BackgroundThe 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.ResultsIn 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.ConclusionsOur 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.

BackgroundEfficient xylose alcoholic fermentation is one of the key to a successful lignocellulosic ethanol production. However, regulation of this process in the native xylose-fermenting yeasts is poorly understood. In this work, we paid attention to the transcriptional factor Cat8 and its possible role in xylose alcoholic fermentation in Ogataea (Hansenula) polymorpha. In Saccharomyces cerevisiae, organism, which does not metabolize xylose, gene CAT8 encodes a Zn-cluster transcriptional activator necessary for expression of genes involved in gluconeogenesis, respiration, glyoxylic cycle and ethanol utilization. Xylose is a carbon source that could be fermented to ethanol and simultaneously could be used in gluconeogenesis for hexose synthesis. This potentially suggests involvement of CAT8 in xylose metabolism.ResultsHere, the role of CAT8 homolog in the natural xylose-fermenting thermotolerant yeast O. polymorpha was characterized. The CAT8 ortholog was identified in O. polymorpha genome and deleted both in the wild-type strain and in advanced ethanol producer from xylose. Constructed cat8Δ strain isolated from wild strain showed diminished growth on glycerol, ethanol and xylose as well as diminished respiration on the last substrate. At the same time, cat8Δ mutant isolated from the best available O. polymorpha ethanol producer showed only visible defect in growth on ethanol. CAT8 deletant was characterized by activated transcription of genes XYL3, DAS1 and RPE1 and slight increase in the activity of several enzymes involved in xylose metabolism and alcoholic fermentation. Ethanol production from xylose in cat8Δ mutants in the background of wild-type strain and the best available ethanol producer from xylose increased for 50 and 30%, respectively. The maximal titer of ethanol during xylose fermentation was 12.5 g ethanol/L at 45 °C. Deletion of CAT8 did not change ethanol production from glucose. Gene CAT8 was also overexpressed under control of the strong constitutive promoter GAP of glyceraldehyde-3-phosphate dehydrogenase. Corresponding strains showed drop in ethanol production in xylose medium whereas glucose alcoholic fermentation remained unchanged. Available data suggest on specific role of Cat8 in xylose alcoholic fermentation.ConclusionsThe CAT8 gene is one of the first identified genes specifically involved in regulation of xylose alcoholic fermentation in the natural xylose-fermenting yeast O. polymorpha.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0652-6) contains supplementary material, which is available to authorized users.

This review summarizes progress in the construction of efficient yeast ethanol producers from glucose/sucrose and lignocellulose. Saccharomyces cerevisiae is the major industrial producer of first-generation ethanol. The different approaches to increase ethanol yield and productivity from glucose in S. cerevisiae are described. Construction of the producers of secondgeneration ethanol is described for S. cerevisiae, one of the best natural xylose fermenters, Scheffersomyces stipitis and the most thermotolerant yeast known Ogataea polymorpha. Each of these organisms has some advantages and drawbacks. S. cerevisiae is the primary industrial ethanol producer and is the most ethanol tolerant natural yeast known and, however, cannot metabolize xylose. S. stipitis can effectively ferment both glucose and xylose and, however, has low ethanol tolerance and requires oxygen for growth. O. polymorpha grows and ferments at high temperatures and, however, produces very low amounts of ethanol from xylose. Review describes how the mentioned drawbacks could be overcome.

BackgroundOgataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve high-temperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol.ResultsInvolvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30–40 times more ethanol than is produced by the wild-type strain.ConclusionsOur results indicate the importance of the peroxisomal enzymes, transketolase (dihydroxyacetone synthase, Das1), and transaldolase (Tal2), in the xylose alcoholic fermentation of O. polymorpha.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1203-z) contains supplementary material, which is available to authorized users.