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Influence of Altered NADH Metabolic Pathway on the Respiratory-deficient Mutant of Rhizopus oryzae and its L-lactate Production

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Abstract

Respiratory-deficient mutants of Rhizopus oryzae (R. oryzae) AS 3.3461 were acquired by ultraviolet (UV) irradiation to investigate changes in intracellular NADH metabolic pathway and its influence on the fermentation characteristics of the strain. Compared with R. oryzae AS 3.3461, the intracellular ATP level of the respiratory-deficient strain UV-1 decreased by 52.7 % and the glucose utilization rate rose by 8.9 %; When incubated for 36 h, the activities of phosphofructokinase (PFK), hexokinase (HK), and pyruvate kinase (PK) in the mutant rose by 74.2, 7.2, and 12.0 %, respectively; when incubated for 48 h, the intracellular NADH/NAD+ ratio of the mutant rose by 14.6 %; when a mixed carbon source with a glucose/gluconic acid ratio of 1:1 was substituted to culture the mutant, the NADH/NAD+ ratio decreased by 4.6 %; the ATP content dropped by 27.6 %; the lactate dehydrogenase (LDH) activity rose by 22.7 %; and the lactate yield rose by 11.6 %. These results indicated that changes to the NADH metabolic pathway under a low-energy charge level can effectively increase the glycolytic rate and further improve the yield of L-lactate of R. oryzae.

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References

  1. Skory, C. D. (2000). Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae. Applied and Environmental Microbiology, 66, 2343–2348.

    Article  CAS  Google Scholar 

  2. Phrueksawan, P., Kulpreecha, S., Sooksai, S., & Thongchul, N. (2012). Direct fermentation of L(+)-lactic acid from cassava pulp by solid state culture of Rhizopus oryzae. Bioprocess and Biosystems Engineering, 35, 1429–1436.

    Article  CAS  Google Scholar 

  3. Bisson, L. F., Coons, D. M., Kruckeberg, A. L., & Lewis, D. A. (1993). Yeast sugar transporters. Critical Reviews in Biochemistry and Molecular Biology, 28, 259–308.

    Article  CAS  Google Scholar 

  4. Elbing, K., Larsson, C., Bill, R. M., Albers, E., Snoep, J. L., Boles, E., Hohmann, S., & Gustafsson, L. (2004). Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 70, 5323–5330.

    Article  CAS  Google Scholar 

  5. Pearce, A. K., Crimmins, K., Groussac, E., Hewlins, M. J., Dickinson, J. R., Francois, J., Booth, I. R., & Brown, A. J. (2001). Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions. Microbiology, 147, 391–401.

    CAS  Google Scholar 

  6. Zhang, X., Agrawal, A., & San, K. Y. (2012). Improving fatty acid production in Escherichia coli through the overexpression of malonyl coA-Acyl carrier protein transacylase. Biotechnology Progress, 28, 60–65.

    Article  Google Scholar 

  7. Thitiprasert, S., Sooksai, S., & Thongchul, N. (2011). In vivo regulation of alcohol dehydrogenase and lactate dehydrogenase in Rhizopus oryzae to improve L-lactic acid fermentation. Applied Biochemistry and Biotechnology, 164, 1305–1322.

    Article  CAS  Google Scholar 

  8. Thitiprasert, S., Songserm, P., Boonkong, W., Sooksai, S., Kodama, K., & Thongchul, N. (2014). Manipulating pyruvate decarboxylase by addition of enzyme regulators during fermentation of Rhizopus oryzae to enhance lactic acid production. Applied Biochemistry and Biotechnology, 174, 1795–1809.

    Article  CAS  Google Scholar 

  9. Larsson, C., Pahlman, I. L., & Gustafsson, L. (2000). The importance of ATP as a regulator of glycolytic flux in Saccharomyces cerevisiae. Yeast, 16, 797–809.

    Article  CAS  Google Scholar 

  10. Ataullakhanov, F. I., Vitvitsky, V. M., Zhabotinsky, A. M., Pichugin, A. V., Platonova, O. V., Kholodenko, B. N., & Ehrlich, L. I. (1981). The regulation of glycolysis in human erythrocytes. The dependence of the glycolytic flux on the ATP concentration. Eeuropean Journal of Biochemistry, 115, 359–365.

