Applied Microbiology and Biotechnology

, Volume 102, Issue 13, pp 5557–5567 | Cite as

Identification and characterization from Candida glycerinogenes of hexose transporters having high efficiency at high glucose concentrations

  • Zhanbin Liang
  • Di Liu
  • Xinyao Lu
  • Hong Zong
  • Jian Song
  • Bin ZhugeEmail author
Biotechnologically relevant enzymes and proteins


During high gravity fermentation, a set of hexose transporters in yeasts plays an important role in efficient sugar transport. However, hexose transporters have been studied mainly in the Saccharomyces cerevisiae model and at low or moderate sugar concentrations. The hexose transporters are still poorly understood in the industrial glycerol producer Candida glycerinogenes, which assimilates sugar efficiently at high glucose concentration. To explore these hexose transporters, 14 candidates were identified using a hidden Markov model and characterized. Five of these functioned as hexose transporters when expressed in S. cerevisiae. In particular, CgHxt4 showed the highest efficiency of glucose transport at elevated glucose concentration among a group of transporters including Hxt1 and Hxt7 from S. cerevisiae. qRT-PCR in C. glycerinogenes revealed that transcription of CgHXT4 was induced by high glucose concentrations while fluorescence localization analysis indicated that CgHxt4 remained relatively stable on the membrane under these conditions. In addition, site-directed mutagenesis revealed that the asparagine 329 from CgHxt4, located in the YYX(T/P) conserved motif of hexose transporters, promoted an increased glucose transport. Overexpressing CgHXT4 in S. cerevisiae enhanced glucose consumption and ethanol production more effectively at high glucose concentrations than ScHXT1, the most significant native transporter from S. cerevisiae. These results indicate that CgHxt4 plays an important role in the fermentation process as a hexose transporter with strong transport activity and efficient expression regulation at high glucose concentrations.


Candida glycerinogenes Expression regulation Hexose transporter High glucose concentration Transport efficiency 



We thank Prof. Eckhard Boles for providing us the S. cerevisiae strain EBY.VW4000. This work was supported by the Collaborative Innovation Center of Jiangsu Modern Industrial Fermentation.

Funding information

This work was supported by the National Natural Science Foundation of China (Nos. 31570052, 31601456, 21708016). This work was supported by national first-class discipline program of Light Industry Technology and Engineering (LITE2018-01).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2018_9027_MOESM1_ESM.docx (1.7 mb)
ESM 1 (DOCX 1700 kb)


