Anaerobic poly-3-D-hydroxybutyrate production from xylose in recombinant Saccharomyces cerevisiae using a NADH-dependent acetoacetyl-CoA reductase.

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 Scheffersomyces 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.

Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library.

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 enantioselectivity 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 E 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.

Saccharomyces cerevisiae: a potential host for carboxylic acid production from lignocellulosic feedstock?

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.

Re-evaluation of the impact of BUD21 deletion on xylose utilization by Saccharomyces cerevisiae.

Various rational metabolic engineering and random approaches have been applied to introduce and improve xylose utilization and ethanol productivity by Saccharomyces cerevisiae. Among them, the BUD21 gene was identified as an interesting candidate for enhancing xylose consumption as its deletion appeared to be sufficient to improve growth, substrate utilization and ethanol productivity on xylose, even in a laboratory strain lacking a heterologous xylose pathway. The present study aimed at studying the influence of BUD21 deletion in recombinant strains carrying heterologous oxido-reductive xylose utilization pathway. The positive effect of BUD21 gene deletion on aerobic growth and xylose utilization could not be confirmed in two non-engineered laboratory strains (BY4741 and CEN.PK 113-7D) that were grown in YP rich medium with 20 g/L xylose as sole carbon source, despite the fact that effective deletion of BUD21 gene was confirmed using both genotypic (colony PCR) and phenotypic (heat sensitive phenotype of the BUD21 deletion mutant) control experiments. Therefore, the effect of BUD21 deletion on xylose fermentation might be strain- or medium-dependent.

Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of alpha-substituted esters.

A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of alpha-substituted p-nitrophenyl esters by directed evolution. The E values of this variant for 7 different esters was 45-276, which is a large improvement compared to 2-20 for the wild type. The broad substrate scope of this enzyme variant is of synthetic use, and hydrolysis of the tested substrates proceeded with an enantiomeric excess between 95-99%. A 30-fold increase in activity was also observed for most substrates. The developed enzyme variant shows (R)-selectivity, which is reversed compared to the wild type that is (S)-selective for most substrates.

Influence of delta-functional groups on the enantiorecognition of secondary alcohols by Candida antarctica lipase B.

The selectivity of acetylation of delta-functionalized secondary alcohols catalyzed by Candida antarctica lipase B has been examined by molecular dynamics. The results from the simulation show that a delta-alcohol functionality forms a hydrogen bond with the carbonyl group of Thr 40. This interaction stabilizes the tetrahedral intermediate and thus leads to selective acetylation of the R enantiomer. A stabilizing interaction of the delta-(R)-acetoxy group with the peptide NH of alanine 282 was also observed. No stabilizing interaction could be found for the delta-keto functionality, and it is proposed that this is the reason for the experimentally observed decrease in enantioselectivity. From these results, it was hypothesized that the enantioselectivity could be restored by mutating the alanine in position 281 for serine. The mutation was made experimentally, and the results show that the E value increased from 9 to 120.

Exploring D-xylose oxidation in Saccharomyces cerevisiae through the Weimberg pathway.

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.

Exploring xylose metabolism in Spathaspora species: XYL1.2 from Spathaspora passalidarum as the key for efficient anaerobic xylose fermentation in metabolic engineered Saccharomyces cerevisiae.

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 10155(T)), 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 gCDW (-1) and 0.33 vs 0.18 g gCDW (-1) 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.

Engineering of Saccharomyces cerevisiae for the production of poly-3-d-hydroxybutyrate from xylose.

Poly-3-d-hydroxybutyrate (PHB) is a promising biopolymer naturally produced by several bacterial species. In the present study, the robust baker's yeast Saccharomyces cerevisiae was engineered to produce PHB from xylose, the main pentose found in lignocellulosic biomass. The PHB pathway genes from the well-characterized PHB producer Cupriavidus necator were introduced in recombinant S. cerevisiae strains already capable of pentose utilization by introduction of the fungal genes for xylose utilization from the yeast Scheffersomyces stipitis. PHB production from xylose was successfully demonstrated in shake-flasks experiments, with PHB yield of 1.17 ± 0.18 mg PHB g(-1) xylose. Under well-controlled fully aerobic conditions, a titer of 101.7 mg PHB L(-1) was reached within 48 hours, with a PHB yield of 1.99 ± 0.15 mg PHB g(-1) xylose, thereby demonstrating the potential of this host for PHB production from lignocellulose.

