Crystal Structure of a Sucrose-6-phosphate Hydrolase from Lactobacillus gasseri with Potential Applications in Fructan Production and the Food Industry.

Fructooligosaccharides (FOSs) are polymers of fructose with a prebiotic activity because of their production and fermentation by bacteria that inhabit the gastrointestinal tract and are widely used in the industry and new functional foods. Lactobacillus gasseri stands out as an important homofermentative microorganism related to FOS production, and its potential applications in the industry are undeniable. In this study, we report the production and characterization of a sucrose-6-phosphate hydrolase from L. gasseri belonging to the GH32 family. Apo-LgAs32 and LgAs32 complexed with ß-d-fructose structures were determined at a resolution of 1.94 and 1.84 Å, respectively. The production of FOS, fructans, 1-kestose, and nystose by the recombinant LgAs32, using sucrose as a substrate, shown in this study is very promising. When compared to its homologous enzyme from Lactobacillus reuteri, the production of 1-kestose by LgAs32 is increased; thus, LgAs32 can be considered as an alternative in fructan production and other industrial applications.

The cytosolic invertase NI6 affects vegetative growth, flowering, fruit set, and yield in tomato.

Sucrose metabolism is important for most plants, both as the main source of carbon and via signaling mechanisms that have been proposed for this molecule. A cleaving enzyme, invertase (INV) channels sucrose into sink metabolism. Although acid soluble and insoluble invertases have been largely investigated, studies on the role of neutral invertases (A/N-INV) have lagged behind. Here, we identified a tomato A/N-INV encoding gene (NI6) co-localizing with a previously reported quantitative trait locus (QTL) largely affecting primary carbon metabolism in tomato. Of the eight A/N-INV genes identified in the tomato genome, NI6 mRNA is present in all organs, but its expression was higher in sink tissues (mainly roots and fruits). A NI6-GFP fusion protein localized to the cytosol of mesophyll cells. Tomato NI6-silenced plants showed impaired growth phenotype, delayed flowering and a dramatic reduction in fruit set. Global gene expression and metabolite profile analyses of these plants revealed that NI6 is not only essential for sugar metabolism, but also plays a signaling role in stress adaptation. We also identified major hubs, whose expression patterns were greatly affected by NI6 silencing; these hubs were within the signaling cascade that coordinates carbohydrate metabolism with growth and development in tomato.

Characterization of raffinose metabolism genes uncovers a wild Arachis galactinol synthase conferring tolerance to abiotic stresses.

Raffinose family oligosaccharides (RFOs) are implicated in plant regulatory mechanisms of abiotic stresses tolerance and, despite their antinutritional proprieties in grain legumes, little information is available about the enzymes involved in RFO metabolism in Fabaceae species. In the present study, the systematic survey of legume proteins belonging to five key enzymes involved in the metabolism of RFOs (galactinol synthase, raffinose synthase, stachyose synthase, alpha-galactosidase, and beta-fructofuranosidase) identified 28 coding-genes in Arachis duranensis and 31 in A. ipaënsis. Their phylogenetic relationships, gene structures, protein domains, and chromosome distribution patterns were also determined. Based on the expression profiling of these genes under water deficit treatments, a galactinol synthase candidate gene (AdGolS3) was identified in A. duranensis. Transgenic Arabidopsis plants overexpressing AdGolS3 exhibited increased levels of raffinose and reduced stress symptoms under drought, osmotic, and salt stresses. Metabolite and expression profiling suggested that AdGolS3 overexpression was associated with fewer metabolic perturbations under drought stress, together with better protection against oxidative damage. Overall, this study enabled the identification of a promising GolS candidate gene for metabolic engineering of sugars to improve abiotic stress tolerance in crops, whilst also contributing to the understanding of RFO metabolism in legume species.

Genome-wide analysis of the invertase gene family from maize.

