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Cloning novel sugar transporters from Scheffersomyces (Pichia) stipitis allowing d-xylose fermentation by recombinant Saccharomyces cerevisiae

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Abstract

Objectives

Since uptake of xylose limits its fermentation, we aimed to identify novel sugar transporters from Scheffersomyces stipitis that allow xylose uptake and fermentation by engineered Saccharomyces cerevisiae.

Results

An hxt-null S. cerevisiae strain, lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) but having high xylose reductase, xylitol dehydrogenase and xylulokinase activities, was transformed with a genomic DNA library from S. stipitis. Four plasmids allowing growth on xylose contained three genes encoding sugar transporters: the previously characterized XUT1 permease, and two new genes (HXT2.6 and QUP2) not previously identified as xylose transporters. High cell density fermentations with the recombinant strains showed that the XUT1 gene allowed ethanol production from xylose or xylose plus glucose as carbon sources, while the HXT2.6 permease produced both ethanol and xylitol, and the strain expressing the QUP2 gene produced mainly xylitol during xylose consumption.

Conclusions

Cloning novel sugar transporters not previously identified in the S. stipitis genome using an hxt-null S. cerevisiae strain with a high xylose-utilizing pathway provides novel promising target genes for improved lignocellulosic ethanol production by yeasts.

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References

  • Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1995) Short protocols in molecular biology, 3rd edn. Wiley & Sons, New York

    Google Scholar 

  • Bertilsson M, Andersson J, Líden G (2008) Modeling simultaneous glucose and xylose uptake in Saccharomyces cerevisiae from kinetics and gene expression of sugar transporters. Bioprocess Biosyst Eng 31:369–377

    Article  CAS  PubMed  Google Scholar 

  • Cadete RM, Santos RO, Melo MA, Mouro A, Golçalves DL, Stambuk BU, Rosa CA (2009) Spathaspora arborariae sp. nov., a d-xylose fermenting yeast species isolated from rotting wood in Brazil. FEMS Yeast Res 9:1338–1342

    Article  CAS  PubMed  Google Scholar 

  • Cadete RM, Melo MA, Zilli JE, Vital MJ, Mouro A, Prompt AH, Gomes FC, Stambuk BU, Lachance MA, Rosa CA (2013) Spathaspora brasiliensis sp. nov., Spathaspora sushii sp. nov., Spathaspora roraimanensis sp. Nov. and Spathaspora xylofermentans sp. nov., four novel (d)-xylose-fermenting yeast species from Brazilian Amazonian forest. Anton Leeuw 103:421–431

    Article  CAS  Google Scholar 

  • Cai Z, Zhang B, Li Y (2012) Engineering Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: reflections and perspectives. Biotechnol J 7:34–36

    Article  CAS  PubMed  Google Scholar 

  • Caspeta L, Buijs NAA, Nielsen J (2013) The role of biofuels in the future energy supply. Energy Environ Sci 6:1077–1082

    Article  CAS  Google Scholar 

  • Diao L, Liu Y, Qian F, Yang J, Jiang Y, Yang S (2013) Construction of fast xylose-fermenting yeast based on industrial ethanol-producing diploid Saccharomyces cerevisiae by rational design and adaptive evolution. BMC Biotechnol 13:1–9

    Article  Google Scholar 

  • Does AL, Bisson LF (1989) Characterization of xylose uptake in the yeasts Pichia heedie and Pichia stipitis. Appl Environ Microbiol 55:159–164

    PubMed Central  CAS  PubMed  Google Scholar 

  • Du J, Li S, Zhao H (2010) Discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis. Mol BioSyst 6:2150–2156

    Article  CAS  PubMed  Google Scholar 

  • Farias DR, de Andrade R, Maugeri-Filho F (2014) Kinetic modeling of ethanol production by Scheffersomyces stipitis from xylose. Appl Biochem Biotechnol 172:361–379

