Fatty Acid Photodecarboxylase Is an Interfacial Enzyme That Binds to Lipid-Water Interfaces to Access Its Insoluble Substrate.

Fatty acid photodecarboxylase (FAP), one of the few natural photoenzymes characterized so far, is a promising biocatalyst for lipid-to-hydrocarbon conversion using light. However, the optimum supramolecular organization under which the fatty acid (FA) substrate should be presented to FAP has not been addressed. Using palmitic acid embedded in phospholipid liposomes, phospholipid-stabilized microemulsions, and mixed micelles, we show that FAP displays a preference for FAs present in liposomes and at the surface of microemulsions. The kinetics of adsorption onto phospholipid and galactolipid monomolecular films further suggests the ability of FAP to bind to and penetrate into membranes, with a higher affinity in the presence of FAs. The FAP structure reveals a potential interfacial recognition site with clusters of hydrophobic and basic residues surrounding the active site entrance. The resulting dipolar moment suggests the orientation of FAP at negatively charged interfaces. These findings provide important clues about the mode of action of FAP and the development of FAP-based bioconversion processes.

Engineering Fatty Acid Photodecarboxylase to Enable Highly Selective Decarboxylation of trans Fatty Acids.

Due to the high risk of heart disease caused by the intake of trans fatty acids, a method to eliminate trans fatty acids from foods has become a critical issue. Herein, we engineered fatty acid photo-decarboxylase from Chlorella variabilis (CvFAP) to selectively catalyze the decarboxylation of trans fatty acids to yield readily-removed hydrocarbons and carbon dioxide, while cis fatty acids remained unchanged. An efficient protein engineering based on FRISM strategy was implemented to intensify the electronic interaction between the residues and the double bond of the substrate that stabilized the binding of elaidic acid in the channel. For the model compounds, oleic acid and elaidic acid, the best mutant, V453E, showed a one-thousand-fold improvement in the trans-over-cis (ToC) selectivity compared with wild type (WT). As the first report of the direct biocatalytic decarboxylation resolution of trans/cis fatty acids, this work offers a safe, facile, and eco-friendly process to eliminate trans fatty acids from edible oils.

Light-driven decarboxylative deuteration enabled by a divergently engineered photodecarboxylase.

Despite the well-established chemical processes for C-D bond formation, the toolbox of enzymatic methodologies for deuterium incorporation has remained underdeveloped. Here we describe a photodecarboxylase from Chlorella variabilis NC64A (CvFAP)-catalyzed approach for the decarboxylative deuteration of various carboxylic acids by employing D2O as a cheap and readily available deuterium source. Divergent protein engineering of WT-CvFAP is implemented using Focused Rational Iterative Site-specific Mutagenesis (FRISM) as a strategy for expanding the substrate scope. Using specific mutants, several series of substrates including different chain length acids, racemic substrates as well as bulky cyclic acids are successfully converted into the deuterated products (>40 examples). In many cases WT-CvFAP fails completely. This approach also enables the enantiocomplementary kinetic resolution of racemic acids to afford chiral deuterated products, which can hardly be accomplished by existing methods. MD simulations explain the results of improved catalytic activity and stereoselectivity of WT CvFAP and mutants.

Light-Driven Enzymatic Decarboxylation of Dicarboxylic Acids.

Photodecarboxylase from Chlorella variabillis (CvFAP) is one of the three known light-activated enzymes that catalyzes the decarboxylation of fatty acids into the corresponding C1-shortened alkanes. Although the substrate scope of CvFAP has been altered by protein engineering and decoy molecules, it is still limited to mono-fatty acids. Our studies demonstrate for the first time that long chain dicarboxylic acids can be converted by CvFAP. Notably, the conversion of dicarboxylic acids to alkanes still represents a chemically very challenging reaction. Herein, the light-driven enzymatic decarboxylation of dicarboxylic acids to the corresponding (C2-shortened) alkanes using CvFAP is described. A series of dicarboxylic acids is decarboxylated into alkanes in good yields by means of this approach, even for the preparative scales. Reaction pathway studies show that mono-fatty acids are formed as the intermediate products before the final release of C2-shortened alkanes. In addition, the thermostability, storage stability, and recyclability of CvFAP for decarboxylation of dicarboxylic acids are well evaluated. These results represent an advancement over the current state-of-the-art.

Mechanism and dynamics of fatty acid photodecarboxylase.

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

A Reconstructed Common Ancestor of the Fatty Acid Photo-decarboxylase Clade Shows Photo-decarboxylation Activity and Increased Thermostability.

