Identification and characterization from Candida glycerinogenes of hexose transporters having high efficiency at high glucose concentrations
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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.
KeywordsCandida 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.
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).
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Boles E, Hollenberg CP (1997) The molecular genetics of hexose transport in yeasts. FEMS Microbiol Rev 21(1):85–111. https://doi.org/10.1111/j.1574-6976.1997.tb00346.x CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1093/nar/gkv1344 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1128/AEM.02056-15 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1042/0264-6021:3390299 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1093/bioinformatics/btm404 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1111/j.1432-1033.1997.00324.x CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1128/AEM.02148-16 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1002/bit.26322 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1186/s13068-016-0564-4 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/j.jbiosc.2012.03.004 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1016/j.ymben.2015.04.007 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.3389/fmicb.2017.01391 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/S0014-5793(99)01698-1 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1016/j.apenergy.2017.05.105 CrossRefGoogle Scholar