scholarly journals Functional characterization of a highly specific l-arabinose transporter from Trichoderma reesei

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Sami Havukainen ◽  
Jonai Pujol-Giménez ◽  
Mari Valkonen ◽  
Matthias A. Hediger ◽  
Christopher P. Landowski
Keyword(s):  
Trichoderma Reesei ◽  
Soluble Sugars ◽  
Functional Characterization ◽  
Value Added ◽  
High Specificity ◽  
Biofuel Production ◽  
Primary Metabolism ◽  
High Affinity ◽  
Xylose Transporter

Abstract Background Lignocellulose biomass has been investigated as a feedstock for second generation biofuels and other value-added products. Some of the processes for biofuel production utilize cellulases and hemicellulases to convert the lignocellulosic biomass into a range of soluble sugars before fermentation with microorganisms such as yeast Saccharomyces cerevisiae. One of these sugars is l-arabinose, which cannot be utilized naturally by yeast. The first step in l-arabinose catabolism is its transport into the cells, and yeast lacks a specific transporter, which could perform this task. Results We identified Trire2_104072 of Trichoderma reesei as a potential l-arabinose transporter based on its expression profile. This transporter was described already in 2007 as d-xylose transporter XLT1. Electrophysiology experiments with Xenopus laevis oocytes and heterologous expression in yeast revealed that Trire2_104072 is a high-affinity l-arabinose symporter with a Km value in the range of $$\sim$$ ∼  0.1–0.2 mM. It can also transport d-xylose but with low affinity (Km$$\sim$$ ∼  9 mM). In yeast, l-arabinose transport was inhibited slightly by d-xylose but not by d-glucose in an assay with fivefold excess of the inhibiting sugar. Comparison with known l-arabinose transporters revealed that the expression of Trire2_104072 enabled yeast to uptake l-arabinose at the highest rate in conditions with low extracellular l-arabinose concentration. Despite the high specificity of Trire2_104072 for l-arabinose, the growth of its T. reesei deletion mutant was only affected at low l-arabinose concentrations. Conclusions Due to its high affinity for l-arabinose and low inhibition by d-glucose or d-xylose, Trire2_104072 could serve as a good candidate for improving the existing pentose-utilizing yeast strains. The discovery of a highly specific l-arabinose transporter also adds to our knowledge of the primary metabolism of T. reesei. The phenotype of the deletion strain suggests the involvement of other transporters in l-arabinose transport in this species.

scholarly journals Functional Characterization of a High-Affinity Choline Transport System in Human Keratinocytes

2002 ◽  
Vol 119 (1) ◽  
pp. 118-121 ◽  
Author(s):  
Kathrin Hoffmann ◽  
Franziska Grafe ◽  
Wolfgang Wohlrab ◽  
Reinhard H. Neubert ◽  
Matthias Brandsch
Keyword(s):  
Transport System ◽  
Functional Characterization ◽  
Human Keratinocytes ◽  
Choline Transport ◽  
High Affinity

scholarly journals A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Minyeong Yoo ◽  
Gwenaelle Bestel-Corre ◽  
Christian Croux ◽  
Antoine Riviere ◽  
Isabelle Meynial-Salles ◽  
...  
Keyword(s):  
Steady State ◽  
Metabolic Engineering ◽  
Clostridium Acetobutylicum ◽  
Functional Characterization ◽  
Scale Model ◽  
Primary Metabolism ◽  
Scale Analysis ◽  
Content Type ◽  
Commercial Process

ABSTRACTEngineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacteriumClostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model,iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering ofC. acetobutylicumto develop a commercial process for the production ofn-butanol.IMPORTANCECurrently, there is a resurgence of interest inClostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to producen-butanol for use both as a bulk chemical and as a renewable alternative transportation fuel. To develop a commercial process for the production ofn-butanol via a metabolic engineering approach, it is necessary to better characterize both the primary metabolism ofC. acetobutylicumand its regulation. Here, we apply a quantitative system-scale analysis to acidogenic, solventogenic, and alcohologenic steady-stateC. acetobutylicumcells and report for the first time quantitative transcriptomic, proteomic, and fluxomic data. This approach allows for a better understanding of the regulation of primary metabolism and for the functional characterization of numerous enzymes involved in primary metabolism.

scholarly journals Functional characterization of two human sulphotransferase cDNAs that encode monoamine- and phenol-sulphating forms of phenol sulphotransferase: substrate kinetics, thermal-stability and inhibitor-sensitivity studies

1994 ◽  
Vol 302 (2) ◽  
pp. 497-502 ◽  
Author(s):  
M E Veronese ◽  
W Burgess ◽  
X Zhu ◽  
M E McManus
Keyword(s):  
Thermal Stability ◽  
Human Liver ◽  
Functional Characterization ◽  
Stability Studies ◽  
Functional Relationships ◽  
High Affinity ◽  
Electrophoretic Mobilities ◽  
Sensitivity Studies ◽  
Substrate Kinetics

