Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae TMB3001, harboring the Pichia stipitis genes XYL1 and XYL2 (xylose reductase and xylitol dehydrogenase, respectively) and the endogenous XKS1(xylulokinase), can convert xylose to ethanol. About 30% of the consumed xylose, however, is excreted as xylitol. Enhanced ethanol yield has previously been achieved by disrupting the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase, but at the expense of the xylose consumption. This is probably the result of reduced NADPH-mediated xylose reduction. In the present study, we increased the xylose reductase (XR) activity 4-19 times in both TMB3001 and the ZWF1-disrupted strain TMB3255. The xylose consumption rate increased by 70% in TMB3001 under oxygen-limited conditions. In the ZWF1-disrupted background, the increase in XR activity fully restored the xylose consumption rate. Maximal specific growth rates on glucose were lower in the ZWF1-disrupted strains, and the increased XR activity also negatively affected the growth rate in these strains. Addition of methionine resulted in 70% and 50% enhanced maximal specific growth rates for TMB3255 (zwfl Delta) and TMB3261 (PGK1-XYL1, zwf1 Delta), respectively. Enhanced XR activity did not have any negative effect on the maximal specific growth rate in the control strain. Enhanced glycerol yields were observed in the high-XR-activity strains. These are suggested to result from the observed reductase activity of the purified XR for dihydroxyacetone phosphate.     

Variability of the response of Saccharomyces cerevisiae strains to lignocellulose hydrolysate

The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory lignocellulose hydrolysates. In the current work, the fermentation performance of six selected strains of Saccharomyces cerevisiae in dilute-acid spruce hydrolysate was compared using two different modes of fermentation; either single pulse addition of hydrolysate to exponentially growing cells or continuous feeding of the same amount of hydrolysate in a controlled fed-batch fermentation was made. All strains performed better in fed-batch mode than when all hydrolysate was added at once. However, the difference between strain performances varied significantly in the two fermentation modes. Large differences were observed between strains during the fed-batch experiments in the in vitro ability to reduce the furan compounds furfural and 5-hydroxymethyl furfural (HMF). A common feature among the strains was the induction of NADPH-coupled reduction of furfural and HMF, with the exception of strain CBS 8066. This strain also performed relatively poorly in both batch and fed-batch fermentations. Strain TMB3000--previously isolated from spent sulphite liquor fermentation--was by far the most efficient strain with respect to specific fermentation rate in both pulse addition and fed-batch mode. This strain was the only strain showing a significant constitutive NADH-coupled in vitro reduction of HMF. The ability to induce NADPH-coupled reduction together with the level of the apparently constitutive NADH-coupled reduction appeared to be key factors for selecting a suitable strain for fed-batch conversion of lignocellulose hydrolysate.     

Flavonoids as inhibitors of human carbonyl reductase 1

Human carbonyl reductase 1 (CBR1), that is one of the enzymes responsible for the reduced efficiency of treatments by the antineoplastic agents anthracyclines, was functionally expressed in Saccharomyces cerevisiae. CBR1 was purified and kinetically characterised using daunorubicin as substrate. CBR1-catalysed reduction of daunorubicin followed an apparent Michaelis-Menten kinetics with K(M)=85.2+/-26.7microM and V(max)=3490+/-220micromol/(mingprotein). The type of inhibition for the flavonoid compound rutin was determined by studying initial reaction rates in the presence of rutin. The inhibition kinetics was found to follow an apparent mixed inhibition with K(ic)=1.8+/-1.2microM and K(iu)=2.8+/-1.6microM. IC50-values were also determined for a set of flavonoids in order to identify essential structure for inhibition activity. Computational docking experiments of the four best inhibitors to the catalytic site of CBR1 showed that the flavonoid skeleton structure was the binding part of the molecule. The presence of a sugar moiety in 1 and 2, or a sugar mimicking part in 9, directed the orientation of the flavonoid so that the sugars were pointing outwards, giving rise to a stabilising effect to the binding. Finally, additional binding epitopes that interacted with various parts of the flavonoid ligand were identified and could potentially be targeted for further improvement of inhibition activity. These included; hydrogen-binding sites surrounding Ser139 and Cys226, Met234 and Tyr193 or Trp229; aromatic-aromatic interaction with Tyr193, Trp229 or NADPH; van der Waals interactions with Ile140.     

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

Background  

Xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis are the two enzymes most commonly used in recombinant Saccharomyces cerevisiae 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 P. stipitis XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference.