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Background: Poly-3-d-hydroxybutyrate (PHB) that is a promising precursor for bioplastic with similar physical properties as polypropylene, is naturally produced by several bacterial species. The bacterial pathway is comprised of the three enzymes ß-ketothiolase, acetoacetyl-CoA reductase (AAR) and PHB synthase, which all together convert acetyl-CoA into PHB. Heterologous expression of the pathway genes from Cupriavidus necator has enabled PHB production in the yeast Saccharomyces cerevisiae from glucose as well as from xylose, after introduction of the fungal xylose utilization pathway from Schefersomyces stipitis including xylose reductase (XR) and xylitol dehydrogenase (XDH). However PHB titers are still low. Results: In this study the acetoacetyl-CoA reductase gene from C. necator (CnAAR), a NADPH-dependent enzyme, was replaced by the NADH-dependent AAR gene from Allochromatium vinosum (AvAAR) in recombinant xylose-utilizing S. cerevisiae and PHB production was compared. A. vinosum AAR was found to be active in S. cerevisiae and able to use both NADH and NADPH as cofactors. This resulted in improved PHB titers in S. cerevisiae when xylose was used as sole carbon source (5-fold in aerobic conditions and 8.4-fold under oxygen limited conditions) and PHB yields (4-fold in aerobic conditions and up to 5.6-fold under oxygen limited conditions). Moreover, the best strain was able to accumulate up to 14% of PHB per cell dry weight under fully anaerobic conditions. Conclusions: This study reports a novel approach for boosting PHB accumulation in S. cerevisiae by replacement of the commonly used AAR from C. necator with the NADH-dependent alternative from A. vinosum. Additionally, to the best of our knowledge, it is the first demonstration of anaerobic PHB synthesis from xylose. [ABSTRACT FROM AUTHOR]

Background: The production of ethanol and other fuels and chemicals from lignocellulosic materials is dependent of efficient xylose conversion. Xylose fermentation capacity in yeasts is usually linked to xylose reductase (XR) accepting NADH as cofactor. The XR from Scheffersomyces stipitis, which is able to use NADH as cofactor but still prefers NADPH, has been used to generate recombinant xylose-fermenting Saccharomyces cerevisiae. Novel xylose-fermenting yeasts species, as those from the Spathaspora clade, have been described and are potential sources of novel genes to improve xylose fermentation in S. cerevisiae. Results: Xylose fermentation by six strains from different Spathaspora species isolated in Brazil, plus the Sp. passalidarum type strain (CBS 10155T), was characterized under two oxygen-limited conditions. The best xylose-fermenting strains belong to the Sp. passalidarum species, and their highest ethanol titers, yields, and productivities were correlated to higher XR activity with NADH than with NADPH. Among the different Spathaspora species, Sp. passalidarum appears to be the sole harboring two XYL1 genes: XYL1.1, similar to the XYL1 found in other Spathaspora and yeast species and XYL1.2, with relatively higher expression level. XYL1.1p and XYL1.2p from Sp. passalidarum were expressed in S. cerevisiae TMB 3044 and XYL1.1p was confirmed to be strictly NADPH-dependent, while XYL1.2p to use both NADPH and NADH, with higher activity with the later. Recombinant S. cerevisiae strains expressing XYL1.1p did not show anaerobic growth in xylose medium. Under anaerobic xylose fermentation, S. cerevisiae TMB 3504, which expresses XYL1.2p from Sp. passalidarum, revealed significant higher ethanol yield and productivity than S. cerevisiae TMB 3422, which harbors XYL1p N272D from Sc. stipitis in the same isogenic background (0.40 vs 0.34 g g-1 CDW and 0.33 vs 0.18 g g-1 CDW h-1, respectively). Conclusion: This work explored a new clade of xylose-fermenting yeasts (Spathaspora species) towards the engineering of S. cerevisiae for improved xylose fermentation. The new S. cerevisiae TMB 3504 displays higher XR activity with NADH than with NADPH, with consequent improved ethanol yield and productivity and low xylitol production. This meaningful advance in anaerobic xylose fermentation by recombinant S. cerevisiae (using the XR/XDH pathway) paves the way for the development of novel industrial pentose-fermenting strains. [ABSTRACT FROM AUTHOR]