Engineering yeast hexokinase 2 for improved tolerance toward xylose-induced inactivation.

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.

Directed evolution of Candida antarctica lipase A using an episomaly replicating yeast plasmid.

We herein report the first directed evolution of Candida antarctica lipase A (CalA), employing a combinatorial active-site saturation test (CAST). Wild-type CalA has a modest E-value of 5.1 in kinetic resolution of 4-nitrophenyl 2-methylheptanoate. Enzyme variants were expressed in Pichia pastoris by using the episomal vector pBGP1 which allowed efficient secretory expression of the lipase. Iterative rounds of CASTing yielded variants with good selectivity toward both the (S)- and the (R)-enantiomer. The best obtained enzyme variants had E-values of 52 (S) and 27 (R).

X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation.

In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-A resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic alpha/beta hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation.

Prediction of the Candida antarctica lipase A protein structure by comparative modeling and site-directed mutagenesis.

A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity.

Ab initio and NMR studies on the effect of hydration on the chemical shift of hydroxy protons in carbohydrates using disaccharides and water/methanol/ethers as model systems.

Density functional theory (DFT) and Hartree-Fock (HF) quantum mechanical calculations have been performed on the disaccharides, [small beta]-l-Fucp-(1[rightward arrow]4)-[small alpha]-d-Galp-OMe, [small beta]-l-Fucp-(1[rightward arrow]4)-[small alpha]-d-Glcp-OMe, and [small beta]-l-Fucp-(1[rightward arrow]3)-[small alpha]-d-Glcp-OMe. The [capital Delta][small delta]-values (difference between the chemical shift in the disaccharide and the corresponding monosaccharide methyl glycoside) for the exchangeable hydroxy protons have been calculated and compared to experimental values previously measured by NMR spectroscopy for samples in aqueous solutions. The calculations performed on molecules in vacuum showed that hydroxy protons hydrogen bonded to the neighboring ring oxygens have large positive [capital Delta][small delta]-values, indicating that they are deshielded relative to those in the corresponding methyl glycoside. The NMR experiments showed instead that these hydroxy protons close to the neighboring ring oxygens were shielded. This discrepancy between calculated and experimental data was attributed to solvent effects, and this hypothesis has been confirmed in this work by monitoring the chemical shift of the hydroxy proton of methanol in water, ethers and water/ether solutions. Shielding of the hydroxy proton of methanol is observed for increased ether concentrations, whereas deshielding is observed for increased concentration of water. The shielding observed for hydroxy protons in disaccharides is a consequence of reduced hydration due to intermolecular hydrogen bonding or steric effects. In strongly hydrated systems such as carbohydrates, the hydration state of a hydroxy proton is the key factor determining the value of the chemical shift of its NMR signal, and the [capital Delta][small delta] will be a direct measure of the change in hydration state.

T lymphocyte expression of thrombospondin-1 and adhesion to extracellular matrix components.

The mechanisms controlling the formation of pseudopodia and other active cell edges in T lymphocytes are not understood. We show here that T lymphocytes express thrombospondin-1 (TSP-1). TSP-1 in T lymphocytes has a high turnover as shown by the fact that brefeldin and monensin rapidly increase while cycloheximide tend to decrease the cellular TSP-1 content. T cell TSP-1 is preferentially stored intracellularly and shows variable cell surface expression. T lymphocyte adhesion to fibronectin and collagen type IV induces TSP-1 expression on the cell surface via a brefeldin sensitive mechanism. A monoclonal antibody to TSP-1 inhibits the flattening and pseudopodia formation of the adherent T cells. Furthermore, the same antibody to TSP-1 also exerts an inhibitory effect on T cell migration in the absence of exogenous TSP-1. These results indicate that endogenous TSP-1 is part of an adhesion-dependent mechanism controlling cytoplasmic spreading and migration in T lymphocytes.