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Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

Mekonnen M Demeke12, Heiko Dietz3, Yingying Li12, María R Foulquié-Moreno12, Sarma Mutturi4, Sylvie Deprez5, Tom Den Abt12, Beatriz M Bonini12, Gunnar Liden4, Françoise Dumortier12, Alex Verplaetse5, Eckhard Boles3 and Johan M Thevelein12*

Author Affiliations

1 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium

2 Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven, Heverlee, Flanders, Belgium

3 Institute of Molecular Biosciences, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany

4 Department of Chemical Engineering, Lund University, P.O. Box 124, 22100 Lund, Sweden

5 Laboratory of Enzyme, Fermentation and Brewing Technology, KAHO Sint-Lieven University College, KU Leuven Association, Gebroeders De Smetstraat 1, 9000, Ghent, Flanders, Belgium

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Biotechnology for Biofuels 2013, 6:89  doi:10.1186/1754-6834-6-89

Published: 21 June 2013

Abstract

Background

The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production.

Results

An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate.

Conclusions

An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates.

Keywords:
Bioethanol; Lignocellulose; D-xylose fermentation; D-xylose isomerase; Inhibitor tolerance; Saccharomyces cerevisiae; Evolutionary engineering