Light-dependent enzymes are a rare type of biocatalyst with high potential for research and biotechnology. A recently discovered fatty acid photo-decarboxylase from Chlorella variabilis NC64A (CvFAP) converts fatty acids to the corresponding hydrocarbons only when irradiated with blue light (400 to 520 nm). To expand the available catalytic diversity for fatty acid decarboxylation, we reconstructed possible ancestral decarboxylases from a set of 12 extant sequences that were classified under the fatty acid decarboxylases clade within the glucose-methanol choline (GMC) oxidoreductase family. One of the resurrected enzymes (ANC1) showed activity in the decarboxylation of fatty acids, showing that the clade indeed contains several photo-decarboxylases. ANC1 has a 15 °C higher melting temperature (Tm ) than the extant CvFAP. Its production yielded 12-fold more protein than this wild type decarboxylase, which offers practical advantages for the biochemical investigation of this photoenzyme. Homology modelling revealed amino acid substitutions to more hydrophilic residues at the surface and shorter flexible loops compared to the wild type. Using ancestral sequence reconstruction, we have expanded the existing pool of confirmed fatty acid photo-decarboxylases, providing access to a more robust catalyst for further development via directed evolution.

Toxic mechanism of three azole fungicides and their mixture to green alga Chlorella pyrenoidosa.

Currently, few studies have investigated the joint toxicity mechanism of azole fungicides at different exposure times and mixed at the relevant environmental concentrations. In this study, three common azole fungicides, namely, myclobutanil (MYC), propiconazole (PRO), and tebuconazole (TCZ), were used in studying the toxic mechanisms of a single substance and its ternary mixture exposed to ambient concentrations of Chlorella pyrenoidosa. Superoxide dismutase (SOD), catalase (CAT), chlorophyll a (Chla), and total protein (TP), were used as physiological indexes. Results showed that three azole fungicides and ternary mixture presented obvious time-dependent toxicities at high concentrations. MYC induced a hormetic effect on algal growth, whereas PRO and TCZ inhibit algal growth in the entire range of the tested concentrations. The toxicities of the three azole fungicides at 7 days followed the order PRO > TCZ > MYC. Three azole fungicides and their ternary mixture induced different levels of SOD and CAT activities in algae at high concentrations. The ternary mixture showed additive effects after 4 and 7 days exposure, but no effect was observed at actual environmental concentrations. The toxic mechanisms may be related to the continuous accumulation of reactive oxygen species, which not only affected protein structures and compositions but also damaged thylakoid membranes, hindered the synthesis of proteins and chlorophyll a, and eventually inhibited algal growth. These findings increase the understanding of the ecotoxicity of azole fungicides and use of azole fungicides in agricultural production.

Mechanism analysis for the process-dependent driven mode of NaHCO3 in algal antibiotic removal: efficiency, degradation pathway and metabolic response.

This work provided a comprehensive perspective to investigate the performance of NaHCO3-driving effect and mechanism including the antibiotic removal, degradation pathway and metabolites analysis, and the algal physiological response during the removal process. Cefuroxime sodium was selected as the target antibiotic. Our results showed that NaHCO3 did not facilitate self-decomposition of the target antibiotic, while drove the improvement on the removal capacity of every algal cell, which then attributed to the total removal efficiency. After 24 h, there was an improvement on the removal rate of the target antibiotic (from 10.21% to 92.89%) when NaHCO3 was added. The degradation pathway of the target antibiotic was confirmed by the formation of three main products (M1, M2 and M3), and the degradation process, that from M1 to M2 and M2 to M3, was accelerated by the existence of NaHCO3. Besides, a 4-stage model illustrated the relationship between NaHCO3 and antibiotic removal process. Moreover, algal culture that supplemented with NaHCO3 demonstrated a better growth capacity. A large increase in the content of chlorophyll a and a moderate increase in the activity of two carbon metabolic enzymes (RuBisCO and CA) might be viewed as a positive response of the algae during the NaHCO3-driving process.

The combined toxicity influence of microplastics and nonylphenol on microalgae Chlorella pyrenoidosa.