    Article  CAS  Google Scholar 

  11. White, A. T., & Schenk, S. (2012). NAD (+)/NADH and skeletal muscle mitochondrial adaptations to exercise. American Journal of Physiology-endocrinology and Metabolism, 303, 308–321.

    Article  Google Scholar 

  12. Zhao, C., Lin, Q. S., Yang, L., & Hu, M. Y. (2010). Breeding and screening of chlorpyrifos-degrading strain Cladosporium cladosporioides by ultraviolet ray mutation. Journal of South China Agricultural University, 31, 44–48 (in Chinese).

    Google Scholar 

  13. Vettraino, M., Manerba, M., Govoni, M., & Di Stefano, G. (2013). Galloflavin suppresses lactate dehydrogenase activity and causes MYC downregulation in Burkitt lymphoma cells through NAD/NADH-dependent inhibition of sirtuin-1. Anti-Cancer Drugs, 24, 862–870.

    Article  CAS  Google Scholar 

  14. Herrero, E., Ros, J., Bellí, G., & Cabiscol, E. (2008). Redox control and oxidative stress in yeast cells. Biochimica et Biophysica Acta (BBA)-General Subjects, 1780, 1217–1235.

    Article  CAS  Google Scholar 

  15. Jain, V. K., Divol, B., Prior, B. A., & Bauer, F. F. (2012). Effect of alternative NAD+-regenerating pathways on the formation of primary and secondary aroma compounds in a Saccharomyces cerevisiae glycerol-defective mutant. Applied Microbiology and Biotechnology, 93, 131–141.

    Article  Google Scholar 

  16. Heux, S., Cachon, R., & Dequin, S. (2006). Cofactor engineering in Saccharomyces cerevisiae: expression of a H2O-forming NADH oxidase and impact on redox metabolism. Metabolic Engineering, 8, 303–314.

    Article  CAS  Google Scholar 

  17. Berrios-riveva, S. J., Sanchez, A. M., Bennett, G. N., & San, K. Y. (2004). Effect of different levels of NADH availability on metabolite distribution in Escherichia coli fermentation in minimal and complex media. Applied Microbiology and Biotechnology, 65, 426–432.

    Article  Google Scholar 

  18. San, K. Y., Bennett, G. N., Berrios-Rivera, S. J., Vadali, R. V., Yang, Y. T., Horton, E., Rudolph, F. B., Sariyar, B., & Blackwood, K. (2002). Metabolic engineering through cofactor manipulation and its effects on metabolic redistribution in Escherichia coli. Metabolic Engineering, 4, 182–192.

    Article  CAS  Google Scholar 

  19. Zhou, J. W., Dong, Z. Y., Liu, L. M., Du, G. C., & Chen, J. (2010). Effect of sodium gluconate on energy metabolism and the accumulation of pyruvate in Torulopsis glabrata CCTCC M202019. Journal of Huazhong Agricultural University, 29, 527–532 (in Chinese).

    CAS  Google Scholar 

  20. Koebmann, B. J., Westerhoff, H. V., Snoep, J. L., Nilsson, D., & Jensen, P. R. (2002). The glycolytic flux in Escherichia coli is controlled by the demand for ATP. Journal of Bacteriology, 184, 3909–3916.

    Article  CAS  Google Scholar 

  21. Liu, L. M., Chen, J., Li, H. Z., & Li, Y. (2005). The influence of oxidative phosphorylation inhibitors on the glycolytic rate of Torulopsis glabrata. Progress in Biochemistry and Biophysics, 32, 25l–257. (in Chinese).

    Google Scholar 

  22. Du, J., Bai, W., Song, H., & Yuan, Y. J. (2013). Combinational expression of sorbose/sorbosone dehydrogenases and metabolic engineering pyrroloquinoline quinone increases 2-keto-l-gulonic acid production in Ketogulonigenium vulgare-Bacillus cereus consortium. Metabolic Engineering, 5, 50–56.