  1. Arshad M, Hussain T, Iqbal M, Abbas M (2017) Enhanced ethanol production at commercial scale from molasses using high gravity technology by mutant S. cerevisiae. Braz J Microbiol 48(3):403–409. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Boles E, Hollenberg CP (1997) The molecular genetics of hexose transport in yeasts. FEMS Microbiol Rev 21(1):85–111. CrossRefPubMedGoogle Scholar
  3. Chao B, Liu R, Zhang X, Zhang X, Tan T (2017) Tannin extraction pretreatment and very high gravity fermentation of acorn starch for bioethanol production. Bioresour Technol 241:900–907. CrossRefPubMedGoogle Scholar
  4. Dimarco AA, Romano AH (1985) D-glucose transport system of Zymomonas mobilis. Appl Environ Microbiol 49(1):151–157PubMedPubMedCentralGoogle Scholar
  5. Duskova M, Ferreira C, Lucas C, Sychrova H (2015) Two glycerol uptake systems contribute to the high osmotolerance of Zygosaccharomyces rouxii. Mol Microbiol 97(3):541–559. CrossRefPubMedGoogle Scholar
  6. Eddy SR (2009) A new generation of homology search tools based on probabilistic inference. Genome Inform 23(1):205–211PubMedGoogle Scholar
  7. Finley D, Ulrich HD, Sommer T, Kaiser P (2012) The ubiquitin-proteasome system of Saccharomyces cerevisiae. Genetics 192(2):319–360. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44(D1):D279–D285. CrossRefPubMedGoogle Scholar
  9. Greatrix BW, van Vuuren HJ (2006) Expression of the HXT13, HXT15 and HXT17 genes in Saccharomyces cerevisiae and stabilization of the HXT1 gene transcript by sugar-induced osmotic stress. Curr Genet 49(4):205–217. CrossRefPubMedGoogle Scholar
  10. Hairu J, Huiying F, Jian Z (2003) By-product formation by a novel glycerol-producing yeast, Candida glycerinogenes, with different O2 supplies. Biotechnol Lett 25:311–314. CrossRefGoogle Scholar
  11. Heiland S, Radovanovic N, Höfer M, Winderickx J, Lichtenberg H (2000) Multiple hexose transporters of Schizosaccharomyces pombe. J Bacteriol 182(8):2153–2162. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Ji H, Lu X, Wang C, Zong H, Fang H, Sun J, Zhuge J, Zhuge B (2014) Identification of a novel HOG1 homologue from an industrial glycerol producer Candida glycerinogenes. Curr Microbiol 69(6):909–914. CrossRefPubMedGoogle Scholar
  13. Ji H, Zhuge B, Zong H, Lu X, Fang H, Zhuge J (2016) Role of CgHOG1 in stress responses and glycerol overproduction of Candida glycerinogenes. Curr Microbiol 73(6):827–833. CrossRefPubMedGoogle Scholar
  14. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kruckeberg AL, Ye L, Berden JA, Van DK (1999) Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein. Biochem J 339(2):299–307. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. CrossRefPubMedGoogle Scholar
  17. Leandro MJ, Fonseca C, Goncalves P (2009) Hexose and pentose transport in ascomycetous yeasts: an overview. FEMS Yeast Res 9(4):511–525. CrossRefPubMedGoogle Scholar
  18. Li H, Schmitz O, Alper HS (2016) Enabling glucose/xylose co-transport in yeast through the directed evolution of a sugar transporter. Appl Microbiol Biotechnol 100(23):10215–10223. CrossRefPubMedGoogle Scholar
  19. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC T method. Methods 25(4):402–408. CrossRefPubMedGoogle Scholar
  20. Malinska K, Malinsky J, Opekarova M, Tanner W (2003) Visualization of protein compartmentation within the plasma membrane of living yeast cells. Mol Biol Cell 14(11):4427–4436. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Nijland JG, Shin HY, Boender LGM, de Waal PP, Klaassen P, Driessen AJM (2017) Improved xylose metabolism by a CYC8 mutant of Saccharomyces cerevisiae. Appl Environ Microbiol 83(11):e00095–e00017. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Nijland JG, Vos E, Shin HY, de Waal PP, Klaassen P, Driessen AJ (2016) Improving pentose fermentation by preventing ubiquitination of hexose transporters in Saccharomyces cerevisiae. Biotechnol Biofuels 9:158. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Özcan S, Johnston M (1999) Function and regulation of yeast hexose transporters. Microbiol Mol Biol Rev 63(3):554–569PubMedPubMedCentralGoogle Scholar
  24. Pereira I, Madeira A, Prista C, Loureirodias MC, Leandro MJ (2014) Characterization of new polyol/H+ symporters in Debaryomyces hansenii. PLoS One 9(2):e88180. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Reifenberger E, Boles E, Ciriacy M (1997) Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. Eur J Biochem 245(2):324–333. CrossRefPubMedGoogle Scholar
  26. Reis TFD, Menino JF, Bom VLP, Brown NA, Colabardini AC, Savoldi M, Goldman MHS, Rodrigues F, Goldman GH (2013) Identification of glucose transporters in Aspergillus nidulans. PLoS One 8(11):e81412. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Rossi G, Sauer M, Porro D, Branduardi P (2010) Effect of HXT1 and HXT7 hexose transporter overexpression on wild-type and lactic acid producing Saccharomyces cerevisiae cells. Microb Cell Factories 9:15. CrossRefGoogle Scholar