Microplastics and nonylphenol (NP) are considered as emerging pollutant and have attracted wide attention, while their combined toxicity on aquatic organisms is barely researched. Therefore, the combined toxicity influence of NP with three types of microplastics containing polyethylene (PE1000, 13 µm and PE, 150 µm), polyamide (PA1000, 13 µm and PA, 150 µm) polystyrene (PS, 150 µm) on microalgae Chlorella pyrenoidosa was analyzed. Both growth inhibition, chlorophyll fluorescence, superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were determined. We found that single microplastics and NP both inhibited algal growth, thereby causing oxidative stress. The order of inhibition effect in single microplastics experiment was PE1000 > PA1000 > PE ≈ PS > PA. The combined toxicity experiment results indicated that the presence of microplastics had positive effect in terms of alleviating NP toxicity to C. pyrenoidosa, and the microplastics adsorption capacity to NP was the dominant contributing factor for this effect. According to the independent action model, the combined toxicity was antagonistic. Because the negative effect of smaller size microplastics on algal growth was aggravated with prolonged exposure time, the optimum effect of microplastics alleviated NP toxicity was PA1000 at 48 h, while this effect was substituted by PA at 96 h during combined toxicity. Thus, the toxicity of smaller size microplastics has a nonnegligible influence on combined toxicity. This study confirms that microplastics significantly affected the toxicity of organic pollutants on microalgae. Further research on the combined toxicity of smaller size microplastics with pollutants in chronic toxicity is needed.

Whole-Cell Photoenzymatic Cascades to Synthesize Long-Chain Aliphatic Amines and Esters from Renewable Fatty Acids.

Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer-Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g-1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.

Glucose-6-Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis.

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose-6-phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19-fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09-fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose-6-phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.

Light Elicits Astaxanthin Biosynthesis and Accumulation in the Fermented Ultrahigh-Density Chlorella zofinginesis.

The growth and astaxanthin production of Chlorella zofingiensis were examined under both heterotrophic and photoautotrophic conditions, and it was found that, in comparison to the photoautotrophic mode, the heterotrophic mode led to high algal densities but attenuated intracellular astaxanthin accumulation. Following the heterotrophy-photoautotrophy transition, a considerable increase in the astaxanthin content was observed, accompanied by the upregulation of key carotenogenic genes, including phytoene synthase (PSY), ß-carotenoid hydroxylase (CHYb), ß-carotenoid ketolase 1 (BKT1), and ß-carotenoid ketolase 2 (BKT2). In contrast, the astaxanthin content and carotenogenic genes underwent an opposite change following the photoautotrophy-heterotrophy transition, suggesting the key role of light in stimulating astaxanthin biosynthesis. To improve the astaxanthin production by C. zofingiensis, a novel heterotrophy-photoinduction culture strategy without dilution was developed and evaluated. The astaxanthin content and productivity reached 2.7 mg g-1 of dry weight and 9.9 mg L-1 day-1, respectively, which were 4.0- and 2.5-fold higher than that obtained under the heterotrophic condition.

Light-Driven Kinetic Resolution of α-Functionalized Carboxylic Acids Enabled by an Engineered Fatty Acid Photodecarboxylase.

Chiral α-functionalized carboxylic acids are valuable precursors for a variety of medicines and natural products. Herein, we described an engineered fatty acid photodecarboxylase (CvFAP)-catalyzed kinetic resolution of α-amino acids and α-hydroxy acids, which provides the unreacted R-configured substrates with high yields and excellent stereoselectivity (ee up to 99 %). This efficient light-driven process requires neither NADPH recycling nor prior preparation of esters, which were required in previous biocatalytic approaches. The structure-guided engineering strategy is based on the scanning of large amino acids at hotspots to narrow the substrate binding tunnel. To the best of our knowledge, this is the first example of asymmetric catalysis by an engineered CvFAP.

Enzyme-Modulated Anaerobic Encapsulation of Chlorella Cells Allows Switching from O2 to H2 Production.

Single-cell encapsulation has become an effective strategy in cell surface engineering; however, the construction of cell wall-like layers that allow the switching of the inherent functionality of the engineered cell is still rare. In this study, we show a universal way to create an enzyme-modulated oxygen-consuming sandwich-like layer by using polydopamine, laccase, and tannic acid as building blocks, which then could generate an anaerobic microenvironment around the cell. This layer protected the encapsulated C. pyrenoidosa cell against external stresses and enabled it to switch from normal photosynthetic O2 production to photobiological H2 production. The layer showed an smaller effect on the PSII activity, which contributed a significant enhancement on the rate (0.32 µmol H2 h-1 (mg chlorophyll)-1 ) and the duration (7 d) of H2 production. This strategy is expected to provide a pathway for modulating the functionality of cells and for breakthroughs in the development of green energy alternatives.

A novel switchable fluorescent sensor for facile and highly sensitive detection of alkaline phosphatase activity in a water environment with gold/silver nanoclusters.