KEY MESSAGE: The recent release of the maize genome (AGPv4) contains annotation errors of invertase genes and therefore the enzymes are bestly curated manually at the protein level in a comprehensible fashion The synthesis, transport and degradation of sucrose are determining factors for biomass allocation and yield of crop plants. Invertase (INV) is a key enzyme of carbon metabolism in both source and sink tissues. Current releases of the maize genome correctly annotates only two vacuolar invertases (ivr1 and ivr2) and four cell wall invertases (incw1, incw2 (mn1), incw3, and incw4). Our comprehensive survey identified 21 INV isogenes for which we propose a standard nomenclature grouped phylogenetically by amino acid similarity: three vacuolar (INVVR), eight cell wall (INVCW), and ten alkaline/neutral (INVAN) isogenes which form separate dendogram branches due to distinct molecular features. The acidic enzymes were curated for the presence of the DPN tripeptide which is coded by one of the smallest exons reported in plants. Particular attention was placed on the molecular role of INV in vascular tissues such as the nodes, internodes, leaf sheath, husk leaves and roots. We report the expression profile of most members of the maize INV family in nine tissues in two developmental stages, R1 and R3. INVCW7, INVVR2, INVAN8, INVAN9, INVAN10, and INVAN3 displayed the highest absolute expressions in most tissues. INVVR3, INVCW5, INVCW8, and INVAN1 showed low mRNA levels. Expressions of most INVs were repressed from stage R1 to R3, except for INVCW7 which increased significantly in all tissues after flowering. The mRNA levels of INVCW7 in the vegetative stem correlated with a higher transport rate of assimilates from leaves to the cob which led to starch accumulation and growth of the female reproductive organs.

The riddle of mitochondrial alkaline/neutral invertases: A novel Arabidopsis isoform mainly present in reproductive tissues and involved in root ROS production.

Alkaline/neutral invertases (A/N-Inv), glucosidases that irreversibly hydrolyze sucrose into glucose and fructose, play significant roles in plant growth, development, and stress adaptation. They occur as multiple isoforms located in the cytosol or organelles. In Arabidopsis thaliana, two mitochondrial A/N-Inv genes (A/N-InvA and A/N-InvC) have already been investigated. In this study, we functionally characterized A/N-InvH, a third Arabidopsis gene coding for a mitochondrial-targeted protein. The phenotypic analysis of knockout mutant plants (invh) showed a severely reduced shoot growth, while root development was not affected. The emergence of the first floral bud and the opening of the first flower were the most affected stages, presenting a significant delay. A/N-InvH transcription is markedly active in reproductive tissues. It is also expressed in the elongation and apical meristem root zones. Our results show that A/N-InvH expression is not evident in photosynthetic tissues, despite being of relevance in developmental processes and mitochondrial functional status. NaCl and mannitol treatments increased A/N-InvH expression twofold in the columella root cap. Moreover, the absence of A/N-InvH prevented ROS formation, not only in invh roots of salt- and ABA-treated seedlings but also in invh control roots. We hypothesize that this isoform may take part in the ROS/sugar (sucrose or its hydrolysis products) signaling pathway network, involved in reproductive tissue development, cell elongation, and abiotic stress responses.

Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM)

Abstract The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30 °C, 6% (v/v), inoculum size and 150 rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.

Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM).

The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30°C, 6% (v/v), inoculum size and 150rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.

Characterization of the alkaline/neutral invertase gene in Dendrobium officinale and its relationship with polysaccharide accumulation.

Dendrobium officinale is one of the most well-known traditional Chinese medicines, and polysaccharide is its main active ingredient. Many studies have investigated the synthesis and accumulation mechanisms of polysaccharide, but until recently, little was known about the molecular mechanism of how polysaccharide is synthesized because no related genes have been cloned. In this study, we cloned an alkaline/neutral invertase gene from D. officinale (DoNI) by the rapid amplification of cDNA ends (RACE) method. DoNI was 2231 bp long and contained an open reading frame that predicted a 62.8-kDa polypeptide with 554-amino acid residues. An alkaline/neutral invertase conserved domain was predicted from this deduced amino acid sequence, and DoNI had a similar deduced amino acid sequence to Setaria italica and Oryza brachyantha. We also found that DoNI expression in different tissues was closely related to DoNI activity, and more importantly, polysaccharide level. Our results indicate that DoNI is associated with polysaccharide accumulation in D. officinale.

