Peroxisomes and peroxisomal transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha

Abstract

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.

Engineering of sugar transporters for improvement of xylose utilization during high-temperature alcoholic fermentation in Ogataea polymorpha yeast

“…The reasons of this phenomenon are not known, however, as methods of molecular genetics are well developed for this organism and genome sequence is publicly available, O. polymorpha is considered as a promising model organism for construction of the efficient thermotolerant ethanol producer. A combination of metabolic engineering and classical selection approaches was successfully used for improvement of parameters of xylose alcoholic fermentation in O. polymorpha [2,3,16]. However, even recombinant strains with improved ethanol production up to 25-30 times were characterized by incomplete xylose utilization during fermentation.…”

“…The DNA fragment harboring the gene coding for the green fluorescent protein (GFP) was amplified using primers Ko819 and Ko820 from the plasmid pGFP-SLK [3]. The backbone plasmids containing HXT1 or HXT1_ N358A_K was amplified with the primers Ko821/Ko822 from the plasmids pUC19/zeo/HXT1 or pUC19_prGAP_ NTC_HXT1_ N358A_K.…”

“…The Advanced O. polymorpha ethanol producers from xylose were obtained by a combination of the methods of metabolic engineering and classical selection [2,3]. Although such recombinant strains were characterized by 25-30-fold improved ethanol production from xylose as compared to the wild-type strain, xylose uptake was slow and incomplete.…”

Engineering of sugar transporters for improvement of xylose utilization during high-temperature alcoholic fermentation in Ogataea polymorpha yeast

“…The DNA fragment harboring the gene coding for the green fluorescent protein (GFP) was amplified using primers Ko819 and Ko820 from the plasmid pGFP-SLK [3]. The backbone plasmids containing HXT1 or HXT1_N358A_K was amplified with the primers Ko821/Ko822 from the plasmids pUC19/zeo/HXT1 or pUC19_prGAP_NTC_HXT1_ N358A_K.…”

“…Advanced O. polymorpha ethanol producers from xylose were obtained by a combination of the methods of metabolic engineering and classical selection [2,3]. Although such recombinant strains were characterized by 25-30-fold improved ethanol production from xylose as compared to the wildtype strain, xylose uptake was slow and incomplete.…”