title: Metabolic engineering and classical selection of the methylotrophic thermotolerant yeast Hansenula polymorpha for improvement of high-temperature xylose alcoholic fermentation.
abstract: BACKGROUND
  The methylotrophic yeast, Hansenula polymorpha is an industrially important microorganism, and belongs to the best studied yeast species with well-developed tools for molecular research. The complete genome sequence of the strain NCYC495 of H. polymorpha is publicly available. Some of the well-studied strains of H. polymorpha are known to ferment glucose, cellobiose and xylose to ethanol at elevated temperature (45 - 50°C) with ethanol yield from xylose significantly lower than that from glucose and cellobiose. Increased yield of ethanol from xylose was demonstrated following directed metabolic changes but, still the final ethanol concentration achieved is well below what is considered feasible for economic recovery by distillation.
  
  
  RESULTS
  In this work, we describe the construction of strains of H. polymorpha with increased ethanol production from xylose using an ethanol-non-utilizing strain (2EthOH-) as the host. The transformants derived from 2EthOH- overexpressing modified xylose reductase (XYL1m) and native xylitol dehydrogenase (XYL2) were isolated. These transformants produced 1.5-fold more ethanol from xylose than the original host strain. The additional overexpression of XYL3 gene coding for xylulokinase, resulted in further 2.3-fold improvement in ethanol production with no measurable xylitol formed during xylose fermentation. The best ethanol producing strain obtained by metabolic engineering approaches was subjected to selection for resistance to the known inhibitor of glycolysis, the anticancer drug 3-bromopyruvate. The best mutant selected had an ethanol yield of 0.3 g/g xylose and produced up to 9.8 g of ethanol/l during xylose alcoholic fermentation at 45°C without correction for ethanol evaporation.
  
  
  CONCLUSIONS
  Our results indicate that xylose conversion to ethanol at elevated temperature can be significantly improved in H. polymorpha by combining methods of metabolic engineering and classical selection.
authors.forename: Olena O
authors.initials: OO
authors.lastname: Kurylenko
authors.forename: Justyna
authors.initials: J
authors.lastname: Ruchala
authors.forename: Orest B
authors.initials: OB
authors.lastname: Hryniv
authors.forename: Charles A
authors.initials: CA
authors.lastname: Abbas
authors.forename: Kostyantyn V
authors.initials: KV
authors.lastname: Dmytruk
authors.affiliation: Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine. sibirny@cellbiol.lviv.ua.
authors.forename: Andriy A
authors.initials: AA
authors.lastname: Sibirny
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects; D000449:Aldehyde Reductase / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D050542:D-Xylulose Reductase / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation / Q000187:drug effects; D060847:Metabolic Engineering / Q000379:methods*; D008697:Methane / Q000378:metabolism*; D010843:Pichia / Q000187:drug effects / Q000201:enzymology / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011773:Pyruvates / Q000494:pharmacology; D013696:Temperature; D014170:Transformation, Genetic / Q000187:drug effects; D014993:Xylitol / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D011773:Pyruvates; D000431:Ethanol; C017092:bromopyruvate; D014994:Xylose; D000449:Aldehyde Reductase; D050542:D-Xylulose Reductase; D008697:Methane; D014993:Xylitol