Screening of Key Genes Responsible for Ethanol Fermentation in Xylose-Metabolizing Yeast Strains

Abstract:

Based on comparison analysis of transcriptome sequencing data, potential genes responsible for xylose fermentation to ethanol were screened and chosen from two model yeast strains of Candida tropicalis and Candida shehatae. Thereafter, key genes were indentified from these candidate genes by the combined method of quantitative real-time PCR detection and metabolism pathway analysis. These genes regulatory mechanism on sugar metabolic pathway were further investigated and illustrated. Four key genes responsible for xylose fermentation to ethanol are determined and confirmed finally as follows: xylose reductase, xylitol dehydrogenase, high-affinity glucose transportor and pyruvate kinase.

• Periodical: Applied Mechanics and Materials (Volumes 448-453)
• Pages: 1592-1598
• DOI: https://doi.org/10.4028/www.scientific.net/AMM.448-453.1592
• Online since: October 2013
• Authors: Peng Cai*, Yuan Yuan Miao, Xu Jie Xiong, Yong Xu
• Keywords: Ethanol Fermentation, Key Genes, Real-Time PCR, Transcriptome Sequencing

References

[1] ZALDIVAR J, NIELSEN J, OLSSON L. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration . Appl Microbiol Biotechnol，2001, 56 (1): 17-34.

DOI: 10.1007/s002530100624

[2] Xu Y, Shen C, Qiu X T, et al. Screening and Analysis of Genes Related to Xylose Fermentation to Ethanol in Candida tropiclis. China Biotechnology, 2012, 32 (11): 61-69.

[3] ZHANG W, GENG A L. Improved ethanol production by a xylose fermenting recombinant yeast strain constructed through a modified genome shuffling method . Biotechnol Biofuel, 2012, 5 (1): 46.

DOI: 10.1186/1754-6834-5-46

[4] PARAMJIT K, BAJWA A, DOMINIC P, et al. Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling . J Microbiol Methods, 2010, 81 (2): 179-186.

DOI: 10.1016/j.mimet.2010.03.009

[5] ZHOU H, CHENG J S, WANG B L, et al. Xylose isomerase overexpression along with engineering of the pentose Phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae . Metabol Engin, 2012, 14 (6): 611-622.

DOI: 10.1016/j.ymben.2012.07.011

[6] MOROZOVA O, MARRA M A. Applications of next-generation sequencing technologies in functional genomics . Genomics, 2008, 92: 255–264.

DOI: 10.1016/j.ygeno.2008.07.001

[7] Xiong X J, Cai P, Xu Y, et al. Transcript profile of converting xylose and glucose to ethanol by Candida shehatae. Acta Microbiologica Sinica, 2013, 53 (4): 338-345.

[8] Tania N, Rebecca E H, Stephen A B. Quantification of mRNA using real-time RT-PCR . Nat Protocol，2006，1: 1559-1582.

[9] Thomas D S, Kenneth J L. Analyzing real-time PCR data by the comparative CT method . Nat Protocol， 2008, 3: 1101-1108.

[10] Kenneth JL, Thomas DS. Analysis of Relative Gene Expression Data Using Real- Time Quantitative PCR and the 2-△△Ct. Method, 2001, 25: 402–408.

DOI: 10.1006/meth.2001.1262

[11] Xu Y, Zhang B, Yong Q, et al. Analysis of Polygenetic affinities among yeast strains with random amplified polymorphism DNA analysis. Chemistry and Industry of Forest Products, 2005, 25(1): 1-4.

[12] Xiong X J, Xu Y, Zhang X Y, et al. Ethanol Fermentation Performance by Candida shehatae. Journal of Microbiology, 2011, 31(6): 43-48.

[13] Wang N, Yuan W J, Bai F W, et al. Research Progress On Xylose Transporters in Yeast. Journal of Agricultural Science and Technology, 2012, 14 (4): 24-30.

[14] KILIAN S G, UDEN N V. Transport of xylose and glucose in the xylose fermentationyeast Pichia stipitis. Appl Microbiol Biotechnol, 1988, 27(5): 545-548.

DOI: 10.1007/bf00451629

[15] Dana J W, ALAN K, TREY K S, et al. Comparative genomics of xylose fermentationfungi for enhanced biofuel production. PNAS, 2011, 108 (32): 13212–13217.

[16] ELIASSON A, CHRISTENSSON C, WAHLBOM C F, et al. Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures. Appl Environ Microbiol, 2000, 66(8): 3381-3386.

DOI: 10.1128/aem.66.8.3381-3386.2000

[17] WALFRIDSSON M，ANDERUND M，BAO X，et al. Expression of different levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its effects on product formation during xylose utilization. Appl Microbiol Biotechnol, 1997, 48(2): 218-224.

DOI: 10.1007/s002530051041

[18] BENJAPHOKEE S, KOEDRITH P, AUESUKAREE C, et al. CDC19 encoding pyruvate kinase is important for high temperature tolerance in Saccharomyces cerevisiae. New Biotechnol, 2012, 29(2): 166–176.

DOI: 10.1016/j.nbt.2011.03.007