Synergistic action of thermophilic pectinases for pectin bioconversion into D-galacturonic acid.

Large amounts of pectin-rich biomass are generated worldwide yearly, which can be hydrolysed by pectinases to obtain bio-based chemical building blocks such as D-galacturonic acid (GalA). The aim of this work was to investigate thermophilic pectinases and explore their synergistic application in the bioconversion of pectic substrates into GalA. Two exo-polygalacturonases (exo-PGs) from Thermotoga maritima (TMA01) and Bacillus licheniformis (BLI04) and two pectin methylesterases (PMEs) from Bacillus licheniformis (BLI09) and Streptomyces ambofaciens (SAM10) were cloned and expressed in Escherichia coli BL21 (DE3), purified and fully characterised. These pectinases exhibited optimum activity at temperatures above 50 °C and good stability at high temperature (40-90 °C) for up to 24 h. Exo-PGs preferred non-methylated substrates, suggesting that previous pectin demethylation by PMEs was necessary to achieve an efficient pectin monomerisation into GalA. Synergistic activity between PMEs and exo-PGs was tested using pectin from apple, citrus and sugar beet. GalA was obtained from apple and citrus pectin in a concentration of up to 2.5 mM after 4 h reaction at 50 °C, through the combined action of BLI09 PME with either TMA01 or BLI04 exo-PGs. Overall, this work contributes to expand the knowledge of pectinases from thermophiles and provides further insights into their application in the initial valorisation of sustainable pectin-rich biomass feedstocks.

Probing binding specificity of the sucrose transporter AtSUC2 with fluorescent coumarin glucosides.

The phloem sucrose transporter, AtSUC2, is promiscuous with respect to substrate recognition, transporting a range of glucosides in addition to sucrose, including naturally occurring coumarin glucosides. We used the inherent fluorescence of coumarin glucosides to probe the specificity of AtSUC2 for its substrates, and determined the structure-activity relationships that confer phloem transport in vivo using Arabidopsis seedlings. In addition to natural coumarin glucosides, we synthesized new compounds to identify key structural features that specify recognition by AtSUC2. Our analysis of the structure-activity relationship revealed that the presence of a free hydroxyl group on the coumarin moiety is essential for binding by AtSUC2 and subsequent phloem mobility. Structural modeling of the AtSUC2 substrate-binding pocket explains some important structural requirements for the interaction of coumarin glucosides with the AtSUC2 transporter.

An integrated biorefinery concept for conversion of sugar beet pulp into value-added chemicals and pharmaceutical intermediates.

Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including d-glucose (Glu), l-arabinose (Ara) and d-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for the fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial yeast strain to produce bioethanol at a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into l-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. The current work is addressing the upgrading of the remaining SBP monomer, GalAc, and the modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA).

Transport Function of Rice Amino Acid Permeases (AAPs).

The transport function of four rice (Oryza sativa) amino acid permeases (AAPs), OsAAP1 (Os07g04180), OsAAP3 (Os06g36180), OsAAP7 (Os05g34980) and OsAAP16 (Os12g08090), was analyzed by expression in Xenopus laevis oocytes and electrophysiology. OsAAP1, OsAAP7 and OsAAP16 functioned, similarly to Arabidopsis AAPs, as general amino acid permeases. OsAAP3 had a distinct substrate specificity compared with other rice or Arabidopsis AAPs. OsAAP3 transported the basic amino acids lysine and arginine well but selected against aromatic amino acids. The transport of basic amino acids was further analyzed for OsAAP1 and OsAAP3, and the results support the transport of both neutral and positively charged forms of basic amino acids by the rice AAPs. Cellular localization using the tandem enhanced green fluorescent protein (EGFP)-red fluorescent protein (RFP) reporter pHusion showed that OsAAP1 and OsAAP3 localized to the plasma membrane after transient expression in onion epidermal cells or stable expression in Arabidopsis.

Arabidopsis sucrose transporter AtSUC1 is important for pollen germination and sucrose-induced anthocyanin accumulation.