    Article  CAS  PubMed  Google Scholar 

  • Gárdonyi M, Jeppsson M, Lidén G, Gorwa-Grauslund MF, Hahn-Hägerdal B (2003) Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 82:818–824

    Article  PubMed  Google Scholar 

  • Gonçalves DL, Matsushika A, Sales BB, Goshima T, Bon EPS, Stambuk BU (2014) Xylose and xylose/glucose co-fermentation by recombinant Saccharomyces cerevisiae strains expressing individual hexose transporters. Enzyme Microb Technol 63:13–20

    Article  PubMed  Google Scholar 

  • Ha SJ, Kim H, Lin Y, Jang UM, Galazka JM, Kim TJ, Cate JHM, Jin YS (2013) Single amino acid substitutions in Hxt2.4 from Scheffersomyces stipitis lead to improved cellobiose fermentation by engineered Saccharomyces cerevisiae. Appl Environ Microbiol 79:1500–1507

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hamacher T, Becker J, Gardonyi M, Hahn-Hägerdal B, Boles E (2002) Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology 148:2783–2788

    Article  CAS  PubMed  Google Scholar 

  • Hector RE, Qureshi N, Hughes SR, Cotta MA (2008) Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption. Appl Microbiol Biotechnol 80:675–684

    Article  CAS  PubMed  Google Scholar 

  • Jeffries TW, Van Vleet JRH (2009) Pichia stipitis genomics, transcriptomics, and gene clusters. FEMS Yeast Res 9:793–807

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Jeffries TW, Grigoriev IV, Grimwood J, Laplaza JM, Aertis A, Salamov A, Schmutz J, Lindquist E, Dehal P, Shapiro H, Jin YSU, Passoth V, Richardson PM (2007) Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis. Nat Biotechnol 25:319–326

    Article  CAS  PubMed  Google Scholar 

  • Jin Y, Alper H, Yang Y, Stephanopoulos G (2005) Improvement of xylose uptake and ethanol production in recombinant Saccharomyces cerevisiae through an inverse metabolic engineering approach. Appl Environ Microbiol 71:8249–8256

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kang Y, Kane J, Kujan J, Stadel JM, Tipper DJ (1990) Effects of expression of mammalian GeL and hybrid mammalian-yeast Ga proteins on the yeast pheromone response signal transduction pathway. Mol Cell Biol 10:2582–2590

    PubMed Central  CAS  PubMed  Google Scholar 

  • Katahira S, Ito M, Takema H, Fujita Y, Tanino T, Tanaka T, Fukuda H, Kondo A (2008) Improvement of ethanol productivity during xylose and glucose co-fermentation by xylose-assimilating S. cerevisiae via expression of glucose transporter Sut1. Enzyme Microb Technol 43:115–119

    Article  CAS  Google Scholar 

  • Kilian SG, Van Uden N (1988) Transport of xylose and glucose in the xylose fermenting yeast Pichia stipitis. Appl Microbiol Biotechnol 27:545–548

    Article  CAS  Google Scholar 

  • Kim SR, Ha SJ, Wei N, Oh EJ, Jin YS (2012) Simultaneous co-fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol. Trends Biotechnol 30:274–282

    Article  PubMed  Google Scholar 

  • Kötter P, Ciriacy M (1993) Xylose fermentation by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 38:776–783

    Article  Google Scholar 

  • Laluce C, Schenberg ACG, Gallardo JCM, Coradello LFC, Pombeiro-Sponchiado SR (2012) Advances and developments in strategies to improve strains of Saccharomyces cerevisiae and processes to obtain the lignocellulosic ethanol—a review. Appl Biochem Biotechnol 166:1908–1926

    Article  CAS  PubMed  Google Scholar 

  • Lee WJ, Kim MD, Ryu YW, Bisson LF, Seo JH (2002) Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 60:186–191

    Article  CAS  PubMed  Google Scholar 

  • Lin S, Tanaka S (2006) Ethanol fermentation from biomass resources: current state and prospects. Appl Microbiol Biotechnol 69:627–642