A novel fluorescent sensor based on bovine serum albumin stabilized gold/silver nanoclusters (BSA-Au/Ag NCs) was developed for sensitive and facile detection of alkaline phosphatase (ALP) activity. For this fluorescent sensor, ascorbic acid 2-phosphate (AAP) was decomposed into ascorbic acid (AA) and phosphate by catalysis with ALP. The initial red fluorescence of the BSA-Au/Ag NCs was effectively quenched by KMnO4 and then the fluorescence was recovered by addition of AA. The mechanism of interaction between BSA-Au/Ag NCs and KMnO4 and AA was studied with use of the fluorescence lifetime and UV-vis absorption spectra. The results indicated that the oxidation/reduction modulated by KMnO4/AA led to surface structure destruction/restoration of the BSA-Au/Ag NCs, resulting in fluorescence quenching/recovery. The proposed fluorescence-based method based on a dark background was used to detect ALP and had excellent sensitivity, with a detection limit of 0.00076 U/L. Moreover, the method was applied to the determination of added analytes, with satisfactory recoveries (97.0-105.0 %). In a simulated eutrophic water body, this method successfully detected ALP in actual water samples and could monitor the dynamic changes of ALP activity through visual observation. More importantly, the proposed fluorescent sensor not only has the advantages of simple operation and high sensitivity but has also been successfully used on filter paper to establish a rapid and visual test paper for ALP.

Newly Identified Essential Amino Acids Affecting Chlorella ellipsoidea DGAT1 Function Revealed by Site-Directed Mutagenesis.

Diacylglycerol acyltransferase (DGAT) is a rate-limiting enzyme in the synthesis of triacylglycerol (TAG), the most important form of energy storage in plants. Some residues have previously been proven to be crucial for DGAT1 activity. In this study, we used site-directed mutagenesis of the CeDGAT1 gene from Chlorella ellipsoidea to alter 16 amino acids to investigate effects on DGAT1 function. Of the 16 residues (L482R, E542R, Y553A, G577R, R579D, Y582R, R596D, H603D, H609D, A624R, F629R, S632A, W650R, A651R, Q658H, and P660R), we newly identified 5 (L482, R579, H603, A651, and P660) as being essential for DGAT1 function and 7 (E542, G577, R596, H609, A624, S632, and Q658) that significantly affect DGAT1 function to different degrees, as revealed by heterologous expression of the mutants in yeast strain INVSc1. Importantly, compared with CeDGAT1, expression of the mutant CeDGAT1Y553A significantly increased the total fatty acid and TAG contents of INVSc1. Comparison among CeDGAT1Y553A, GmDGAT1Y341A, AtDGAT1Y364A, BnDGAT1Y347A, and BoDGAT1Y352A, in which tyrosine at the position corresponding to the 553rd residue in CeDGAT1 is changed into alanine, indicated that the impact of changing Y to A at position 553 is specific for CeDGAT1. Overall, the results provide novel insight into the structure and function of DGAT1, as well as a mutant gene with high potential for lipid improvement in microalgae and plants.

Colorimetric determination of ß-1,2-glucooligosaccharides for an enzymatic assay using 3-methyl-2-benzothiazolinonehydrazone.

A colorimetric determination method measuring the reducing ends of sugars is usually used for quantitative evaluation of polysaccharide-degrading activity of endo-type enzymes. However, no appropriate colorimetric method has been established for enzymatic assay of ß-1,2-glucanases, which produce ß-1,2-glucooligosaccharides from ß-1,2-glucans. The Anthon-MBTH method has been potentially the most adaptable for color development of ß-1,2-glucooligosaccharides among various known colorimetric methods for detecting the reducing power of oligosaccharides, since the difference between sophorose and other ß-1,2-glucooligosaccharides in absorbance is relatively small. Almost the same color development was obtained for ß-1,2-glucooligosaccharides when the heating time with the Anthon-MBTH method was changed. The kind of base and concentration of dithiothreitol did not markedly affect the color development. Most buffer components, salts and a chelating reagent used for usual enzymatic experiments did not inhibit color development. Furthermore, assay was performed successfully for a ß-1,2-glucanase using the modified MBTH method.

Application of microalgae hydrolysate as a fermentation medium for microbial production of 2-pyrone 4,6-dicarboxylic acid.

