Skip to main content

Advertisement

SpringerLink
Log in

A Procedure to Design One-Pot Multi-enzyme System for Industrial CDP-Choline Production

  • Original Article
  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Fermentation and chemical methods for industrial cytidine diphosphate choline (CDP-choline) catalytic production both suffer from several disadvantages such as relatively low efficiency and productivity. To overcome these problems, we applied the concept of synthetic biology to develop a new one-pot multi-enzyme system to produce CDP-choline from orotic acid. Enzymes from different sources were selected and optimized as building blocks of the system, and parameters such as oxygen supply were also optimized. This system shows a titer of 37.6 ± 1.1 mM and a reaction rate of 1.6 mM L-1 h−1, both increase 66 % from traditional processes. It also has an efficiency of energy of 25.4%, improves 2-folds. This new one-pot CDP-choline-producing system has a potential for industrial use, and the procedure to design one-pot multi-enzyme system can be applied to build other one-pot system producing energy-rich compounds.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data Availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

G-6-P :

Glucose 6-phosphate

PRPP:

Phosphoribosyl pyrophosphate

OMP:

Orotidine 5′-monophosphate

UMP:

Uridine 5′-monophosphate

UDP:

Uridine 5′-diphosphate

UTP:

Uridine 5′-triphosphate

CTP:

Cytidine 5′-triphosphate

OPRT:

Orotate phosphoribosyltransferase

ODC:

Orotidine-5′-phosphate decarboxylase

UMK:

UMP kinase

NDK:

NDP kinase

CTPS:

CTP synthetase

CKI:

Choline kinase

CCT:

Choline-phosphate cytidylyltransferase

EMP pathway:

Embden-Meyerhof-Parnas pathway

PP pathway:

Pentose phosphate pathway

References

  1. Akira, K., Makoto, M., & Tatsurokuro, T. (1971). Fermentative production of CDP-choline by yeasts: part III. Some additional results on the production of CDP-choline byBrewer’s yeast. Agr BioI Chem., 35(12), 1955–1960.

    Google Scholar 

  2. Bornscheuer, U. T., Huisman, G. W., Kazlauskas, R. J., Lutz, S., Moore, J. C., & Robins, K. (2012). Engineering the third wave of biocatalysis. Nature., 485(7397), 185–194.

    Article  CAS  Google Scholar 

  3. Calderone, T. L., Stevens, R. D., & Oas, T. G. (1996). High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli. J Mol Biol., 262(4), 407–412.

    Article  CAS  Google Scholar 

  4. Guzman, L. M., Belin, D., Carson, M. J., & Beckwith, J. O. N. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol., 177(14), 4121–4130.

    Article  CAS  Google Scholar 

  5. Hannig, G., & Makrides, S. C. (1998). Strategies for optimizing heterologous protein expression in Escherichia coli. Trends in Biotechnology., 16(2), 54–60.

    Article  CAS  Google Scholar 

  6. Hosaka, K., Kodaki, T., & Yamashita, S. (1989). Cloning and characterization of the yeast CKI gene encoding choline kinase and its expression in Escherichia coli. J BiolChem., 264(4), 2053–2059.

    CAS  Google Scholar 

  7. Huisman, G. W., Liang, J., & Krebber, A. (2010). Practical chiral alcohol manufacture using ketoreductases. Curr. Opin. Chem. Biol., 14(2), 122–129.

    Article  CAS  Google Scholar 

  8. II’chenko, A. P., Shishkanova, N. V., Chernyavskaya, O. G., & Finogenova, T. V. (1998). Oxygen concentration as a factor controlling central metabolism and citric acid biosynthesis in the yeast Yarrowia lipolytica grown on ethanol. Microbiology., 67, 241–244.

    Google Scholar 

  9. Kane, J. F. (1996). Effects of rare codon clusters on high-level expression ofheterologousproteins in Escherichia coli. Curr. Opin. Biotech., 6, 494–500.

    Article  Google Scholar 

  10. Kim, S. Y., Kim, J. H., & Oh, D. K. (1997). Improvement of xylitol production by controlling oxygen supply. In Candida parapsilosis.JFermentBioeng (Vol. 83, pp. 267–270).

    Google Scholar 

  11. Kimura, A., Morita, M., & Tochikura, T. (1971). Fermentative production of CDP-choline byyeasts (III) some additional results on the production of CDP-choline by brewer's yeast. Agric. Biol. Chem., 35(12), 1955–1960.

    CAS  Google Scholar 

  12. Kurland, C., & Gallant, J. (1996). Errors of heterologous protein expression. Curr. Opin. Biotech., 7(5), 489–493.

    Article  CAS  Google Scholar 

  13. Fujio, T., & Maruyama, A. (1997). Enzymatic production of pyrimidine nucleotides using Corynebacterium ammoniagenes cells and recombinant Escherichia coli cells: enzymatic production of CDP-choline from orotic acid and choline chloride (part I). Biosci. Biotechnol. Biochem., 61(6), 956–959.

    Article  CAS  Google Scholar 

  14. Liu, Y., Wang, J., Xu, C., Chen, Y., Yang, J., Liu, D., Niu, H., Jiang, Y., Yang, S., & Ying, H. (2017). Efficient multi-enzyme-catalyzed CDP-choline production driven by an ATP donor module. Applied microbiology and biotechnology, 101(4), 1409–1417.

    Article  CAS  Google Scholar 

  15. MIkio, Honjo, Y. F. (1968). Method for production of cytidine (or deoxycytidine)-5’-diphosphate choline and intermediates therefor. US Patent 3666748.

  16. Miyake, H., Hayakawa, I., & Takakura, K. (1964). Treatment of head injuries with intermediate substances of the metabolic cycle of the brain. 1. The use of CDP-choline. No to shinkei, 16, 873–878.

    CAS  PubMed  Google Scholar 

  17. Rajiv, K., Yogendra, C., Vilas, D., Vijay, T., & Atul, W. A process for preparing pure citicoline (CDP-choline). WO, 2013/128393.

  18. Rick, W. Y., Tao, W., Bedzyk, L., Young, T., Chen, M., & Li, L. (2000). Global gene expressionprofiles of Bacillus subtilis grown under anaerobic conditions. JBacteriol, 182, 4458–4465.

    Google Scholar 

  19. Sorensen, H. P., & Mortensen, K. K. (2005). Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol., 115(2), 113–128.

    Article  CAS  Google Scholar 

  20. Serrano, L. (2007). Synthetic biology: promises and challenges. Molecular systems biology., 3(1), 158.

    Article  Google Scholar 

  21. Tang, J., Yao, Y., Ying, H., Xiong, J., Zhang, L., Li, Z., Bai, J., Zhang, Y., & Ouyang, P. (2009). Effect of NH4+ and glycerol on cytidine 5’-diphosphocholine synthesis in Saccharomyces cerevisiae. Bioresoure Technol., 100(20), 4848–4853.

    Article  CAS  Google Scholar 

  22. Tanakamaru, S. (1964). Experimental and clinical studies on CDP-choline therapy for brain injuries. Kumamoto Igakkai zasshi., 38, 45–176.

    Google Scholar 

  23. Tsuchida, T., Nagai, M., Hoshino, T., Kamano, S., & Miyake, H. (1967). Treatment of head injuries with intermediate substances in the metabolic cycle of the brain. 2. Basicstudy on the metabolism of cytidine diphosphate choline. No to shinkei., 19(10), 1041–1045.

    CAS  PubMed  Google Scholar 

  24. Tsai, P. S., Hatzimanikatis, V., & Bailey, J. E. (1996). Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism. Biotechnol Bioeng, 49(2), 139–150.

