholar 

  3. Hough, L. and B. E. Stacey (1963) The occurrence of D-ribohexulose in Itea ilicifolialtea virginica, and Itea yunnanensis. Phytochemistry. 2: 315–320.

    Article  CAS  Google Scholar 

  4. Chung, M. Y., D. K. Oh, and K. W. Lee (2012) Hypoglycemic health benefits of D-psicose. J. Agric. Food Chem. 60: 863–869.

    Article  CAS  PubMed  Google Scholar 

  5. Matsuo, T., T. Tanaka, M. Hashiguchi, K. Izumori, and H. Suzuki (2003) Metabolic effects of D-psicose in rats: studies on faecal and urinary excretion and caecal fermentation. Asia Pac. J. Clin. Nutr. 12: 225–231.

    CAS  PubMed  Google Scholar 

  6. Tiefenbacher, K. F. (2017) The Technology of Wafers and Waffles: Vol. 1. Operational Aspects. Elsevier, Amsterdam, Netherlands.

    Google Scholar 

  7. Lê, K. A., F. Robin, and O. Roger (2016) Sugar replacers: from technological challenges to consequences on health. Curr. Opin. Clin. Nutr. Metab. Care. 19: 310–315.

    Article  PubMed  CAS  Google Scholar 

  8. Nagata, Y., A. Kanasaki, S. Tamaru, and K. Tanaka (2015) D-psicose, an epimer of D-fructose, favorably alters lipid metabolism in Sprague-Dawley rats. J. Agric. Food Chem. 63: 3168–3176.

    Article  CAS  PubMed  Google Scholar 

  9. Hossain, A., F. Yamaguchi, T. Matsuo, I. Tsukamoto, Y. Toyoda, M. Ogawa, Y. Nagata, and M. Tokuda (2015) Rare sugar D-allulose: potential role and therapeutic monitoring in maintaining obesity and type 2 diabetes mellitus. Pharmacol. Ther. 155: 49–59.

    Article  CAS  PubMed  Google Scholar 

  10. Hossain, M. A., S. Kitagaki, D. Nakano, A. Nishiyama, Y. Funamoto, T. Matsunaga, I. Tsukamoto, F. Yamaguchi, K. Kamitori, Y. Dong, Y. Hirata, K. Murao, Y. Toyoda, and M. Tokuda (2011) Rare sugar D-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Biochem. Biophys. Res. Commun. 405: 7–12.

    Article  CAS  PubMed  Google Scholar 

  11. Hossain, A., F. Yamaguchi, T. Matsunaga, Y. Hirata, K. Kamitori, Y. Dong, L. Sui, I. Tsukamoto, M. Ueno, and M. Tokuda (2012) Rare sugar D-psicose protects pancreas β-islets and thus improves insulin resistance in OLETF rats. Biochem. Biophys. Res. Commun. 425: 717–723.

    Article  CAS  PubMed  Google Scholar 

  12. Matsuo, T. and K. Izumori (2006) Effects of dietary D-psicose on diurnal variation in plasma glucose and insulin concentrations of rats. Biosci. Biotechnol. Biochem. 70: 2081–2085.

    Article  CAS  PubMed  Google Scholar 

  13. Iida, T., Y. Kishimoto, Y. Yoshikawa, N. Hayashi, K. Okuma, M. Tohi, K. Yagi, T. Matsuo, and K. Izumori (2008) Acute D-psicose administration decreases the glycemic responses to an oral maltodextrin tolerance test in normal adults. J. Nutr. Sci. Vitaminol. (Tokyo). 54: 511–514.

    Article  CAS  PubMed  Google Scholar 

  14. Hayashi, N., T. Iida, T. Yamada, K. Okuma, I. Takehara, T. Yamamoto, K. Yamada, and M. Tokuda (2010) Study on the postprandial blood glucose suppression effect of D-psicose in borderline diabetes and the safety of long-term ingestion by normal human subjects. Biosci. Biotechnol. Biochem. 74: 510–519.

    Article  CAS  PubMed  Google Scholar 

  15. Izumori, K., A. R. Khan, H. Okaya, and T. Tsumura (1993) A new enzyme, D-ketohexose 3-epimerase, from Pseudomonas sp. ST-24. Biosci. Biotechnol. Biochem. 57: 1037–1039.

    Article  CAS  Google Scholar 

  16. Yoshida, H., M. Yamada, T. Nishitani, G. Takada, K. Izumori, and S. Kamitori (2007) Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose. J. Mol. Biol. 374: 443–453.

    Article  CAS  PubMed  Google Scholar 

  17. Zhang, L., W. Mu, B. Jiang, and T. Zhang (2009) Characterization of D-tagatose-3-epimerase from Rhodobacter sphaeroides that converts D-fructose into D-psicose. Biotechnol. Lett. 31: 857–862.

    Article  CAS  PubMed  Google Scholar 

  18. Zhu, Z., C. Li, X. Liu, D. Gao, X. Wang, M. Tanokura, H. M. Qin, and F. Lu (2019) Biochemical characterization and biocatalytic application of a novel D-tagatose 3-epimerase from Sinorhizobium sp. RSC Adv. 9: 2919–2927.

    Article  CAS  Google Scholar 

  19. Tseng, W. C., C. N. Chen, C. T. Hsu, H. C. Lee, H. Y. Fang, M. J. Wang, Y. H. Wu, and T. Y. Fang (2018) Characterization of a recombinant d-allulose 3-epimerase from Agrobacterium sp. ATCC 31749 and identification of an important interfacial residue. Int. J. Biol. Macromol. 112: 767–774.

    Article  CAS  PubMed  Google Scholar 

  20. Kim, H. J., E. K. Hyun, Y. S. Kim, Y. J. Lee, and D. K. Oh (2006) Characterization of an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to D-psicose. Appl. Environ. Microbiol. 72: 981–985.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yoshihara, A., T. Kozakai, T. Shintani, R. Matsutani, K. Ohtani, T. Iida, K. Akimitsu, K. Izumori, and P. K. Gullapalli (2017) Purification and characterization of d-allulose 3-epimerase derived from Arthrobacter globiformis M30, a GRAS microorganism. J. Biosci. Bioeng. 123: 170–176.

    Article  CAS  PubMed  Google Scholar 

  22. Jia, M., W. Mu, F. Chu, X. Zhang, B. Jiang, L. L. Zhou, and T. Zhang (2014) A D-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for D-psicose production: cloning, expression, purification, and characterization. Appl. Microbiol. Biotechnol. 98: 717–725.

    Article  CAS  PubMed  Google Scholar 

  23. Mu, W., F. Chu, Q. Xing, S. Yu, L. Zhou, and B. Jiang (2011) Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10. J. Agric. Food Chem. 59: 7785–7792. (Erratum published 2013, J. Agric. Food Chem. 61: 10408)

    Article  CAS  PubMed  Google Scholar 

  24. Zhang, W., D. Fang, Q. Xing, L. Zhou, B. Jiang, and W. Mu (2013) Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704. PLoS One. 8: e62987. (Erratum published 2014, PLoS One 9: https://doi.org/10.1371/annotation/4bc8d881-0bee-4ccd-8c1a-f73833589134)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mu, W., W. Zhang, D. Fang, L. Zhou, B. Jiang, and T. Zhang (2013) Characterization of a D-psicose-producing enzyme, D-psicose 3-epimerase, from Clostridium sp. Biotechnol. Lett. 35: 1481–1486.

    Article  CAS  PubMed  Google Scholar 

  26. Zhang, W., D. Fang, T. Zhang, L. Zhou, B. Jiang, and W. Mu (2013) Characterization of a metal-dependent D-psicose 3-epimerase from a novel strain, Desmospora sp. 8437. J. Agric. Food Chem. 61: 11468–11476.

    Article  CAS  PubMed  Google Scholar 

  27. Zhang, W., H. Li, T. Zhang, B. Jiang, L. Zhou, and W. Mu (2015) Characterization of a d-psicose 3-epimerase from Dorea sp. CAG317 with an acidic pH optimum and a high specific activity. J. Mol. Catal. B Enzym. 120: 68–74.

    Article  CAS  Google Scholar 

  28. Park, C. S., T. Kim, S. H. Hong, K. C. Shin, K. R. Kim, and D. K. Oh (2016) D-allulose production from D-fructose by permeabilized recombinant cells of Corynebacterium glutamicum cells expressing D-allulose 3-epimerase Flavonifractor plautii. PLoS One. 11: e0160044.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Yang, J., C. Tian, T. Zhang, C. Ren, Y. Zhu, Y. Zeng, Y. Men, Y. Sun, and Y. Ma (2019) Development of food-grade expression system for d-allulose 3-epimerase preparation with tandem isoenzyme genes in Corynebacterium glutamicum and its application in conversion of cane molasses to D-allulose. Biotechnol. Bioeng. 116: 745–756.

