Overexpression of smORF YNR034W-A/EGO4 in Saccharomyces cerevisiae increases the fermentative efficiency of Agave tequilana Weber must

  • Naurú Idalia Vargas-Maya
  • Gloria Angélica González-Hernández
  • Israel Enrique Padilla-Guerrero
  • Juan Carlos Torres-Guzmán
Fermentation, Cell Culture and Bioengineering - Original Paper
First Online:
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Accepted:

Abstract

Fermentative processes are widely used to produce food, beverages and biofuels. Saccharomyces cerevisiae is an efficient ethanol-producing microorganism. However, a concentration of high ethanol and other metabolites can affect yeast viability and decrease the ethanol yield. Many studies have focused on improving the fermentative efficiency, mostly through the genetic engineering of genes that have a direct impact on specific metabolic pathways. In the present study, we characterized a small open reading frame encoding a protein with an unknown function and biological role termed YNR034W-A. We analyzed the expression profile of the YNR034W-A gene during growth and glucose treatment, finding that it is expressed during the diauxic shift and stationary phase and is negatively regulated by glucose. We overexpressed the YNR034W-A gene in the BY4741 laboratory strain and a wild-type yeast strain (AR5) isolated during the Tequila fermentation process. Transformant derivatives of the AR5 strain showed an improved fermentative efficiency during fermentation of Agave tequilana Weber juice. We suggest that the improved fermentative efficiency is the result of a higher stress tolerance response in the YNR034W-A overexpressing transformant.

Keywords

Saccharomyces Fermentation Agave tequilana smORF Tequila 
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Notes

Acknowledgements

We express our thanks to PhD. Bernardo Franco for expert assistance and technical support in the flow cytometry analysis. We also thank QFB. Miguel Roa Castañeda from Tequilera Corralejo, S.A. de C.V. for providing the Agave juice. Financial support is greatly acknowledged from the following institutions: Universidad de Guanajuato Grants: UGTO2011, UGTO 415/2014, UGTO 511/2015, UGTO 641/2015. Apoyo al Fortalecimiento de la Excelencia Académica Grants: 005/2014; 003/2015. Consejo Nacional de Ciencia y Tecnología (CONACyT): CB-2007-01. 84394; Ciencia básica: 220780, 388394; Consejo de Ciencia y Tecnología del Estado de Guanajuato (CONCyTEG): FINNOVATEC 2105, 00079. Tequilera Corralejo, S.A. de C.V., Grant: 01-2016. Naurú Idalia Vargas Maya received a scholarship from CONACyT.

