Expression of an extremophilic xylanase in Nicotiana benthamiana and its use for the production of prebiotic xylooligosaccharides.

A gene construct encoding a xylanase, which is active in extreme conditions of temperature and alkaline pH (90 °C, pH 10.5), has been transitorily expressed with high efficiency in Nicotiana benthamiana using a viral vector. The enzyme, targeted to the apoplast, accumulates in large amounts in plant tissues in as little as 7 days after inoculation, without detrimental effects on plant growth. The properties of the protein produced by the plant, in terms of resistance to temperature, pH, and enzymatic activity, are equivalent to those observed when Escherichia coli is used as a host. Purification of the plant-produced recombinant xylanase is facilitated by exporting the protein to the apoplastic space. The production of this xylanase by N. benthamiana, which avoids the hindrances derived from the use of E. coli, namely, intracellular production requiring subsequent purification, represents an important step for potential applications in the food industry in which more sustainable and green products are continuously demanded. As an example, the use of the enzyme producing prebiotic xylooligosdaccharides from xylan is here reported.

Dissecting mRNA decay and translation inhibition during iron deficiency.

Iron participates as a vital cofactor in multiple metabolic pathways. Despite its abundance, iron bioavailability is highly restricted in aerobic and alkaline environments. Therefore, living organisms have evolved multiple adaptive mechanisms to respond to iron scarcity. These strategies include a global remodeling of iron metabolism directed to optimize iron utilization. In the baker's yeast Saccharomyces cerevisiae, this metabolic reorganization is accomplished to a large extent by an mRNA-binding protein called Cth2. Yeast Cth2 belongs to a conserved family of tandem zinc finger containing proteins that specifically bind to transcripts with AU-rich elements and promote their turnover. A recent study has revealed that Cth2 also inhibits the translation of its target mRNAs (Ramos-Alonso et al., PLoS Genet 14:e1007476, https://doi.org/10.1371/journal.pgen.1007476 , 2018). Interestingly, the mammalian Cth2 ortholog known as tristetraprolin (aka TTP/TIS11/ZFP36), which is also implicated in controlling iron metabolism, promotes the decay and prevents the translation of its regulated transcripts. These observations open the possibility to study the relative contribution of altering mRNA stability and translation to the physiological adaptation to iron deficiency, the function played by the different domains within the mRNA-binding protein, and the potential factors implicated in coordinating both post-transcriptional events.

Enzymatic Synthesis of a Novel Pterostilbene α-Glucoside by the Combination of Cyclodextrin Glucanotransferase and Amyloglucosidase.

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙⁺ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

Molecular and structural modeling of the Phanerochaete flavido-alba extracellular laccase reveals its ferroxidase structure.

The fungus Phanerochaete flavido-alba is highly efficient in the oxidation of olive oil wastewater-derived polyphenols. This capability is largely due to the action of a multicopper-oxidase (MCO), encoded by the pfaL gene. We describe the sequence and organization of pfaL gene and the biochemical characterization and predicted 3D structural model of the encoded protein. pfaL gene organization and peptide sequence are highly similar to those of P. chrysosporium MCOs. However, PfaL is the first MCO in the Phanerochaete genus to show evident laccase activity. Phylogenetic analysis places PfaL in a differentiated sub-branch of ferroxidases. Protein structure analysis reveals close similarity of PfaL and ferroxidases and provides clues about the differences of activity between both types of enzymes. To summarize, P. flavido-alba laccase is the first enzyme in the novel and biochemically poorly defined group of "ferroxidases/laccases" that shows efficacious oxidation of laccase substrates, biotechnologically exploitable in bioremediation approaches.

Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved (509)VSG(511) tripeptide.

A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial saturation mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved (509)VSG(511) tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The K(m)(O)(2) value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the (509)VSG(511) tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme.

Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis, and phylogenetic relationships.

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 degrees C, pH 4.5, yielded values of 26, 0.43, and 0.45 microm and 18, 660, and 1175 s(-1), respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.

Cloning and molecular characterization of the delta6-desaturase from two echium plant species: production of GLA by heterologous expression in yeast and tobacco.

The synthesis of GLA (delta6,9,12-1-8:3) is carried out in a number of plant taxa by introducing a double bond at the delta6 position of its precursor, linoleic acid (delta9,12-18:2), through a reaction catalyzed by a delta6-desaturase enzyme. We have cloned genes encoding the delta6-desaturase (D6DES) from two different Macaronesian Echium species, E. pitardii and E. gentianoides (Boraginaceae), which are characterized by the accumulation of high amounts of GLA in their seeds. The Echium D6DES genes encode proteins of 438 amino acids bearing the prototypical cytochrome b(5) domain at the N-terminus. Cladistic analysis of desaturases from higher plants groups the Echium D6DES proteins together with other delta6-desaturases in a different cluster from that of the highly related delta8-desaturases. Expression analysis carried out in E. pitardii shows a positive correlation between the D6DES transcript level and GLA accumulation in different tissues of the plant. Although a ubiquitous expression in all organs is observed, the transcript is particularly abundant in developing fruits, whereas a much lower level is present in mature leaves. Functional characterization of the D6DES gene from E. gentianoides has been achieved by heterologous expression in tobacco plants and in the yeast Saccharomyces cerevisiae. In both cases, overexpression of the gene led to the synthesis of GLA. Biotechnological application of these results can be envisaged as an initial step toward the generation of transgenic oleaginous plants producing GLA.