Actual biomass of microalgae was tested as a fermentation substrate for microbial production of 2-pyrone 4,6-dicarboxylic acid (PDC). Acid-hydrolyzed green microalgae Chlorella emersonii (algae hydrolysate) was diluted to adjust the glucose concentration to 2 g/L and supplemented with the nutrients of Luria-Bertani (LB) medium (tryptone 10 g/L and yeast extract 5 g/L). When the algae hydrolysate was used as a fermentation source for recombinant Escherichia coli producing PDC, 0.43 g/L PDC was produced with a yield of 20.1% (mol PDC/mol glucose), whereas 0.19 g/L PDC was produced with a yield of 8.6% when LB medium supplemented with glucose was used. To evaluate the potential of algae hydrolysate alone as a fermentation medium for E. coli growth and PDC production, the nutrients of LB medium were reduced from the algae hydrolysate medium. Interestingly, 0.17 g/L PDC was produced even without additional nutrient, which was comparable to the case using pure glucose medium with nutrients of LB medium. When using a high concentration of hydrolysate without additional nutrients, 1.22 g/L PDC was produced after a 24-h cultivation with the yield of 16.1%. Overall, C. emersonii has high potential as cost-effective fermentation substrate for the microbial production of PDC.

A novel galactolipase from a green microalga Chlorella kessleri: purification, characterization, molecular cloning, and heterologous expression.

We have identified an enzyme, galactolipase (ckGL), which hydrolyzes the acyl ester bond of galactolipids such as digalactosyldiacylglycerol (DGDG), in the microalga Chlorella kessleri. Following purification of the enzyme to electrophoretic homogeneity from cell-free extract, the maximum activity toward DGDG was observed at pH 6.5 and 37 °C. ckGL was Ca2+-dependent enzyme and displayed an apparent molecular mass of approx. 53 kDa on SDS-PAGE. The substrate specificity was in the order: DGDG (100%) > monogalactosyldiacylglycerol ≈ phosphatidylglycerol (~ 40%) > sulfoquinovosyldiacylglycerol (~ 20%); the enzyme exhibited almost no activity toward glycerides and other phospholipids. Gas chromatography analysis demonstrated that ckGL preferably hydrolyzed the sn-1 acyl ester bond in the substrates. The genomic DNA sequence (5.6 kb) containing the ckGL gene (designated glp1) was determined and the cDNA was cloned. glp1 was composed of 10 introns and 11 exons, and the 1608-bp full-length cDNA encoded a mature ckGL containing 475 amino acids (aa), with a presequence (60 aa) containing a potential chloroplast transit peptide. Recombinant functional ckGL was produced in Escherichia coli. Although the deduced aa sequence of ckGL contained the typical GXSXG motif of serine hydrolases together with conserved histidine and aspartate residues which would form part of the catalytic triad of α/ß-hydrolases, ckGL showed no significant overall similarity with known lipases including GLs from Chlamydomonas reinhardtii and Aspergillus japonicus, indicating that ckGL is a novel GL. ckGL, with high specificity for DGDG, could be applicable to food processing as an enzyme capable of improving material textures.

Multisubstrate specific flavin containing monooxygenase from Chlorella pyrenoidosa with potential application for phenolic wastewater remediation and biosensor application.

Microbial degradation of phenolic pollutants in industrial wastewater is dependent on enzymatic pathway comprising a cascade of phenol metabolizing enzymes. Phenol hydroxylase is the first enzyme of the pathway catalysing the initial attack on phenol in green algae Chlorella pyrenoidosa. The present work reports cost-effective production of partially purified microalgal phenol hydroylase by single-step purification and characterization of its kinetic properties with the view of application for enzyme-based remediation of phenolic wastewater or in phenolic biosensor. The enzyme with a molecular weight of 25 kDa shows all characteristics of phenol hydroxylases, that is, hydroxylation of phenol to catechol (confirmed by HPLC), substrate-dependent NADPH oxidation, absorption spectrum typical of flavoproteins and peptide mass fingerprint corresponding to flavoprotein hydroxylase. The enzyme utilizes phenol with apparent Michealis constant (Km) of 1.71 µM, maximal velocity (Vmax) of 0.4 µM/min with optimal activity at pH 7 and 35°C. Fe2+chelators (Phenanthroline and sodium arsenate), heavy metals, denaturants and oxidizing agents showed inhibitory effect on phenol hydroxylation activity of the enzyme. The enzyme has broad substrate specificity against isomeric diphenols, isomeric methylphenols, halogen-substituted phenols, amino-substituted phenols, nitrophenols, hydroxybenzaldehyde and hydroxylbenzoic acid. The enzyme shows remarkable storage stability at room temperature and at 4°C. The multisubstrate specificity coupled to remarkable storage stability of the microalgal phenol hydroxylase opens up avenues for its application in remediation of a wide range of phenolics released in industrial wastewater or phenolic biosensor application.