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Construction of engineered Saccharomyces cerevisiae strain to improve that whole-cell biocatalytic production of melibiose from raffinose

  • Food Biotechnology & Probiotics - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

There are excessive by-products in the biocatalysis process of this whole-cell biocatalytic production of melibiose from raffinose with current Saccharomyces cerevisiae strains. To solve this problem, we constructed engineered strains based on a liquor yeast (S. cerevisiae) via gene deletion (mel1 gene), heterologous integration (fsy1 or/and ffzi1 gene from Candida magnoliae), and gene overexpression (gcr1 gene). Functional verification showed that deletion of the mel1 gene led to elimination of the reactions catalyzed by α-galactosidase, as well as elimination of the degradation of melibiose and the formation of galactose by-product. Insertion of the fsy1 or/and ffzi1 gene and overexpression of the gcr1 gene could contribute to fructose transport for enhancing the biopurification rate of the fructose by-product. Compared with the wild-type strain, the optimal engineered strain of MP8 (Δmel1::fsy1 cm ::ffzi1 cm ::gcr1 sc ) had improved about 30% on yield, 31% on productivity, and 36% on purity of the melibiose product.

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References

  1. Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485(7397):185–194. doi:10.1038/nature11117

    Article  CAS  PubMed  Google Scholar 

  2. Cao P, Wang L, Zhou G, Wang Y, Chen Y (2014) Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus. Plasmid 76:40–46. doi:10.1016/j.plasmid.2014.09.002

    Article  CAS  PubMed  Google Scholar 

  3. de Carvalho CC (2011) Enzymatic and whole cell catalysis: finding new strategies for old processes. Biotechnol Adv 29(1):75–83. doi:10.1016/j.biotechadv.2010.09.001

    Article  PubMed  Google Scholar 

  4. de Jong BW, Shi S, Valle-Rodríguez JO, Siewers V, Nielsen J (2015) Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration. J Ind Microbiol Biotechnol 42(3):477–486. doi:10.1007/s10295-014-1540-2

    Article  PubMed  Google Scholar 

  5. Deminoff SJ, Santangelo GM (2001) Rap1p requires Gcr1p and Gcr2p homodimers to activate ribosomal protein and glycolytic genes, respectively. Genetics 158(1):133–143

    CAS  PubMed  PubMed Central  Google Scholar 

  6. De Sousa HR, Spencer-Martins I, Gonçalves P (2004) Differential regulation by glucose and fructose of a gene encoding a specific fructose/H+ symporter in Saccharomyces sensu stricto yeasts. Yeast 21(6):519–530. doi:10.1002/yea.1118

    Article  Google Scholar 

  7. Diezemann A, Boles E (2003) Functional characterization of the Frt1 sugar transporter and of fructose uptake in Kluyveromyces lactis. Curr Genet 43(4):281–288. doi:10.1007/s00294-003-0392-5

    Article  CAS  PubMed  Google Scholar 

  8. Ford CW (1972) Oligosaccharides from Phaseolus atropurpureus. Aust J Chem 25(4):889–892. doi:10.1071/CH9720889

    Article  CAS  Google Scholar 

  9. Galeote V, Novo M, Salema-Oom M, Brion C, Valério E, Gonçalves P, Dequin S (2010) FSY1, a horizontally transferred gene in the Saccharomyces cerevisiae EC1118 wine yeast strain, encodes a high-affinity fructose/H+ symporter. Microbiology 156(12):3754–3761. doi:10.1099/mic.0.041673-0

    Article  CAS  PubMed  Google Scholar 

  10. Gonçalves P, de Sousa HR, Spencer-Martins I (2000) FSY1, a novel gene encoding a specific fructose/H+ symporter in the type strain of Saccharomyces carlsbergensis. J Bacteriol 182(19):5628–5630. doi:10.1128/JB.182.19.5628-5630.2000