    Article  Google Scholar 

  23. Mao, W. J., Liu, Z. Z., Zhu, H., Zhu, R. R., Sun, X. Y., Yao, S. D., & Wang, S. L. (2009). Selection of the respiration deficiency mutant yeast irritated by laser and optimization of the fermentation condition. Journal of Radiation Research and Radiation Processing, 27, 1–4 (in Chinese).

    CAS  Google Scholar 

  24. Wei, Y., Zhang, S. S., Chen, D., Lu, Q., Chen, Y., & Huang, R. B. (2012). Breeding of two respiration-impaired mutants of Saccharomyces cerevisiae with enhanced sugar metabolism capacity. Biotechnology Bulletin, 7, 158–162 (in Chinese).

    Google Scholar 

  25. Hu, Y. H., Jiang, S. T., Luo, S. Z., Zheng, Z., & Gu, C. Y. (2008). 60 Co-γ irradiation and screening of heat-resistant Rhizopus oryzae mutant producing L-lactic acid. Food Science, 29, 452–456 (in Chinese).

    CAS  Google Scholar 

  26. Dou, C., Xu, Q., Song, P., Jiang, L., & Li, S. (2011). Metabolism of Rhizopus oryzae with xylose or glucose as carbon resource. Wei Sheng Wu Xue Bao, 51, 468–473.

    CAS  Google Scholar 

  27. Ge, C. M., Pan, R. R., Zhang, J., Cai, J. M., & Yu, Z. L. (2013). Effect of ZnSO4 on L-lactic acid production by Rhizopus oryzae. Wei Sheng Wu Xue Bao, 53, 515–520.

    CAS  Google Scholar 

  28. Luo, S. Z. (2012). Breeding of Rhizopus oryzae producing L-lactate based on SR soft X-ray radiation. Ph.D. Theis. Hefei: Hefei university of technology. (in Chinese).

    Google Scholar 

  29. Liu, L. M., Li, H. Z., Li, Y., & Chen, J. (2005). Torulopsis glaabrata neomycin-resistant mutant abolishes pyruvate production with enhancement of glucose consumption rate. Wei Sheng Wu Xue Bao, 45, 617–620.

    CAS  Google Scholar 

  30. Liu, L. M., Chen, J., Li, H. Z., & Li, Y. (2004). The decrease of the activity of electron transfer chain of Torulopsis glabrata enhanced pyruvate productivity. Wei Sheng Wu Xue Bao, 44, 800–804.

    CAS  Google Scholar 

  31. Barnard, E. A. (1975). Hexokinase from yeast. Methods in Enzymology, 42, 6–20.

    Article  CAS  Google Scholar 

  32. Qin, Y., Liu, L. M., Li, C. H., Xu, S., & Chen, J. (2010). Accelerating glycolytic flux of Torulopsis glabrata CCTCC M202019 at high oxidoreduction potential created using potassium ferricyanide. Biotechnology Progress, 26, 1551–1557.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The study was funded by the National Natural Science Foundation of China (No. 31171741) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (No. 2010JYLH0837).

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Authors and Affiliations

  1. School of Biotechnology and Food Engineering, Hefei University of Technology, 193 Tunxi road, Hefei, 230009, People’s Republic of China

    Chang Shu, Chenchen Guo, Shuizhong Luo, Shaotong Jiang & Zhi Zheng

  2. Key Laboratory for Agricultural Products Processing of Anhui Province, Hefei University of Technology, 193 Tunxi road, Hefei, 230009, People’s Republic of China

    Shuizhong Luo, Shaotong Jiang & Zhi Zheng

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  1. Chang Shu
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  2. Chenchen Guo
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Correspondence to Zhi Zheng.

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Shu, C., Guo, C., Luo, S. et al. Influence of Altered NADH Metabolic Pathway on the Respiratory-deficient Mutant of Rhizopus oryzae and its L-lactate Production. Appl Biochem Biotechnol 176, 2053–2064 (2015). https://doi.org/10.1007/s12010-015-1700-5

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