Isolation of xylose isomerases by sequence- and function-based screening from a soil metagenomic library

<p>Abstract</p> <p>Background</p> <p>Xylose isomerase (XI) catalyses the isomerisation of xylose to xylulose in bacteria and some fungi. Currently, only a limited number of XI genes have been functionally expressed in <it>Saccharomyces cerevisiae</it>, the microorganism of choice for lignocellulosic ethanol production. The objective of the present study was to search for novel XI genes in the vastly diverse microbial habitat present in soil. As the exploitation of microbial diversity is impaired by the ability to cultivate soil microorganisms under standard laboratory conditions, a metagenomic approach, consisting of total DNA extraction from a given environment followed by cloning of DNA into suitable vectors, was undertaken.</p> <p>Results</p> <p>A soil metagenomic library was constructed and two screening methods based on protein sequence similarity and enzyme activity were investigated to isolate novel XI encoding genes. These two screening approaches identified the <it>xym1 </it>and <it>xym2 </it>genes, respectively. Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups. When <it>xym1 </it>and <it>xym2 </it>were overexpressed in a <it>xylA</it>-deficient <it>Escherichia coli </it>strain, similar growth rates to those in which the <it>Piromyces </it>XI gene was expressed were obtained. However, expression in <it>S. cerevisiae </it>resulted in only one-fourth the growth rate of that obtained for the strain expressing the <it>Piromyces </it>XI gene.</p> <p>Conclusions</p> <p>For the first time, the screening of a soil metagenomic library in <it>E. coli </it>resulted in the successful isolation of two active XIs. However, the discrepancy between XI enzyme performance in <it>E. coli </it>and <it>S. cerevisiae </it>suggests that future screening for XI activity from soil should be pursued directly using yeast as a host.</p

Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Xylose reductase (XR) and xylitol dehydrogenase (XDH) from <it>Pichia stipitis </it>are the two enzymes most commonly used in recombinant <it>Saccharomyces cerevisiae </it>strains engineered for xylose utilization. The availability of NAD⁺for XDH is limited during anaerobic xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of <it>P. stipitis </it>XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference.</p> <p>Results</p> <p>XR variants were evaluated in <it>S. cerevisiae </it>strains with the following genetic modifications: overexpressed native <it>P. stipitis </it>XDH, overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deleted GRE3 gene encoding an NADPH dependent aldose reductase. All overexpressed genes were chromosomally integrated to ensure stable expression. Crude extracts of four different strains overexpressing genes encoding native <it>P. stipitis </it>XR, K270M and K270R mutants, as well as <it>Candida parapsilosis </it>XR, were enzymatically characterized. The physiological effects of the mutations were investigated in anaerobic xylose fermentation. The strain overexpressing <it>P. stipitis </it>XR with the K270R mutation gave an ethanol yield of 0.39 g (g consumed sugars)^-1, a xylitol yield of 0.05 g (g consumed xylose)^-1and a xylose consumption rate of 0.28 g (g biomass)^-1h^-1in continuous fermentation at a dilution rate of 0.12 h^-1, with 10 g l^-1glucose and 10 g l^-1xylose as carbon sources.</p> <p>Conclusion</p> <p>The cofactor preference of <it>P. stipitis </it>XR was altered by site-directed mutagenesis. When the K270R XR was combined with a metabolic engineering strategy that ensures high xylose utilization capabilities, a recombinant <it>S. cerevisiae </it>strain was created that provides a unique combination of high xylose consumption rate, high ethanol yield and low xylitol yield during ethanolic xylose fermentation.</p

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

<p>Abstract</p> <p>Background</p> <p>Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant <it>Saccharomyces cerevisiae </it>strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in <it>S. cerevisiae </it>to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.</p> <p>Results</p> <p>The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1h^-1than the xylose isomerase strain, which only reached 0.03 h^-1and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1compared with 0.18 g g^-1for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1compared with 0.32 g g^-1for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1h^-1compared with 0.01 g (g cells)^-1h^-1for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.</p> <p>Conclusion</p> <p>The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.</p

PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>In <it>Saccharomyces cerevisiae </it>galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene <it>PGM2 </it>encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of <it>PGM2 </it>has previously been shown to enhance aerobic growth of <it>S. cerevisiae </it>in galactose medium.</p> <p>Results</p> <p>In the present study we show that overexpression of <it>PGM2 </it>under control of the <it>HXT7'</it>promoter from an integrative plasmid increased the PGM activity 5 to 6 times, which significantly reduced the lag phase of glucose-pregrown cells in an anaerobic galactose culture. <it>PGM2 </it>overexpression also increased the anaerobic specific growth rate whereas ethanol production was less influenced. When <it>PGM2 </it>was overexpressed from a multicopy plasmid instead, the PGM activity increased almost 32 times. However, this increase of PGM activity did not further improve aerobic galactose fermentation as compared to the strain carrying <it>PGM2 </it>on the integrative plasmid.</p> <p>Conclusion</p> <p><it>PGM2 </it>overexpression in <it>S. cerevisiae </it>from an integrative plasmid is sufficient to reduce the lag phase and to enhance the growth rate in anaerobic galactose fermentation, which results in an overall decrease in fermentation duration. This observation is of particular importance for the future development of stable industrial strains with enhanced PGM activity.</p

Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway

<p>Abstract</p> <p>Background</p> <p>Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. <it>Saccharomyces cerevisiae </it>is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation.</p> <p>Results</p> <p>A new recombinant <it>S. cerevisiae </it>strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized.</p> <p>Conclusion</p> <p>Pentose fermenting recombinant <it>S. cerevisiae </it>strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar.</p

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

BACKGROUND: Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. RESULTS: We describe the engineering of laboratory and industrial S. cerevisiae strains to co-ferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. CONCLUSION: Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production

Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae

BACKGROUND: Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. RESULTS: In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. CONCLUSION: Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed

Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)

<p>Abstract</p> <p>Background</p> <p><it>Pichia stipitis </it>xylose reductase (Ps-XR) has been used to design <it>Saccharomyces cerevisiae </it>strains that are able to ferment xylose. One example is the industrial <it>S. cerevisiae </it>xylose-consuming strain TMB3400, which was constructed by expression of <it>P. stipitis </it>xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial <it>S. cerevisiae </it>strain USM21.</p> <p>Results</p> <p>In this study, we demonstrate that strain TMB3400 not only converts xylose, but also displays higher tolerance to lignocellulosic hydrolysate during anaerobic batch fermentation as well as 3 times higher <it>in vitro </it>HMF and furfural reduction activity than the control strain USM21. Using laboratory strains producing various levels of Ps-XR, we confirm that Ps-XR is able to reduce HMF both <it>in vitro </it>and <it>in vivo</it>. Ps-XR overexpression increases the <it>in vivo </it>HMF conversion rate by approximately 20%, thereby improving yeast tolerance towards HMF. Further purification of Ps-XR shows that HMF is a substrate inhibitor of the enzyme.</p> <p>Conclusion</p> <p>We demonstrate for the first time that xylose reductase is also able to reduce the furaldehyde compounds that are present in undetoxified lignocellulosic hydrolysates. Possible implications of this newly characterized activity of Ps-XR on lignocellulosic hydrolysate fermentation are discussed.</p

Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering

<p>Abstract</p> <p>Background</p> <p>Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by <it>Saccharomyces cerevisiae </it>requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced.</p> <p>Results</p> <p>Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial <it>Saccharomyces cerevisiae </it>strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways.</p> <p>Conclusion</p> <p>To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant <it>S. cerevisiae </it>strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.</p