    Article  CAS  PubMed  Google Scholar 

  30. Zhu, Y., Y. Men, W. Bai, X. Li, L. Zhang, Y. Sun, and Y. Ma (2012) Overexpression of D-psicose 3-epimerase from Ruminococcus sp. in Escherichia coli and its potential application in D-psicose production. Biotechnol. Lett. 34: 1901–1906.

    Article  PubMed  Google Scholar 

  31. Zhu, Z., D. Gao, C. Li, Y. Chen, M. Zhu, X. Liu, M. Tanokura, H. M. Qin, and F. Lu (2019) Redesign of a novel D-allulose 3-epimerase from Staphylococcus aureus for thermostability and efficient biocatalytic production of D-allulose. Microb. Cell Fact. 18: 59.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Zhang, W., T. Zhang, B. Jiang, and W. Mu (2016) Biochemical characterization of a D-psicose 3-epimerase from Treponema primitia ZAS-1 and its application on enzymatic production of D-psicose. J. Sci. Food Agric. 96: 49–56.

    Article  CAS  PubMed  Google Scholar 

  33. Itoh, H., T. Sato, and K. Izumori (1995) Preparation of d-psicose from d-fructose by immobilized d-tagatose 3-epimerase. J. Ferment. Bioeng. 80: 101–103.

    Article  CAS  Google Scholar 

  34. Takeshita, K., A. Suga, G. Takada, and K. Izumori (2000) Mass production of D-psicose from d-fructose by a continuous bioreactor system using immobilized D-tagatose 3-epimerase. J. Biosci. Bioeng. 90: 453–455.

    Article  CAS  PubMed  Google Scholar 

  35. Oh, D. K., H. J. Kim, Y. J. Lee, S. H. Song, S. W. Park, J. H. Kim, and S. B. Kim (2011) D-psicose production method by D-psicose epimerase. US Patent 8,030,035 B2.

  36. Lim, B. C., H. J. Kim, and D. K. Oh (2009) A stable immobilized d-psicose 3-epimerase for the production of d-psicose in the presence of borate. Process Biochem. 44: 822–828.

    Article  CAS  Google Scholar 

  37. Choi, J. G., Y. H. Ju, S. J. Yeom, and D. K. Oh (2011) Improvement in the thermostability of D-psicose 3-epimerase from Agrobacterium tumefaciens by random and site-directed mutagenesis. Appl. Environ. Microbiol. 77: 7316–7320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Narayan Patel, S., V. Singh, M. Sharma, R. S. Sangwan, N. K. Singhal, and S. P. Singh (2018) Development of a thermo-stable and recyclable magnetic nanobiocatalyst for bioprocessing of fruit processing residues and D-allulose synthesis. Bioresour. Technol. 247: 633–639.

    Article  CAS  PubMed  Google Scholar 

  39. Ran, G., D. Tan, J. Zhao, F. Fan, Q. Zhang, X. Wu, P. Fan, X. Fang, and X. Lu (2019) Functionalized polyhydroxyalkanoate nano-beads as a stable biocatalyst for cost-effective production of the rare sugar d-allulose. Bioresour. Technol. 289: 121673.

    Article  CAS  PubMed  Google Scholar 

  40. Tseng, C. W., C. Y. Liao, Y. Sun, C. C. Peng, J. T. Tzen, R. T. Guo, and J. R. Liu (2014) Immobilization of Clostridium cellulolyticum D-psicose 3-epimerase on artificial oil bodies. J. Agric. Food Chem. 62: 6771–6776.

    Article  CAS  PubMed  Google Scholar 

  41. Li, Z., Y. Li, S. Duan, J. Liu, P. Yuan, H. Nakanishi, and X. D. Gao (2015) Bioconversion of D-glucose to D-psicose with immobilized D-xylose isomerase and D-psicose 3-epimerase on Saccharomyces cerevisiae spores. J. Ind. Microbiol. Biotechnol. 42: 1117–1128.

    Article  CAS  PubMed  Google Scholar 

  42. Park, C. S., C. S. Park, K. C. Shin, and D. K. Oh (2016) Production of d-psicose from d-fructose by whole recombinant cells with high-level expression of d-psicose 3-epimerase from Agrobacterium tumefaciens. J. Biosci. Bioeng. 121: 186–190.