Allelic differences in a vacuolar invertase affect Arabidopsis growth at early plant development.

Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-Inv activity was detected in a near isogenic line (NIL) population derived from a cross between two divergent accessions: Landsberg erecta (Ler) and Cape Verde Island (CVI), with the CVI allele conferring both higher Inv activity and longer radicles. The aim of the current work is to understand the mechanism(s) underlying this QTL by analyzing structural and functional differences of vac-Inv from both accessions. Relative transcript abundance analyzed by quantitative real-time PCR (qRT-PCR) showed similar expression patterns in both accessions; however, DNA sequence analyses revealed several polymorphisms that lead to changes in the corresponding protein sequence. Moreover, activity assays revealed higher vac-Inv activity in genotypes carrying the CVI allele than in those carrying the Ler allele. Analyses of purified recombinant proteins showed a similar K m for both alleles and a slightly higher V max for that of Ler. Treatment of plant extracts with foaming to release possible interacting Inv inhibitory protein(s) led to a large increase in activity for the Ler allele, but no changes for genotypes carrying the CVI allele. qRT-PCR analyses of two vac-Inv inhibitors in seedlings from parental and NIL genotypes revealed different expression patterns. Taken together, these results demonstrate that the vac-Inv QTL affects root biomass accumulation and also carbon partitioning through a differential regulation of vac-Inv inhibitors at the mRNA level.

Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties.

Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVAAOX1 and INVBAOX1. The expression levels of INVAAOX1 (1660 U/mg) and INVBAOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVAAOX1 and INVBAOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (kcat/KM) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVAAOX1 and INVBAOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVAAOX1 and D-INVBAOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164 s(-1) mM(-1), respectively), and a 1.3- to 5-fold lower thermal stability than the glycosylated forms at temperatures of 35-45 °C. After deglycosylation no effect was observed in optimal pH, being of 5.5 for INVAAOX1, INVBAOX1, D-INVAAOX1 and D-INVBAOX1. The invertase activity of both enzymes increased in 80% (INVAAOX1) and 20% (INVBAOX1) in the presence of Mn(2+) at 1 mM and 5 mM, respectively. INVAAOX1 and INVBAOX1 were highly active at sucrose concentrations of up to 400 and 300 mM, respectively; however, the tolerance to sucrose decreased to 300 mM for D-INVAAOX1. Our findings suggest that glycosylation of INVAAOX1 and INVBAOX1 plays an important role in their thermal stability, catalytic efficiency, and tolerance to sucrose. In conclusion, the expression of INVA and INVB from Z. mobilis in P. pastoris yields new catalysts with improved catalytic properties, making them suitable candidates for a number of industrial applications or for the improvement of ethanol production from cane molasses.

HbNIN2, a cytosolic alkaline/neutral-invertase, is responsible for sucrose catabolism in rubber-producing laticifers of Hevea brasiliensis (para rubber tree).

In Hevea brasiliensis, an alkaline/neutral invertase (A/N-Inv) is responsible for sucrose catabolism in latex (essentially the cytoplasm of rubber-producing laticifers, the source of natural rubber) and implicated in rubber yield. However, neither the gene encoding this enzyme nor its molecular and biochemical properties have been well documented. Three Hevea A/N-Inv genes, namely HbNIN1, 2 and 3, were first cloned and characterized in planta and in Escherichia coli. Cellular localizations of HbNIN2 mRNA and protein were probed. From latex, active A/N-Inv proteins were purified, identified, and explored for enzymatic properties. HbNIN2 was identified as the major A/N-Inv gene functioning in latex based on its functionality in E. coli, its latex-predominant expression, the conspicuous localization of its mRNA and protein in the laticifers, and its expressional correlation with rubber yield. An active A/N-Inv protein was partially purified from latex, and determined as HbNIN2. The enhancement of HbNIN2 enzymatic activity by pyridoxal is peculiar to A/N-Invs in other plants. We conclude that HbNIN2, a cytosolic A/N-Inv, is responsible for sucrose catabolism in rubber laticifers. The results contribute to the studies of sucrose catabolism in plants as a whole and natural rubber synthesis in particular.

