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Characterization of UGPase from Aureobasidium pullulans NRRL Y-12974 and Application in Enhanced Pullulan Production

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Abstract

UDPG pyrophosphatase (UGPase) plays an important role in carbohydrate metabolism, catalyzing a reversible production of uridine diphosphate glucose (UDPG) and pyrophosphate (PPi) from Glc-1-P and UTP. UGPase gene from Aureobasidium pullulans NRRL Y-12974 was cloned, overexpressed in Escherichia coli. The recombinant UGPase possess molecular mass of 55 KDa and specific activity of 7.33 U/mg protein. The K m values of rUGPase were 5.045 μM against UTP and 3.333 μM against Glc-1-P. The V max values of rUGPase were 3.467 μM min−1against UTP and 2.817 μM min−1 against Glc-1-P. And, it does not catalyze Glc-1-P and ATP, nor galactose-1-P and UTP. Homolgous expression of UGPase in native organism can improve the intracellular UDPG concentration by 4.7-fold time. The yield of pullulan in engineering strain A4 was improved to 18.2 g g−1 cell dry weight which is 1.3-fold time of parent strain. No obvious change of growth was found between engineering strain and parent strain. To the best of our knowledge, this is the first report of improving pullulan yield in A. pullulans using metabolic engineering technique.

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References

  1. Chi, Z., Wang, F., Chi, Z., Yue, L., Liu, G., & Zhang, T. (2009). Bioproducts from Aureobasidium pullulans, a biotechnologically important yeast. Applied and Environmental Microbiology, 82, 793–804.

    CAS  Google Scholar 

  2. Shingel, K. I. (2004). Current knowledge on biosynthesis, biological activity, and chemical modification of the exopolysaccharide, pullulan. Carbohydrate Research, 339, 447–460.

    Article  CAS  Google Scholar 

  3. Duan, X., Chi, Z., Li, H., & Gao, L. (2007). High pullulan yield is related to low UDP-glucose level and high pullulan-related synthases activity in Aureobasidium pullulans Y68. Annals of Microbiology, 57, 243–248.

    Article  CAS  Google Scholar 

  4. Catley, B. J., & McDowel, W. (1982). Lipid-linked saccharides formed during pullulan biosynthesis in Aureobasidium pullulans. Carbohydrate Research, 103, 65–75.

    Article  CAS  Google Scholar 

  5. Kleczkowski, L. A., Geisler, M., Ciereszko, I., & Johansson, H. (2004). UDP-glucose pyrophosphorylase. An old protein with new tricks. Plant Physiology, 134, 912–918.

    Article  CAS  Google Scholar 

  6. Meng, M., Geisler, M., Johansson, H., Harholt, J., Scheller, H. V., Mellerowicz, E. J., & Kleczkowski, L. A. (2009). UDP-glucose pyrophosphorylase is not rate limiting, but is essential in Arabidopsis. Plant Cell Physiology, 50, 998–1011.

    Article  CAS  Google Scholar 

  7. Daran, J. M., Dallies, N., Thines-Sempoux, D., Paquet, V., & Francois, J. (1995). Genetic and biochemical characterization of the UGP1 gene encoding the UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae. European Journal of Biochemistry, 233, 520–530.

    Article  CAS  Google Scholar 

  8. Yang, J., & Guo, L. (2014). Biosynthesis of beta-carotene in engineered E. coli using the MEP and MVA pathways. Microbial Cell Factories, 13, 160–170.

    Article  Google Scholar 

  9. Zhu, Z., Zhang, S., Liu, H., Shen, H., Lin, X., Yang, F., Zhou, Y. J., Jin, G., Ye, M., Zou, H., & Zhao, Z. K. (2012). A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nature Communications, 3, 1112–1123.

    Article  Google Scholar 

  10. Duan, X., Chi, Z., Wang, L., & Wang, X. (2008). Influence of different sugars on pullulan production and activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68. Carbohydrate Polymers, 73, 587–593.

    Article  CAS  Google Scholar 

  11. Chi, Z., Wang, X. X., Ma, Z. C., Buzdar, M. A., & Chi, Z. M. (2012). The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2. Biometals, 25, 219–230.