    Article  CAS  PubMed  Google Scholar 

  • Lobo FP, Gonçalves DL, Alves SL Jr, Gerber AL, Vasconcelos ATR, Basso LC, Franco GR, Soares MA, Cadete RM, Rosa CA, Stambuk BU (2014) Draft genome sequence of the d-xylose-fermenting yeast Spathaspora arborariae UFMG-HM19.1AT. Genome Announc 2:e01163-13

  • Madhavan A, Tamalampudi S, Srivastava A, Fukuda H, Bisaria VS, Kondo A (2009) Alcoholic fermentation of xylose and mixed sugars using recombinant Saccharomyces cerevisiae engineered for xylose utilization. Appl Microbiol Biotechnol 82:1037–1047

    Article  CAS  PubMed  Google Scholar 

  • Matsushika A, Inoue H, Kodaki T, Sawayama S (2009) Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives. Appl Microbiol Biotechnol 84:37–53

    Article  CAS  PubMed  Google Scholar 

  • Moon J, Liu ZL, Ma M, Slininger PJ (2013) New genotypes of industrial yeast Saccharomyces cerevisiae engineered with YXI and heterologous xylose transporters improve xylose utilization and ethanol production. Biocatal Agric Biotechnol 2:247–254

    Google Scholar 

  • Nielsen J, Larsson C, van Maris A, Pronk J (2013) Metabolic engineering of yeast for production of fuels and chemicals. Curr Opin Biotechnol 24:398–404

    Article  CAS  PubMed  Google Scholar 

  • Nigam JN (2001) Ethanol production from wheat straw hemicelluloses by Pichia stipitis. J Biotechnol 87:17–27

    Article  CAS  PubMed  Google Scholar 

  • Nogueira LAH, Moreira JR, Schuchardt U, Goldemberg J (2013) The rationality of biofuels. Energy Policy 61:595–598

    Article  Google Scholar 

  • Parachin NS, Bergdahl B, van Niel EW, Gorwa-Grauslund MF (2011) Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae. Metab Eng 13:508–517

    Article  CAS  PubMed  Google Scholar 

  • Parambil LK, Sarkar D (2014) Probing the bioethanol production potential of Scheffersomyces (Pichia) stipitis using validated genome-scale model. Biotechnol Lett 36:2443–2451

    Article  CAS  PubMed  Google Scholar 

  • Runquist D, Fonseca C, Radstrom P, Spencer-Martins I, Hahn-Hägerdal B (2009) Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae. Appl Microbiol Biotechnol 82:123–130

    Article  CAS  PubMed  Google Scholar 

  • Runquist D, Hahn-Hägerdal B, Radstrom P (2010) Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 3:5

    Article  PubMed Central  PubMed  Google Scholar 

  • Saloheimo A, Rauta J, Stasyk OV, Sibirny AA, Penttilä M, Ruohonen L (2007) Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases. Appl Microbiol Biotechnol 74:1041–1052

    Article  CAS  PubMed  Google Scholar 

  • Sànchez Nogué V, Karhumaa K (2015) Xylose fermentation as a challenge for commercialization of lignocellulosic fuels and chemicals. Biotechnol Lett 37:761–772

    Article  PubMed  Google Scholar 

  • Sarkar N, Ghosh SK, Bannerjee S, Aikat K (2011) Bioethanol production from agricultural wastes: an overview. Renew Energy 37:19–23

    Article  Google Scholar 

  • Sedlak M, Ho NWY (2004) Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast. Yeast 21:671–684

    Article  CAS  PubMed  Google Scholar 

  • Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, Li W, Lopez R, Mcwilliam H, Remmert M, Soding J, Thomson JD, Higgins D (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:1–6

    Google Scholar 

  • Stambuk BU, Eleutherio ECA, Florez-Pardo LM, Souto-Maior AM, Bon EPS (2008) Brazilian potential for biomass ethanol: challenge of using hexose and pentose cofermenting yeast strains. J Sci Ind Res 67:918–926