    Article  PubMed  PubMed Central  Google Scholar 

  11. Guimarães VM, de Rezende ST, Moreira MA, de Barros EG, Felix CR (2001) Characterization of α-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides. Phytochemistry 58(1):67–73. doi:10.1016/S0031-9422(01)00165-0

    Article  PubMed  Google Scholar 

  12. Hegemann JH, Heick SB (2011) Delete and repeat: a comprehensive tool kit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae. In: Strain engineering: methods and protocols, pp 189–206. doi:10.1007/978-1-61779-197-0_12

  13. Heljo VP, Filipe V, Romeijn S, Jiskoot W, Juppo AM (2013) Stability of rituximab in freeze-dried formulations containing trehalose or melibiose under different relative humidity atmospheres. J Pharm Sci 102(2):401–414. doi:10.1002/jps.23392

    Article  CAS  PubMed  Google Scholar 

  14. Hendrix DL, Peelen KK (1987) Artifacts in the analysis of plant tissues for soluble carbohydrates. Crop Sci 27(4):710–715. doi:10.2135/cropsci1987.0011183X002700040022x

    Article  CAS  Google Scholar 

  15. Hossain GS, Li J, Shin HD, Liu L, Wang M, Du G, Chen J (2014) Improved production of α-ketoglutaric acid (α-KG) by a Bacillus subtilis whole-cell biocatalyst via engineering of l-amino acid deaminase and deletion of the α-KG utilization pathway. J Biotechnol 187:71–77. doi:10.1016/j.jbiotec.2014.07.431

    Article  CAS  PubMed  Google Scholar 

  16. Hudson CS, Harding TS (1915) The preparation of melibiose. J Am Chem Soc 37(12):2734–2736. doi:10.1021/ja02177a020

    Article  CAS  Google Scholar 

  17. Kell DB, Swainston N, Pir P, Oliver SG (2015) Membrane transporter engineering in industrial biotechnology and whole cell biocatalysis. Trends Biotechnol 33(4):237–246. doi:10.1016/j.tibtech.2015.02.001

    Article  CAS  PubMed  Google Scholar 

  18. Kim D, Song JY, Hahn JS (2015) Improvement of glucose uptake rate and production of target chemicals by overexpressing hexose transporters and transcriptional activator Gcr1 in Saccharomyces cerevisiae. Appl Environ Microbiol 81(24):8392–8401. doi:10.1128/AEM.02056-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Leandro MJ, Sychrová H, Prista C, Loureiro-Dias MC (2011) The osmotolerant fructophilic yeast Zygosaccharomyces rouxii employs two plasma-membrane fructose uptake systems belonging to a new family of yeast sugar transporters. Microbiology 157(2):601–608. doi:10.1099/mic.0.044446-0

    Article  CAS  PubMed  Google Scholar 

  20. Lee DH, Kim SJ, Seo JH (2014) Molecular cloning and characterization of two novel fructose-specific transporters from the osmotolerant and fructophilic yeast Candida magnoliae JH110. Appl Microbiol Biotechnol 98(8):3569–3578. doi:10.1007/s00253-013-5225-y

    Article  CAS  PubMed  Google Scholar 

  21. Lee GC, Lin CH, Tao YC, Yang JM, Hsu KC, Huang YJ (2015) The potential of lactulose and melibiose, two novel trehalase-indigestible and autophagy-inducing disaccharides, for polyQ-mediated neurodegenerative disease treatment. Neurotoxicology 48:120–130. doi:10.1016/j.neuro.2015.03.009

    Article  CAS  PubMed  Google Scholar 

  22. Naumov GI, Naumova ES, Korshunova IV, Jakobsen M (2002) Yeast comparative genetics: a new mel15 α-galactosidase gene of Saccharomyces cerevisiae. Russ J Genet 38(10):1127–1132