Carboxylic acids are important bulk chemicals that can be used as building blocks for the production of polymers, as acidulants, preservatives and flavour compound or as precursors for the synthesis of pharmaceuticals. Today, their production mainly takes place through catalytic processing of petroleum-based precursors. An appealing alternative would be to produce these compounds from renewable resources, using tailor-made microorganisms. Saccharomyces cerevisiae has already demonstrated its value for bioethanol production from renewable resources. In this review, we discuss Saccharomyces cerevisiae engineering potential, current strategies for carboxylic acid production as well as the specific challenges linked to the use of lignocellulosic biomass as carbon source. [ABSTRACT FROM AUTHOR]

Hexokinase 2 (Hxk2p) from Saccharomyces cerevisiae is a bi-functional enzyme being both a catalyst and an important regulator in the glucose repression signal. In the presence of xylose Hxk2p is irreversibly inactivated through an autophosphorylation mechanism, affecting all functions. Consequently, the regulation of genes involved in sugar transport and fermentative metabolism is impaired. The aim of the study was to obtain new Hxk2p-variants, immune to the autophosphorylation, which potentially can restore the repressive capability closer to its nominal level. In this study we constructed the first condensed, rationally designed combinatorial library targeting the active-site in Hxk2p. We combined protein engineering and genetic engineering for efficient screening and identified a variant with Phe159 changed to tyrosine. This variant had 64% higher catalytic activity in the presence of xylose compared to the wild-type and is expected to be a key component for increasing the productivity of recombinant xylose-fermenting strains for bioethanol production from lignocellulosic feedstocks. [ABSTRACT FROM AUTHOR]

A highly combinatorial structure-based protein engineering method for obtaining enantioselectivity is reported that results in a thorough modification of the substrate binding pocket of Candida antarctica lipase A (CALA). Nine amino acid residues surrounding the entire pocket were simultaneously mutated, contributing to a reshaping of the substrate pocket to give increased enantioselectivity and activity for a sterically demanding substrate. This approach seems to be powerful for developing enantio-selectivity when a complete reshaping of the active site is required. Screening toward ibuprofen ester 1, a substrate for which previously used methods had failed, gave variants with a significantly increased enantioselectivity and activity. Wild-type CALA has a moderate activity with an if value of only 3.4 toward this substrate. The best variant had an E value of 100 and it also displayed a high activity. The variation at each mutated position was highly reduced, comprising only the wild type and an alternative residue, preferably a smaller one with similar properties. These minimal binary variations allow for an extremely condensed protein library. With this highly combinatorial method synergistic effects are accounted for and the protein fitness landscape is explored efficiently. [ABSTRACT FROM AUTHOR]

Time-resolved absorption spectra of the FADF-containing enzyme mercuric reductase were recorded during the catalytic reaction at 25°C, pH 7.3. With an excess of NADPH over Hg2+ there was a rapid (k = 43 s-1) initial formation of a spectral species similar to that previously assigned to an NADPH complex of two-electron- reduced enzyme. EH2-NADPH. This spectrum persisted during the quasisteady-state phase of the reaction suggesting that EH2-NADPH is a true catalytic intermediate and that the rate of catalysis is limited by the oxidation of EH2-NADPH by Hg2+. Also with an excess of Hg2+ over NADPH a spectrum similar to that of EH2-NADPH was rapidly formed. As the NADPH was exhausted, the spectrum of oxidized enzyme, E, did not reappear but rather a spectrum similar to that previously assigned to an NADP+ complex of two-electron-reduced enzyme, EH2-NADP+. These results suggest that EH2-NADP+ cannot rapidly reduce the Hg2+ substrate. However, eventually all reducing equivalents from NADPH added to oxidized, activated enzyme are utilized for the reduction of Hg2+. A mechanism model is proposed that does not involve the free, oxidized enzyme in the catalytic cycle. [ABSTRACT FROM AUTHOR]