    CAS  Google Scholar 

  • Sun L, Zeng X, Yan C, Sun X, Gong X, Rao Y, Yan N (2012) Crystal structure of a bacterial homologue of glucose transporters GLUT1–4. Nature 490:361–368

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Peterson D, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  PubMed Central  CAS  PubMed  Google 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 xylose isomerase pathway. J Biosci Bioeng 114:209–211

    Article  CAS  PubMed  Google Scholar 

  • Toivola A, Yarrow D, Van Den Bosch E, Van Dijken JP, Scheffers WA (1984) Alcoholic fermentation of d-xylose by yeasts. Appl Environ Microbiol 47:1221–1223

    PubMed Central  CAS  PubMed  Google Scholar 

  • Watanabe T, Watanabe I, Yamamoto Y, Ando A, Nakamura TA (2011) UV-induced mutant of Pichia stipitis with increased ethanol production from xylose and selection of a spontaneous mutant with increased ethanol tolerance. Bioresour Technol 102:1844–1848

    Article  CAS  PubMed  Google Scholar 

  • Weierstall T, Hollenberg CP, Boles E (1999) Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipitis. Mol Microbiol 31:871–883

    Article  CAS  PubMed  Google Scholar 

  • Wohlbach DJ, Kuo A, Sato TK, Potts KM, Salamov AA, Labutti KM, Sun H, Clum A, Pangilinan JL, Lindquist EA, Lucas S, Lapidus A, Jin M, Gunawan C, Balan V, Dale BD, Jeffries TW, Zinkel R, Barry KW, Grigoriev C, Gash AP (2011) Comparative genomics of xylose-fermenting fungi for enhanced biofuel production. Proc Natl Acad Sci USA 108:13212–13217

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Young E, Lee SM, Alper H (2010) Optimizing pentose utilization in yeast: the need for novel tools and approaches. Biotechnol Biofuels 3:24

    Article  PubMed Central  PubMed  Google Scholar 

  • 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 77:3311–3319

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Young ME, Comer AD, Huang H, Alper HS (2012) A molecular transporter engineering approach to improving xylose catabolism in Saccharomyces cerevisiae. Metab Eng 14:401–411

    Article  CAS  PubMed  Google Scholar 

  • Young E, Tong A, Bui H, Spofford C, Alper HS (2014) Rewiring yeast sugar transporter preference through modifying a conserved protein motif. Proc Natl Acad Sci USA 111:131–136

    Article  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

This work was supported in part by grants and fellowships from the Brazilian agencies CNPq (Process Nos. 490029/2009-4, 551392/2010-0, 307290/2012-3, and 478841/2013-2), FINEP (Process No. 01.09.0566.00/1421-08) and FAPESC (Process No. 17293/2009-6), and by the Japanese International Cooperation Agency (JICA).

Supporting information

Supplementary Table 1—Growth rates and ethanol yields of the recombinant yeast strains grown on synthetic SC medium with the indicated sugars.

Supplementary Fig. 1—Amino acid sequence alignment of S. stipites sugar transporters.

Author information

Authors and Affiliations

  1. Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brazil

    Belisa B. de Sales, Bruna Scheid, Davi L. Gonçalves, Marilia M. Knychala & Boris U. Stambuk

  2. Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-0046, Japan

    Akinori Matsushika

  3. Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio De Janeiro, RJ, 21941-909, Brazil

    Elba P. S. Bon

Authors
  1. Belisa B. de Sales
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  2. Bruna Scheid
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  3. Davi L. Gonçalves
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  4. Marilia M. Knychala
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  7. Boris U. Stambuk
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Correspondence to Boris U. Stambuk.

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de Sales, B.B., Scheid, B., Gonçalves, D.L. et al. Cloning novel sugar transporters from Scheffersomyces (Pichia) stipitis allowing d-xylose fermentation by recombinant Saccharomyces cerevisiae . Biotechnol Lett 37, 1973–1982 (2015). https://doi.org/10.1007/s10529-015-1893-2

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