    Article  CAS  Google Scholar 

  23. Pina C, Gonçalves P, Prista C, Loureiro-Dias MC (2004) Ffz1, a new transporter specific for fructose from Zygosaccharomyces bailii. Microbiol 150(7):2429–2433. doi:10.1099/mic.0.26979-0

    Article  CAS  Google Scholar 

  24. Silman RW, Black LT, McGhee JE, Bangley EB (1980) Hydrolysis of raffinose in a hollow-fiber reactor using an unrefined mixture of α-galactosidase and invertase. Biotechnol Bioeng 22(3):533–541. doi:10.1002/bit.260220306

    Article  CAS  Google Scholar 

  25. Song C, Zhao L, Ono S, Shimasaki C, Inoue M (2001) Production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) from cottonseed oil and valeric acid in batch culture of Ralstonia sp. strain JC-64. Appl Microbiol Biotechnol 94(2):169–178. doi:10.1385/ABAB:94:2:169

    CAS  Google Scholar 

  26. Tanaka S, Shinoki A, Hara H (2014) Melibiose, a non-digestible saccharide, promotes absorption of quercetin glycosides in rat small intestine with a novel mechanism. FASEB 28(1 Supplement):1044–1049

    Google Scholar 

  27. Tomita K, Nagura T, Okuhara Y, Nakajima-Adachi H, Shigematsu N, Aritsuka T (2007) Dietary melibiose regulates Th cell response and enhances the induction of oral tolerance. Biosci Biotechnol Biochem 71(11):2774–2780. doi:10.1271/bbb.70372

    Article  CAS  PubMed  Google Scholar 

  28. Weber N, Gorwa-Grauslund M, Carlquist M (2014) Exploiting cell metabolism for biocatalytic whole-cell transamination by recombinant Saccharomyces cerevisiae. Appl Microbiol Biotechnol 98(10):4615–4624. doi:10.1007/s00253-014-5576-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Xiao M, Tanaka K, Qian XM, Yamamoto K, Kumagai H (2000) High-yield production and characterization of α-galactosidase from Bifidobacterium breve grown on raffinose. Biotechnol Lett 22(9):747–751. doi:10.1023/A:1005626228056

    Article  CAS  Google Scholar 

  30. Zhou Y, Zhu Y, Dai L, Men Y, Wu J, Zhang J, Sun Y (2016) Efficiency analysis and mechanism Insight of that whole-cell biocatalytic production of melibiose from raffinose with Saccharomyces cerevisiae. Appl Biochem Biotechnol 2016:1–17. doi:10.1007/s12010-016-2220-7

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the National High Technology Research and Development Program of China (No. 2013AA102102) and the National Natural Science Foundation of China (General Program, 31571793).

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Authors and Affiliations

  1. Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education, Key Laboratory of Industrial Microbiology of Tianjin, College of Bioengineering, Tianjin University of Science and Technology, No. 32, 13th. Avenue, Tianjin Economic and Development Area, Tianjin, 300457, China

    Yingbiao Zhou & Juankun Zhang

  2. National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, No. 32, West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China

    Yingbiao Zhou, Yueming Zhu, Yan Men & Yuanxia Sun

  3. Tianjin Key Laboratory on Technology Enabling Development of Clinical Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin, 300070, China

    Caixia Dong

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  1. Yingbiao Zhou
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  2. Yueming Zhu
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  3. Yan Men
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  4. Caixia Dong
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  5. Yuanxia Sun
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Correspondence to Yuanxia Sun or Juankun Zhang.

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We declare that we have no financial or personal relationships with other people or organizations, which would inappropriately influence our work; there is no professional or other personal interest of any nature in any product, service, and/or company that could be construed as influencing the position presented in, or the review of, this manuscript.

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Zhou, Y., Zhu, Y., Men, Y. et al. Construction of engineered Saccharomyces cerevisiae strain to improve that whole-cell biocatalytic production of melibiose from raffinose. J Ind Microbiol Biotechnol 44, 489–501 (2017). https://doi.org/10.1007/s10295-017-1901-8

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