    Article  CAS  Google Scholar 

  25. Watanabe, S., Kitajima, N., Shirota, S., & Takeda, I. (1981). Effects of several factors on CDP-choline production by yeast. J.Ferment.Technol, 59, 197–201.

    CAS  Google Scholar 

  26. Watanabe, S., Shirota, S., Haneda, K., & Takeda, I. (1981). Effects of cultural conditions on CDP-choline production by hydrocarbon assimilating yeasts. J Ferment Technol., 59, 191–195.

    CAS  Google Scholar 

  27. Wildeman, S. M. A. D., Sonke, T., Schoemaker, H. E., & May, O. (2007). Biocatalytic reductions: from lab curiosity to “first choice”. Acc. Chem. Res., 40(12), 1260–1266.

    Article  Google Scholar 

  28. Xu, H., Dou, W., Xu, H., Zhang, X., Rao, Z., Shi, Z., & Xu, Z. (2009). A two-stage oxygen supply strategy for enhanced L-arginine production by Corynebacterium crenatumbased on metabolic fluxes analysis. Biochem Eng J., 43(1), 41–51.

    Article  CAS  Google Scholar 

  29. Yu, W. B., Gao, S. H., Yin, C. Y., Zhou, Y., & Ye, B. C. (2011). Comparative transcriptome analysisof Bacillus subtilis responding to dissolved oxygen in adenosine fermentation. PLoS One, 6, e20092.

    Article  CAS  Google Scholar 

  30. Zhang, Y. H. P. (2015). Production of biofuels and biochemicals by in vitro synthetic biosystems: opportunities and challenges. Biotechnology advances, 33(7), 1467–1483.

    Article  CAS  Google Scholar 

Download references

Code Availability

Not applicable.

Funding

This work was supported by the National High-Tech Research and Development Program of China (863) (2012AA021203), the National Basic Research Program of China (973) (2013CB733602), the Major Research Plan of the National Natural Science Foundation of China (21390204), the National Technology Support Program (2012BAI44G01), the National Natural Science Foundation of China, General Program (2137611), the Program for Changjiang Scholars and Innovative Research Team in university (IRT_14R28), the young investigator grant program of National Natural Science Foundation of China (21506097), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Author information

Author notes

  1. Cheng Zheng and Rongxin Miao contributed equally to this work.

Authors and Affiliations

  1. College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, 211816, Nanjing, China

    Cheng Zheng, Yingmiao Liu, Yang Cao, Dong Liu, Junzhi Wang & Hanjie Ying

  2. State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, 210009, Nanjing, China

    Cheng Zheng, Yingmiao Liu, Yang Cao, Dong Liu, Junzhi Wang & Hanjie Ying

  3. School of Life Sciences, Zhengzhou University, Zhengzhou, China

    Rongxin Miao

  4. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China

    Yingmiao Liu

  5. Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, No. 30, Puzhu South Road, 211816, Nanjing, China

    Dong Liu, Junzhi Wang & Hanjie Ying

Authors
  1. Cheng Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rongxin Miao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yingmiao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yang Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dong Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Junzhi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hanjie Ying
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Chen Zheng, Rongxin Miao,Yingmiao Liu, Yang Cao, Dong Liu, Junzhi Wang, and Hanjie Ying .The manuscript was written by Chen Zheng and Rongxin Miao. All authors commented on previous versions of the manuscript. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Junzhi Wang or Hanjie Ying.

Ethics declarations

Competing Interests

The authors declare no competing interests.

Ethical Approval

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

Consent to Participate

Not applicable.

Consent to Publish

Not applicable.

Additional information

Publisher’s Note

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

Supplementary Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zheng, C., Miao, R., Liu, Y. et al. A Procedure to Design One-Pot Multi-enzyme System for Industrial CDP-Choline Production. Appl Biochem Biotechnol 193, 2769–2780 (2021). https://doi.org/10.1007/s12010-021-03564-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-021-03564-2

Keywords

Search

Navigation