Metabolic engineering and classical selectionof the methylotrophic thermotolerant yeast Hansenulapolymorpha for improvement of high-temperature xylose alcoholicfermentation

Microbial Cell Factories

Research

Charles A Abbas¹  Justyna Ruchala²  Kostyantyn V Dmytruk³  Orest B Hryniv³  Olena O Kurylenko³  Andriy A Sibirny⁴ 

[1] ADM Research, 62526, Decatur, IL, USA;Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601, Rzeszow, Poland;Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street, 14/16, 79005, Lviv, Ukraine;Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street, 14/16, 79005, Lviv, Ukraine;Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601, Rzeszow, Poland;

关键词: 3-Bromopyruvate;    High-temperature fermentation;    Xylose;    Fuel ethanol;    Hansenula polymorpha;   

DOI  :  10.1186/s12934-014-0122-3

 received in 2014-05-17, accepted in 2014-08-12,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundThe methylotrophic yeast, Hansenulapolymorpha is an industrially important microorganism, andbelongs to the best studied yeast species with well-developed tools formolecular research. The complete genome sequence of the strain NCYC495 ofH. polymorpha is publicly available. Someof the well-studied strains of H. polymorphaare known to ferment glucose, cellobiose and xylose to ethanol at elevatedtemperature (45 – 50°C) with ethanol yield from xylose significantly lower thanthat from glucose and cellobiose. Increased yield of ethanol from xylose wasdemonstrated following directed metabolic changes but, still the final ethanolconcentration achieved is well below what is considered feasible for economicrecovery by distillation.ResultsIn this work, we describe the construction of strains of H. polymorpha with increased ethanol productionfrom xylose using an ethanol-non-utilizing strain(2EthOH−) as the host. The transformants derivedfrom 2EthOH− overexpressing modified xylose reductase(XYL1m) and native xylitol dehydrogenase(XYL2) were isolated. These transformantsproduced 1.5-fold more ethanol from xylose than the original host strain. Theadditional overexpression of XYL3 gene codingfor xylulokinase, resulted in further 2.3-fold improvement in ethanol productionwith no measurable xylitol formed during xylose fermentation. The best ethanolproducing strain obtained by metabolic engineering approaches was subjected toselection for resistance to the known inhibitor of glycolysis, the anticancerdrug 3-bromopyruvate. The best mutant selected had an ethanol yield of 0.3 g/gxylose and produced up to 9.8 g of ethanol/l during xylose alcoholicfermentation at 45°C without correction for ethanol evaporation.ConclusionsOur results indicate that xylose conversion to ethanol at elevated temperaturecan be significantly improved in H.polymorpha by combining methods of metabolic engineering andclassical selection.

【 授权许可】

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© Kurylenko et al.; licensee BioMed Central 2014. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproductionin any medium, provided the original work is properly credited. The CreativeCommons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unlessotherwise stated.