The Arabidopsis (Arabidopsis thaliana) sucrose transporter AtSUC1 (At1g71880) is highly expressed in pollen; however, its function has remained unknown. Here, we show that suc1 mutant pollen is defective in vivo, as evidenced by segregation distortion, and also has low rates of germination in vitro. AtSUC1-green fluorescent protein was localized to the plasma membrane in pollen tubes. AtSUC1 is also expressed in roots and external application of sucrose increased AtSUC1 expression in roots. AtSUC1 is important for sucrose-dependent signaling leading to anthocyanin accumulation in seedlings. suc1 mutants accumulated less anthocyanins in response to exogenous sucrose or maltose and microarray analysis revealed reduced expression of many genes important for anthocyanin biosynthesis. The results indicate that AtSUC1 is important for sugar signaling in vegetative tissue and for normal male gametophyte function.

A colorimetric assay for screening transketolase activity.

A tetrazolium red-based colorimetric assay has been devised to screen for transketolase activity with a range of aldehyde acceptors. The colorimetric TK assay is able to detect >8% bioconversion using non-alpha-hydroxylated aldehydes as acceptor substrates and is significantly faster and more convenient to use than chromatographic procedures.

Integrating membrane transport with male gametophyte development and function through transcriptomics.

Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::beta-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K+ homeostasis in pollen development.

A combined bioinformatic and experimental approach is being used to uncover the functions of a novel family of cation/H(+) exchanger (CHX) genes in plants using Arabidopsis as a model. The predicted protein (85-95 kD) of 28 AtCHX genes after revision consists of an amino-terminal domain with 10 to 12 transmembrane spans (approximately 440 residues) and a hydrophilic domain of approximately 360 residues at the carboxyl end, which is proposed to have regulatory roles. The hydrophobic, but not the hydrophilic, domain of plant CHX is remarkably similar to monovalent cation/proton antiporter-2 (CPA2) proteins, especially yeast (Saccharomyces cerevisiae) KHA1 and Synechocystis NhaS4. Reports of characterized fungal and prokaryotic CPA2 indicate that they have various transport modes, including K(+)/H(+) (KHA1), Na(+)/H(+)-K(+) (GerN) antiport, and ligand-gated ion channel (KefC). The expression pattern of AtCHX genes was determined by reverse transcription PCR, promoter-driven beta-glucuronidase expression in transgenic plants, and Affymetrix ATH1 genome arrays. Results show that 18 genes are specifically or preferentially expressed in the male gametophyte, and six genes are highly expressed in sporophytic tissues. Microarray data revealed that several AtCHX genes were developmentally regulated during microgametogenesis. An exciting idea is that CHX proteins allow osmotic adjustment and K(+) homeostasis as mature pollen desiccates and then rehydrates at germination. The multiplicity of CHX-like genes is conserved in higher plants but is not found in animals. Only 17 genes, OsCHX01 to OsCHX17, were identified in rice (Oryza sativa) subsp. japonica, suggesting diversification of CHX in Arabidopsis. These results reveal a novel CHX gene family in flowering plants with potential functions in pollen development, germination, and tube growth.

Protein-protein interactions between sucrose transporters of different affinities colocalized in the same enucleate sieve element.

Suc represents the major transport form for carbohydrates in plants. Suc is loaded actively against a concentration gradient into sieve elements, which constitute the conduit for assimilate export out of leaves. Three members of the Suc transporter family with different properties were identified: SUT1, a high-affinity Suc proton cotransporter; SUT4, a low-affinity transporter; and SUT2, which in yeast is only weakly active and shows features similar to those of the yeast sugar sensors RGT2 and SNF3. Immunolocalization demonstrated that all three SUT proteins are localized in the same enucleate sieve element. Thus, the potential of Suc transporters to form homooligomers was tested by the yeast-based split-ubiquitin system. The results show that both SUT1 and SUT2 have the potential to form homooligomers. Moreover, all three Suc transporters have the potential to interact with each other. As controls, a potassium channel and a monosaccharide transporter, expressed in the plasma membrane, did not interact with the SUTs. The in vivo interaction between the functionally different Suc transporters indicates that the membrane proteins are capable of forming oligomeric structures that, like mammalian Glc transporter complexes, might be of functional significance for the regulation of transport.