The present paper describes the functional characterization of two human aryl sulphotransferase (HAST) cDNAs, HAST1 and HAST3, previously isolated by us from liver and brain, respectively [Zhu, Veronese, Sansom, and McManus (1993) Biochem. Biophys. Res. Commun. 192, 671-676; Zhu, Veronese, Bernard, Sansom and McManus (1993) Biochem. Biophys. Res. Commun. 195, 120-127]. These appear to encode the two major forms of phenol sulphotransferase (PST) characterized in a number of human tissue cytosols, these being the phenolsulphating (P-PST) and monoamine-sulphating (M-PST) forms of phenol sulphotransferase. HAST1 and HAST3 cDNAs were functionally expressed in COS-7 cells and kinetically characterized using the model substrates for P-PST and M-PST, p-nitrophenol and dopamine (3,4-dihydroxyphenethylamine) respectively. COS-expressed HAST1 was shown to be enzymatically active in sulphating p-nitrophenol with high affinity (Km 0.6 microM), whereas dopamine was the preferred substrate for HAST3 (Km 9.7 microM). HAST1 could also sulphate dopamine, as could HAST3 sulphate p-nitrophenol, but the Km for these reactions were at least two orders of magnitude greater than for the preferred substrates. COS-expressed HAST1 and HAST3 displayed inhibition profiles with the ST inhibitor 2,6-dichloro-4-nitrophenol (DCNP), identical with human liver cytosolic P-PST and M-PST activities respectively. Thermal-stability studies with the expressed enzymes showed that HAST1 was considerably more thermostable (TS) than HAST3, which is consistent with P-PST being termed the TS PST and M-PST being termed the thermolabile (TL) PST. Western immunoblot analyses of the expressed PST proteins using an antibody generated to a bacterially expressed rat liver aryl/phenol ST showed that HAST1 and HAST3 migrated as single proteins with different electrophoretic mobilities (32 versus 34 kDa). This is consistent with the differences in electrophoretic mobilities observed for P-PST and M-PST in a variety of tissues reported by other workers. This report on the functional characterization of P-PST and M-PST cDNAs provides important information on the structural as well as functional relationships of human PSTs, which sulphate a vast array of exogenous and endogenous compounds.

Functional characterization of high-affinity Na+/dicarboxylate cotransporter found in Xenopus laevis kidney and heart

2005 ◽  
Vol 289 (5) ◽  
pp. C1159-C1168 ◽  
Author(s):  
Naomi Oshiro ◽  
Ana M. Pajor
Keyword(s):  
Xenopus Laevis ◽  
Cdna Sequence ◽  
Citric Acid Cycle ◽  
Functional Characterization ◽  
Activation Kinetics ◽  
Electrophysiological Properties ◽  
High Affinity ◽  
The Family ◽  
Kidney Liver

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na+/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is ∼70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a Km for succinate of 4 μM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site.

Functional characterization of long-chain prenyl diphosphate synthases from tomato

2013 ◽  
Vol 449 (3) ◽  
pp. 729-740 ◽  
Author(s):  
Matthew O. Jones ◽  
Laura Perez-Fons ◽  
Francesca P. Robertson ◽  
Peter M. Bramley ◽  
Paul D. Fraser
Keyword(s):  
Functional Characterization ◽  
Primary Metabolism ◽  
Virus Induced Gene Silencing ◽  
In Planta ◽  
Constitutive Overexpression ◽  
Prenyl Diphosphate ◽  
And Function ◽  
Leaf Appearance ◽  
Long Chain

The electron transfer molecules plastoquinone and ubiquinone are formed by the condensation of aromatic head groups with long-chain prenyl diphosphates. In the present paper we report the cloning and characterization of two genes from tomato (Solanum lycopersicum) responsible for the production of solanesyl and decaprenyl diphosphates. SlSPS (S. lycopersicum solanesyl diphosphate synthase) is targeted to the plastid and both solanesol and plastoquinone are associated with thylakoid membranes. A second gene [SlDPS (S. lycopersicum solanesyl decaprenyl diphosphate synthase)], encodes a long-chain prenyl diphosphate synthase with a different subcellular localization from SlSPS and can utilize geranyl, farnesyl or geranylgeranyl diphosphates in the synthesis of C45 and C50 prenyl diphosphates. When expressed in Escherichia coli, SlSPS and SlDPS extend the prenyl chain length of the endogenous ubiquinone to nine and ten isoprene units respectively. In planta, constitutive overexpression of SlSPS elevated the plastoquinone content of immature tobacco leaves. Virus-induced gene silencing showed that SlSPS is necessary for normal chloroplast structure and function. Plants silenced for SlSPS were photobleached and accumulated phytoene, whereas silencing SlDPS did not affect leaf appearance, but impacted on primary metabolism. The two genes were not able to complement silencing of each other. These findings indicate a requirement for two long-chain prenyl diphosphate synthases in the tomato.

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