    Article  CAS  PubMed  Google Scholar 

  43. Woodward, J. (1985) Immobilised Cells and Enzymes: A Practical Approach. IRL Press, Oxford, UK.

    Google Scholar 

  44. Seo, M. J., K. C. Shin, and D. K. Oh (2014) Production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by whole recombinant Escherichia coli cells expressing diol synthase from Aspergillus nidulans. Appl. Microbiol. Biotechnol. 98: 7447–7456.

    Article  CAS  PubMed  Google Scholar 

  45. He, W., W. Mu, B. Jiang, X. Yan, and T. Zhang (2016) Construction of a food grade recombinant Bacillus subtilis based on replicative plasmids with an auxotrophic marker for biotransformation of d-fructose to d-allulose. J. Agric. Food Chem. 64: 3243–3250.

    Article  CAS  PubMed  Google Scholar 

  46. Yang, P., X. Zhu, Z. Zheng, D. Mu, S. Jiang, S. Luo, Y. Wu, and M. Du (2018) Cell regeneration and cyclic catalysis of engineered Kluyveromyces marxianus of a D-psicose-3-epimerase gene from Agrobacterium tumefaciens for D-allulose production. World J. Microbiol. Biotechnol. 34: 65.

    Article  PubMed  CAS  Google Scholar 

  47. Park, S. H., H. U. Kim, T. Y. Kim, J. S. Park, S. S. Kim, and S. Y. Lee (2014) Metabolic engineering of Corynebacterium glutamicum for L-arginine production. Nat. Commun. 5: 4618.

    Article  CAS  PubMed  Google Scholar 

  48. Jojima, T., M. Fujii, E. Mori, M. Inui, and H. Yukawa (2010) Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid L-alanine under oxygen deprivation. Appl. Microbiol. Biotechnol. 87: 159–165.

    Article  CAS  PubMed  Google Scholar 

  49. Ault, A. (2004) The monosodium glutamate story: the commercial production of MSG and other amino acids. J. Chem. Educ. 81: 347.

    Article  CAS  Google Scholar 

  50. Becker, J., O. Zelder, S. Häfner, H. Schröder, and C. Wittmann (2011) From zero to hero—design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metab. Eng. 13: 159–168.

    Article  CAS  PubMed  Google Scholar 

  51. Heider, S. A. and V. F. Wendisch (2015) Engineering microbial cell factories: metabolic engineering of Corynebacterium glutamicum with a focus on non-natural products. Biotechnol. J. 10: 1170–1184.

    Article  CAS  PubMed  Google Scholar 

  52. Bayan, N., C. Houssin, M. Chami, and G. Leblon (2003) Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications. J. Biotechnol. 104: 55–67.

    Article  CAS  PubMed  Google Scholar 

  53. Jeong, K. J., M. J. Seo, B. L. Iverson, and G. Georgiou (2007) APEx 2-hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering. Proc. Natl. Acad. Sci. U.S.A. 104: 8247–8252.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Yim, S. S., S. J. An, M. Kang, J. Lee, and K. J. Jeong (2013) Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum. Biotechnol. Bioeng. 110: 2959–2969.

    Article  CAS  PubMed  Google Scholar 

  55. Iida, T., N. Hayashi, T. Yamada, Y. Yoshikawa, S. Miyazato, Y. Kishimoto, K. Okuma, M. Tokuda, and K. Izumori (2010) Failure of d-psicose absorbed in the small intestine to metabolize into energy and its low large intestinal fermentability in humans. Metabolism. 59: 206–214.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021R1A5A8029490) and the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01566401), Rural Development Administration, Republic of Korea.

Author information

Authors and Affiliations

  1. Division of Applied Life Science (BK21 Four), ABC-RLRC, PMBBRC, Gyeongsang National University, Jinju, 52828, Korea

    Seong-Hee Jeong, Moonhyuk Kwon & Seon-Won Kim

Authors
  1. Seong-Hee Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Moonhyuk Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Seon-Won Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Moonhyuk Kwon or Seon-Won Kim.

Ethics declarations

The authors declare no conflict of interest.

Neither ethical approval nor informed consent was required for this study.

Additional information

Publisher’s Note

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

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jeong, SH., Kwon, M. & Kim, SW. Advanced Whole-cell Conversion for D-allulose Production Using an Engineered Corynebacterium glutamicum. Biotechnol Bioproc E 27, 276–285 (2022). https://doi.org/10.1007/s12257-022-0057-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-022-0057-1

Keywords

Search

Navigation