Generation of a library of carbohydrate-active enzymes for plant biomass deconstruction

Áreas de pesquisa: Biochemistry & Molecular Biology ; ChemistryIn nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sourcesinfo:eu-repo/semantics/publishedVersio

A novel a-L-Arabinofuranosidase of Family 43 Glycoside Hydrolase (Ct43Araf ) from Clostridium thermocellum

Articles in International JournalsThe study describes a comparative analysis of biochemical, structural and functional properties of two recombinant derivatives from Clostridium thermocellum ATCC 27405 belonging to family 43 glycoside hydrolase. The family 43 glycoside hydrolase encoding a-L-arabinofuranosidase (Ct43Araf) displayed an N-terminal catalytic module CtGH43 (903 bp) followed by two carbohydrate binding modules CtCBM6A (405 bp) and CtCBM6B (402 bp) towards the C-terminal. Ct43Araf and its truncated derivative CtGH43 were cloned in pET-vectors, expressed in Escherichia coli and functionally characterized. The recombinant proteins displayed molecular sizes of 63 kDa (Ct43Araf) and 34 kDa (CtGH43) on SDS-PAGE analysis. Ct43Araf and CtGH43 showed optimal enzyme activities at pH 5.7 and 5.4 and the optimal temperature for both was 50uC. Ct43Araf and CtGH43 showed maximum activity with rye arabinoxylan 4.7 Umg21 and 5.0 Umg21, respectively, which increased by more than 2-fold in presence of Ca2+ and Mg2+ salts. This indicated that the presence of CBMs (CtCBM6A and CtCBM6B) did not have any effect on the enzyme activity. The thin layer chromatography and high pressure anion exchange chromatography analysis of Ct43Araf hydrolysed arabinoxylans (rye and wheat) and oat spelt xylan confirmed the release of L-arabinose. This is the first report of a-L-arabinofuranosidase from C. thermocellum having the capacity to degrade both pnitrophenol- a-L-arabinofuranoside and p-nitrophenol-a-L-arabinopyranoside. The protein melting curves of Ct43Araf and CtGH43 demonstrated that CtGH43 and CBMs melt independently. The presence of Ca2+ ions imparted thermal stability to both the enzymes. The circular dichroism analysis of CtGH43 showed 48% b-sheets, 49% random coils but only 3% a-helices

Analysis of shared heritability in common disorders of the brain

ience, this issue p. eaap8757 Structured Abstract INTRODUCTION Brain disorders may exhibit shared symptoms and substantial epidemiological comorbidity, inciting debate about their etiologic overlap. However, detailed study of phenotypes with different ages of onset, severity, and presentation poses a considerable challenge. Recently developed heritability methods allow us to accurately measure correlation of genome-wide common variant risk between two phenotypes from pools of different individuals and assess how connected they, or at least their genetic risks, are on the genomic level. We used genome-wide association data for 265,218 patients and 784,643 control participants, as well as 17 phenotypes from a total of 1,191,588 individuals, to quantify the degree of overlap for genetic risk factors of 25 common brain disorders. RATIONALE Over the past century, the classification of brain disorders has evolved to reflect the medical and scientific communities' assessments of the presumed root causes of clinical phenomena such as behavioral change, loss of motor function, or alterations of consciousness. Directly observable phenomena (such as the presence of emboli, protein tangles, or unusual electrical activity patterns) generally define and separate neurological disorders from psychiatric disorders. Understanding the genetic underpinnings and categorical distinctions for brain disorders and related phenotypes may inform the search for their biological mechanisms. RESULTS Common variant risk for psychiatric disorders was shown to correlate significantly, especially among attention deficit hyperactivity disorder (ADHD), bipolar disorder, major depressive disorder (MDD), and schizophrenia. By contrast, neurological disorders appear more distinct from one another and from the psychiatric disorders, except for migraine, which was significantly correlated to ADHD, MDD, and Tourette syndrome. We demonstrate that, in the general population, the personality trait neuroticism is significantly correlated with almost every psychiatric disorder and migraine. We also identify significant genetic sharing between disorders and early life cognitive measures (e.g., years of education and college attainment) in the general population, demonstrating positive correlation with several psychiatric disorders (e.g., anorexia nervosa and bipolar disorder) and negative correlation with several neurological phenotypes (e.g., Alzheimer's disease and ischemic stroke), even though the latter are considered to result from specific processes that occur later in life. Extensive simulations were also performed to inform how statistical power, diagnostic misclassification, and phenotypic heterogeneity influence genetic correlations. CONCLUSION The high degree of genetic correlation among many of the psychiatric disorders adds further evidence that their current clinical boundaries do not reflect distinct underlying pathogenic processes, at least on the genetic level. This suggests a deeply interconnected nature for psychiatric disorders, in contrast to neurological disorders, and underscores the need to refine psychiatric diagnostics. Genetically informed analyses may provide important "scaffolding" to support such restructuring of psychiatric nosology, which likely requires incorporating many levels of information. By contrast, we find limited evidence for widespread common genetic risk sharing among neurological disorders or across neurological and psychiatric disorders. We show that both psychiatric and neurological disorders have robust correlations with cognitive and personality measures. Further study is needed to evaluate whether overlapping genetic contributions to psychiatric pathology may influence treatment choices. Ultimately, such developments may pave the way toward reduced heterogeneity and improved diagnosis and treatment of psychiatric disorders

Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins

International audienceBackgroundGene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression.ResultsHere, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins.ConclusionsAn automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery

The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans

International audienceno abstrac

HPAEC analysis of <i>Ct</i>43Ara<i>f</i> reaction mixture showing released sugars.

<p><b>A</b>) L-arabinose, <b>B</b>) D-xylose, <b>C</b>) rye arabinoxylan, <b>D</b>) wheat arabinoxylan and <b>E</b>) oat spelt xylan. The reaction was carried out at pH 5.7, 50°C for 30 min.</p

The molecular architecture of <i>Ct</i>43Ara<i>f</i> shows modular structure with an N-terminal family 43 glycoside hydrolase (<i>Ct</i>GH43) catalytic module (903 bp), a C-terminal family 6 carbohydrate binding module (<i>Ct</i>CBM6B, 402 bp) and another family 6 Carbohydrate binding module (<i>Ct</i>CBM6A, 405 bp) sandwiched between these two modules.

<p>The molecular architecture of <i>Ct</i>43Ara<i>f</i> shows modular structure with an N-terminal family 43 glycoside hydrolase (<i>Ct</i>GH43) catalytic module (903 bp), a C-terminal family 6 carbohydrate binding module (<i>Ct</i>CBM6B, 402 bp) and another family 6 Carbohydrate binding module (<i>Ct</i>CBM6A, 405 bp) sandwiched between these two modules.</p

Kinetic properties and catalytic efficiencies of <i>Ct</i>43Ara<i>f</i> and <i>Ct</i>GH43 from <i>C. thermocellum.</i>

<p><i>The assays with natural substrates were carried out with 20 mM sodium phosphate buffer (pH 5.7) for <b>^a</b>Ct43Araf and sodium acetate buffer (pH 5.4) for <b>^b</b>CtGH43 at 50°C. The assays were performed in triplicates. The incubation time and other conditions for reducing sugar estimation were as same as described in the Materials and Methods section. <b>^c</b>The assays with synthetic pNP-glycosides were carried out in 20 mM sodium phosphate buffer pH 5.7.</i></p

A) SDS-PAGE (13%) showing over-expression and purification of <i>Ct</i>43Ara<i>f</i>.

<p>Lane 1: Page Ruler protein marker, Lane 2: uninduced <i>Ct</i>43Ara<i>f</i> cells, Lane 3: Induced <i>Ct</i>43Ara<i>f</i> cells, Lane 4: Cell free extract, Lanes 5: Purified <i>Ct</i>43Ara<i>f</i> (63 kDa approx.), <b>B</b>) Effect of pH and temperature on <i>Ct</i>43Ara<i>f</i> activity, where (•) represents pH profile and (▾) represents temperature profile, <b>C</b>) pH and thermal stability analysis of <i>Ct</i>43Ara<i>f,</i> where (▾) represents pH stability and (•) represents thermal stability profile.</p

Far-UV CD spectra of truncated <i>Ct</i>GH43 (15 µM) from <i>Clostridium thermocellum</i> in 20 mM sodium phosphate buffer, pH 7.0.

<p>Far-UV CD spectra of truncated <i>Ct</i>GH43 (15 µM) from <i>Clostridium thermocellum</i> in 20 mM sodium phosphate buffer, pH 7.0.</p