    Article  CAS  Google Scholar 

  12. Finkelman, M. A. J., & Vardanis, A. (1987). Synthesis of β-glucan by cell-free extracts of Aureobasidium pullulans. Canadian Journal of Microbiology, 33, 123–127.

    Article  CAS  Google Scholar 

  13. Strominger, J. L., Elizabeth, S. M., & Kalckar, H. M. (1957). Determination of UDPG and UTP by means of UDPG dehydrogenase. Methods in Enzymology, 3, 974–977.

    Article  Google Scholar 

  14. Dallies, N., Francois, J., & Paquet, V. (1998). A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast, 14, 1297–1306.

    Article  CAS  Google Scholar 

  15. Weissborn, A. C., Qingyun, L., Rumley, M. K., & Kennedy, E. P. (1994). UTP: co-D-glucose-l-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme. Journal of Bacteriology, 176, 2611–2618.

    CAS  Google Scholar 

  16. Annette, R., Plitzko, J. M., Roman, K., Böttcher, U. M. K., Katja, S., Manajit, H.-H., & Andreas, B. (2006). Structural basis for subunit assembly in UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae. Journal of Molecular Biology, 364, 551–560.

    Article  Google Scholar 

  17. Valla, S., Coucheron, D. H., Fjaervik, E., Kjosbakken, J., Weinhouse, H., Ross, P., Amikam, D., & Benziman, M. (1989). Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene. Molecular and General Genetics, 217, 26–30.

    Article  CAS  Google Scholar 

  18. Li, N., Wang, L., Zhang, W., Takechi, K., Takano, H., & Lin, X. (2014). Overexpression of UDP-glucose pyrophosphorylase from Larix gmelinii enhances vegetative growth in transgenic Arabidopsis thaliana. Plant Cell Reports, 33, 779–791.

    Article  CAS  Google Scholar 

  19. Coleman, H., Ellis, D., Gilbert, M., Mansfield, S.D. (2003). Tree Biotechnology, Increased growth and yield by altered carbohydrate allocation. In: B Sundberg (ed), Umeå, Sweden. pp S10.41.

  20. Zhang, G., Qi, J., Xu, J., Niu, X., Zhang, Y., Tao, A., Zhang, L., Fang, P., & Lin, L. (2013). Overexpression of UDP-glucose pyrophosphorylase gene could increase cellulose content in jute (Corchorus capsularis L.). Biochemical and Biophysical Research Communications, 442, 153–158.

    Article  CAS  Google Scholar 

  21. Inoue, S. B., Qadota, H., Arisawa, M., Watanabe, T., & Ohya, Y. (1999). Prenylation of Rho1p is required for activation of yeast 1, 3-beta-glucan synthase. Journal of Biological Chemistry, 274, 38119–38124.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was financially supported by the Natural Scientific Foundation of Zhejiang Province, China (grant number: Y15B060012), Administration of Technology Division of Hangzhou, China, (grant number: KH10365).

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Authors and Affiliations

  1. College of Medicine, Hangzhou Normal University, No.1378 Wenyi Xi Road, Hangzhou, 311121, Zhejiang, People’s Republic of China

    Haifeng Li, Yunyun Gao, Yuanyang Lan & Lifeng Huang

  2. Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, 310051, Zhejiang, People’s Republic of China

    Yunyi Zhang

  3. College of Chemistry and Materials Science, Hangzhou Normal University, Hangzhou, 311121, Zhejiang, People’s Republic of China

    Xiaopu Yin

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  1. Haifeng Li
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  2. Yunyi Zhang
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  3. Yunyun Gao
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  5. Xiaopu Yin
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Correspondence to Haifeng Li.

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Li, H., Zhang, Y., Gao, Y. et al. Characterization of UGPase from Aureobasidium pullulans NRRL Y-12974 and Application in Enhanced Pullulan Production. Appl Biochem Biotechnol 178, 1141–1153 (2016). https://doi.org/10.1007/s12010-015-1934-2

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  • DOI: https://doi.org/10.1007/s12010-015-1934-2

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