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Optimization of the enzymatic one pot reaction for the synthesis of uridine 5′-diphosphogalactose

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Abstract

Five recombinant Escherichia coli extracts harboring overexpressed galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophophorylase, UMP kinase, and acetate kinase (AK) were utilized for the production of UDP-galactose (UDP-Gal). We analyzed the parameters which limit the yield of UDP-Gal in the reaction, and the reaction was optimized by increasing the concentration of AK. AK was used for the ATP regeneration as well as the conversion of UDP to UTP. The activities of four overexpressed enzymes were identically fixed, and then we increased the activity of AK to 20 times higher than others. The extracts catalyzed the production of UDP-Gal from UMP (10 mM), galactose (12 mM), ATP (1 mM), and acetyl phosphate (40 mM). As the result of the reaction, the conversion yield of UDP-Gal reached to 95% from 10 mM UMP.

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Abbreviations

Acetyl Pi:

Acetyl phosphate

ADP:

Adenosine 5′-diphosphate

ATP:

Adenosine 5′-triphosphate

CMP-NeuAc:

Cytidine 5′-monophosphate

dNTP:

Deoxynucleoside-triphosphate

E. coli :

Escherichia coli

Gal:

Galactose

Glc:

Glucose

Pi:

Phosphate

PPi:

Pyrophosphate

UDP:

Uridine 5′-diphosphate

UDP-Gal:

Uridine 5′-diposphogalactose

UDP-Glc:

Uridine 5′-diphosphoglucose

UMP:

Uridine 5′-monophosphate

UTP:

Uridine 5′-triphosphate

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Acknowledgments

This research was supported by a grant (M10417060004-04N1706-00410) from Korea Biotech R&D Group of Next-generation growth engine project of the Ministry of Education, Science and Technology, Republic of Korea and the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. R0A-2007-000-10007-0).

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Authors and Affiliations

  1. Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, 151-742, South Korea

    Jae-Hun Lee, Hwa-Jin Lee, Kyuong-Soon Jang & Byung-Gee Kim

  2. School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742, South Korea

    Seung-Wook Chung & Byung-Gee Kim

  3. Department of Chemical and Biochemical Engineering, Pusan National University, Busan, 609-735, South Korea

    Sun-Gu Lee

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  1. Jae-Hun Lee
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  2. Seung-Wook Chung
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  3. Hwa-Jin Lee
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  4. Kyuong-Soon Jang
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  5. Sun-Gu Lee
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Correspondence to Byung-Gee Kim.

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Lee, JH., Chung, SW., Lee, HJ. et al. Optimization of the enzymatic one pot reaction for the synthesis of uridine 5′-diphosphogalactose. Bioprocess Biosyst Eng 33, 71–78 (2010). https://doi.org/10.1007/s00449-009-0365-2

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