The binding of N3- to Co(II)-substituted bovine carbonic anhydrase III was measured at various pH values by spectrophotometric titrations. The apparent Ki values were found to increase with pH in the studied range between pH 5.8 and 8.9. The inhibition of CO2 hydration by N3- was found to be essentially uncompetitive at all investigated pH values (pH 6.3-8.9). The Ki values for the inhibition of kcat are much smaller than those obtained in the spectrophotometric titrations indicating that an enzyme form with a high affinity for N3-, presumably having a metal-bound H2O, accumulates in the steady state at saturating CO2 concentrations. Assuming that the low pH limit of Ki = 9 µM for the inhibition of kcat represents the affinity of N3- for the Co(II)-OH2 form, a pKa value near 5 can be estimated for Co(II)-bound water from the pH dependence of N3- binding in the absence of CO2. Measurements of time-resolved absorption spectra during CO2 hydration in the presence of a low Nj concentration showed the transient appearance of the characteristic spectrum of the enzyme-N3- adduct clearly demonstrating the accumulation in the steady state of an enzyme form with a high affinity for N3-. In similar experiments without inhibitor the transient formation of a spectral form corresponding to a Co(II)-OH2 species has been demonstrated. This spectral form is rather featureless lacking the absorption maxima at 618 nm and 640 nm characteristic of the Co(II)-OH- species. Our results strongly support the hypothesis that the rate-limiting step in CO2 hydration catalyzed by carbonic anhydrase III is the protolysis of metal-bound water. [ABSTRACT FROM AUTHOR]

Preincubation of the oxidized form of the flavoenzyme mercuric reductase with the reducing substrate, NADPH, or with a high concentration of cysteine (30 mM) results in a substantial increase of the catalytic activity as measured in a standard spectrophotometric assay. Also NADH has some activating effect but NADP+ or EDTA have no effect. In the presence of 1 mM cysteine only one equivalent of NADPH per FAD seems to be required for full activation which occurs after an incubation time of about 10 rain. Activated mercuric reductase appears to be stable under anaerobic conditions but eventually returns to the original level of activity in the presence of oxygen. The activated state seems to be stabilized by 1 mM cysteine. Activation of mercuric reductase does not seem to be correlated with a change in the number of reactive thiol groups. The chemical nature of the activation process is not yet understood. Stopped-flow studies have shown that the nonactivated enzyme is practically inactive prior to contact with the substrates. The enzyme is gradually activated during the assay. The kinetics of activation of the ‘native’ enzyme is biphasic but ‘clipped’ enzyme, lacking an 85-residue N-terminal domain, is activated in a single first-order process. The progress curves obtained with preactivated enzyme are approximately exponential even at saturating concentrations of NADPH (Km = 0.4 μM at 25°C, pH 7.3) and Hg2+ (Km = 3.2 μM in the presence of 1 mM cysteine). The initial rates yield kcat values of about 13 s-1 per FAD molecule (25 °C, pH 7.3). We find no evidence for a thiol-dependent change from a rapid to a slow kinetic phase. The shape of the progress curves presumably depends on product inhibition, but NADP+ is not a sufficiently effective inhibitor to explain the effect fully. [ABSTRACT FROM AUTHOR]

Engineering of the yeast Saccharomyces cerevisiae towards efficient d-xylose assimilation has been a major focus over the last decades since d-xylose is the second most abundant sugar in nature, and its conversion into products could significantly improve process economy in biomass-based processes. Up to now, two different metabolic routes have been introduced via genetic engineering, consisting of either the isomerization or the oxido-reduction of d-xylose to d-xylulose that is further connected to the pentose phosphate pathway and glycolysis. In the present study, cytosolic d-xylose oxidation was investigated instead, through the introduction of the Weimberg pathway from Caulobacter crescentus in S. cerevisiae. This pathway consists of five reaction steps that connect d-xylose to the TCA cycle intermediate α-ketoglutarate. The corresponding genes could be expressed in S. cerevisiae, but no growth was observed on d-xylose indicating that not all the enzymes were functionally active. The accumulation of the Weimberg intermediate d-xylonate suggested that the dehydration step(s) might be limiting, blocking further conversion into α-ketoglutarate. Although four alternative dehydratases both of bacterial and archaeon origins were evaluated, d-xylonate accumulation still occurred. A better understanding of the mechanisms associated with the activity of dehydratases, both at a bacterial and yeast level, appears essential to obtain a fully functional Weimberg pathway in S. cerevisiae. [ABSTRACT FROM AUTHOR]