Compliance with ethical standards

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

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

10295_2016_1871_MOESM1_ESM.pdf (4.7 mb)
Supplementary material 1 Fig. S1 pJRC71 plasmid construction. The YNR034W-A gene was amplified by PCR using the oligonucleotides oliDYNR034W-A and oliRYNR034W-ABamHI. The HXT7 promoter region fragment was amplified from AR5 genomic DNA using oligonucleotides oliDHxt7SacI and oliRHxt7-YNR034W-A. The fusion cassette was constructed using the Double-joint PCR technique using each PCR product (HXT7 promoter region and YNR034W-A gene). The fusion PCR product was utilized in a second amplification reaction with oligonucleotides oliDHxt7SacI and oliRYNR034W-ABamHI. The final PCR fusion product (termed HXT7p-YNR034W-A) was digested with SacI and BamHI and ligated into the pJRC34 centromeric plasmid to obtain the pJRC71 plasmid. Fig. S2 AdhI C-Terminus GFP-Tag. The green fluorescent protein (GPF) and Schizosaccharomyces pombe HIS5 genes were amplified from the pFA6a-link-yEGFP-SpHIS5 plasmid (pKT128) with oligonucleotides F5GFPADH1D and R3GFPADH1R. The GFP-HIS5 cassette was used to transform the BY4741 laboratory strain. The BY4741-ADHcarboxyl-GFP strain was selected in YNB selective media. Correct cassette integration was verified by PCR using genomic DNA of each transformant and oligonucleotides ORFGFPADH1Dir and ORFGFPADH1Rev, designed outside the insertion site. Fig. S3 Ynr034w-a C-Terminus GFP-Tag. The green fluorescent protein (GPF) and Schizosaccharomyces pombe HIS5 genes were amplified from the pFA6a-link-yEGFP-SpHIS5 plasmid (pKT128) with oligonucleotides, F5GFPYNR034W-AD and R3GFPYNR034W-AR. The GFP-HIS5 cassette was used to transform the BY4741 laboratory strain. The BY4741-YNR034W-Acarboxyl-GFP strain was selected in YNB selective media. Correct cassette integration was verified by PCR using genomic DNA of each transformant and oligonucleotides ORFGFPYNR034W-AdDir and ORFGFPYNR034W-ARev, designed outside the insertion site. Fig. S4 Ynr034w A-Terminus GFP-Tag. The green fluorescent protein (GPF) and Schizosaccharomyces pombe HIS5 genes were amplified from the pFA6a-link-yEGFP-SpHIS5 plasmid (pKT128) with oligonucleotides, GFPaminoYNR034W-ADir and GFPaminoYNR034W-ARev. The GFP-HIS5 cassette was used to transform the BY4741 laboratory strain. The BY4741-YNR034W-Aamino-GFP strain was selected in YNB selective media. Correct cassette integration was verified by PCR using genomic DNA of each transformant and oligonucleotides ORFGFPYNR034W-AaminoDir and ORFGFPYNR034W-AaminoRev, designed outside the insertion site. Fig. S5 Flow cytometry analysis of BY4741-YNR034W-Aamino-GFP cells, grown in YPD supplemented with 0.6 M NaCl or 6% ethanol. Each culture was sampled at 0 h and 24 h and analyzed with a MoFlo™ XDP High-Speed Cell Sorter System (Beckman Coulter). Fluorescence was recorded from 100,000 events per sample in the FL1 channel (green fluorescence), and data analysis was performed with Summit 5.2 software. Histograms show the total fluorescence emission by cell population (FL1 channel). Representative data are from 100,000 events from biological replicates. Fig. S6 Subcellular localization of Ynr034w-a GFP. BY4741-YNR034W-Acarboxyl-GFP cells were cultured in liquid YPD for 24 h; 2x106 cells mL−1 from these cultures were inoculated in 20 mL of YPD supplemented with different stress conditions (25% glucose, 0.6 M NaCl or 6% ethanol). Cultures were grown with agitation at 28 °C. Each culture was sampled at 0 h and 24 h. Confocal microscopy images were acquired with an inverted Confocal Laser Scanning Microscope (Carl Zeiss LSM 700) with a 60X objective. Fig. S7 Cell growth of the AR5 (empty triangles), AR5-YNR034W-A (filled triangles), BY4741 (filled squares) and BY4741-YNR034 W (empty squares) yeast strains, grown in liquid YPD media. O.D. Optical Density. Data are presented as the mean of three independent experiments completed in triplicate. Fig. S8 Osmotic stress tolerance. (a) S. cerevisiae BY4741 and BY4741-YNR034W-A strains grown on YPD media plates supplemented with Hygromycin (100 µg mL−1). (b) Growth of the S. cerevisiae strains BY4741 and BY4741-YNR034W-A strains on YPD media plates supplemented with different NaCl concentrations. Fig. S9 Cell growth of the AR5 (filled symbols) and AR5-YNR034W-A (empty symbols) strains in A. tequilana Weber juice containing a sugar concentration of 180 g L−1. Data are presented as the mean of three independent experiments completed in triplicate. Cell growth data were subjected to factorial ANOVA. * Denotes a significant difference between values (p < 0.05). (PDF 4787 kb)
10295_2016_1871_MOESM2_ESM.docx (103 kb)
Supplementary material 2 (DOCX 102 kb)

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Copyright information

© Society for Industrial Microbiology and Biotechnology 2016

Authors and Affiliations

  1. 1.División de Ciencias Naturales y Exactas, Departamento de BiologíaUniversidad de GuanajuatoGuanajuatoMexico

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