  28. Roy A, Kim YB, Cho KH, Kim JH (2014) Glucose starvation-induced turnover of the yeast glucose transporter Hxt1. Biochim Biophys Acta 1840(9):2878–2885. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Sen A, Acosta-Sampson L, Alvaro CG, Ahn JS, Cate JH, Thorner J (2016) Internalization of heterologous sugar transporters by endogenous α-arrestins in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 82(24):7074–7085. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Shin HY, Nijland JG, Waal PP, Driessen AJM (2017) The amino-terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid. Biotechnol Bioeng 114(9):1937–1945. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Sloothaak J, Tamayo-Ramos JA, Odoni DI, Laothanachareon T, Derntl C, Mach-Aigner AR, Martins Dos Santos VA, Schaap PJ (2016) Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei. Biotechnol Biofuels 9:148. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Tanino T, Ito T, Ogino C, Ohmura N, Ohshima T, Kondo A (2012) Sugar consumption and ethanol fermentation by transporter-overexpressed xylose-metabolizing Saccharomyces cerevisiae harboring a xyloseisomerase pathway. J Biosci Bioeng 114(2):209–211. CrossRefPubMedGoogle Scholar
  33. Tomas-Cobos L, Casadome L, Mas G, Sanz P, Posas F (2004) Expression of the HXT1 low affinity glucose transporter requires the coordinated activities of the HOG and glucose signalling pathways. J Biol Chem 279(21):22010–22019. CrossRefPubMedGoogle Scholar
  34. Wang C, Bao X, Li Y, Jiao C, Hou J, Zhang Q, Zhang W, Liu W, Shen Y (2015) Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization. Metab Eng 30:79–88. CrossRefPubMedGoogle Scholar
  35. Wang C, Li Y, Qiu C, Wang S, Ma J, Shen Y, Zhang Q, Du B, Ding Y, Bao X (2017) Identification of important amino acids in Gal2p for improving the L-arabinose transport and metabolism in Saccharomyces cerevisiae. Front Microbiol 8:1391. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Wang M, Yu C, Zhao H (2016) Identification of an important motif that controls the activity and specificity of sugar transporters. Biotechnol Bioeng 113(7):1460–1467. CrossRefPubMedGoogle Scholar
  37. Wieczorke R, Krampe S, Weierstall T, Freidel K, Hollenberg CP, Boles E (1999) Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett 464(3):123–128. CrossRefPubMedGoogle Scholar
  38. Xiao YH, Pei Y (2011) Asymmetric overlap extension PCR method for site-directed mutagenesis. Methods Mol Biol 687:277–282. CrossRefPubMedGoogle Scholar
  39. Xie T, Fang HY, Zhuge B, Zhuge J (2009) Promotional mechanism of high glycerol productivity in the aerobic batch fermentation of Candida glycerinogenes after feeding several amino acids. Appl Biochem Microbiol 45(3):303–308. CrossRefGoogle Scholar
  40. Yongguang Z, Wei S, Zhiming R, Huiying F, Jian Z (2007) Deletion of the CgTPI gene encoding triose phosphate isomerase of Candida glycerinogenes inhibits the biosynthesis of glycerol. Curr Microbiol 55(2):147–151. CrossRefPubMedGoogle Scholar
  41. Young E, Poucher A, Comer A, Bailey A, Alper H (2011) Functional survey for heterologous sugar transport proteins, using Saccharomyces cerevisiae as a host. Appl Environ Microbiol 77(10):3311–3319. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Young EM, Tong A, Bui H, Spofford C, Alper HS (2014) Rewiring yeast sugar transporter preference through modifying a conserved protein motif. Proc Natl Acad Sci U S A 111(1):131–136. CrossRefPubMedGoogle Scholar
  43. Zhang Q, Nurhayati CC-L, Lo Y-C, Nagarajan D, Hu J, Chang J-S, Lee D-J (2017) Ethanol production by modified polyvinyl alcohol-immobilized Zymomonas mobilis and in situ membrane distillation under very high gravity condition. Appl Energy 202:1–5. CrossRefGoogle Scholar
  44. Zhang Q, Wu D, Lin Y, Wang X, Kong H, Tanaka S (2015a) Substrate and product inhibition on yeast performance in ethanol fermentation. Energy Fuel 29(2):1019–1027. CrossRefGoogle Scholar
  45. Zhang W, Cao Y, Gong J, Bao X, Chen G, Liu W (2015b) Identification of residues important for substrate uptake in a glucose transporter from the filamentous fungus Trichoderma reesei. Sci Rep 5:13829. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Zhuge J, Fang HY, Wang ZX, Chen DZ, Jin HR, Gu HL (2001) Glycerol production by a novel osmotolerant yeast Candida glycerinogenes. Appl Microbiol Biotechnol 55(6):686–692. CrossRefPubMedGoogle Scholar
  47. Zong H, Zhang C, Zhuge B, Lu X, Fang H, Sun J (2016) Effects of xylitol dehydrogenase (XYL2) on xylose fermentation by engineered Candida glycerinogenes. Biotechnol Appl Biochem 64(4):590–599. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Zhanbin Liang
    • 1
    • 2
  • Di Liu
    • 1
    • 2
  • Xinyao Lu
    • 1
    • 2
  • Hong Zong
    • 1
    • 2
  • Jian Song
    • 1
    • 2
  • Bin Zhuge
    • 1
    • 2
    Email author
  1. 1.The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina
  2. 2.The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina

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