A novel ß-fructofuranosidase in Coleoptera: Characterization of a ß-fructofuranosidase from the sugarcane weevil, Sphenophorus levis.

ß-fructofuranosidases or invertases (EC 3.2.1.26) catalyze the hydrolysis of sucrose into fructose and glucose. ß-fructofuranosidases have been widely described in microorganisms, but were not known in the animal kingdom until very recently. There are studies reporting lepidopteran ß-fructofuranosidases, but no ß-fructofuranosidase gene sequence or encoding transcript has previously been identified in beetles. Considering the scarcity of functional studies on insect ß-fructofuranosidases and their apparent non-occurrence among coleopterans, the aim of the present study was to investigate the occurrence and characterize a ß-fructofuranosidase transcript identified in a cDNA library from the sugarcane weevil, Sphenophorus levis (Curculionidae). To validate that the ß-fructofuranosidase sequence (herein denominated Sl-ß-fruct) is indeed encoded by the S. levis genome, PCRs were performed using genomic DNA extracted from the larval fat body as well as DNA from the midgut with microbial content. Amplification of Sl-ß-fruct gene using larval fat body DNA indicated its presence in the insect's genomic DNA. The Sl-ß-fruct gene was cloned in Pichia pastoris to produce the recombinant enzyme (rSl-ß-fruct). Molecular weight of the recombinant protein was about 64 kDa, indicating possible glycosylation, since the theoretical weight was 54.8 kDa. The substrate specificity test revealed that rSl-ß-fruct hydrolyzes sucrose and raffinose, but not melibiose or maltose, thereby confirming invertase activity. The pH curve revealed greatest activity at pH 5.0, demonstrating rSl-ß-fruct to be an acidic ß-fructofuranosidase. Quantitative PCR (qRT-PCR) analyses indicated that the production of mRNA only occurs in the midgut and reaches the greatest expression level in 30-day-old larvae, which is the expected pattern for digestive enzymes. Chromatography of glycosidases from S. levis midguts showed two enzymes acting as ß-fructofuranosidase, indicating the presence of a Sl-ß-fruct isoform or a ß-fructofuranosidase from insect intestinal microbiota. Moreover, it was found that α-glucosidases do not act on sucrose hydrolysis. Phylogenetic analyses indicated this enzyme to be similar to enzymes found in other coleopteran and lepidopteran ß-fructofuranosidases, but also closely similar to bacterial enzymes, suggesting potential horizontal gene transfer. Despite this, the enzyme seems to be restricted to different groups of bacteria, which suggests distinct origin events. The present study expands the concept of the occurrence of ß-fructofuranosidase in insects. Despite the few descriptions of this gene in the animal kingdom, it is possible to state that ß-fructofuranosidase is crucial to the establishment of some insects throughout their evolutionary history, especially members of the Lepidoptera and Coleoptera clades.

Gene encoding a novel invertase from a xerophilic Aspergillus niger strain and production of the enzyme in Pichia pastoris.

ß-Fructofuranosidases or invertases (EC 3.2.1.26) are enzymes that are widely used in the food industry, where fructose is preferred over sucrose, because it is sweeter and does not crystallize easily. Since Aspergillus niger GH1, an xerophilic fungus from the Mexican semi-desert, has been reported to be an invertase producer, and because of the need for new enzymes with biotechnological applications, in this work, we describe the gene and amino acid sequence of the invertase from A. niger GH1, and the use of a synthetic gene to produce the enzyme in the methylotrophic yeast Pichia pastoris. In addition, the produced invertase was characterized biochemically. The sequence of the invertase gene had a length of 1770 bp without introns, encodes a protein of 589 amino acids, and presented an identity of 93% and 97% with invertases from Aspergillus kawachi IFO 4308 and A. niger B60, respectively. A 4.2 L culture with the constructed recombinant P. pastoris strain showed an extracellular and periplasmic invertase production at 72 h induction of 498 and 3776 invertase units (U), respectively, which corresponds to 1018 U/L of culture medium. The invertase produced had an optimum pH of 5.0, optimum temperature of 60 °C, and specific activity of 3389 U/mg protein, and after storage for 96 h at 4 °C showed 93.7% of its activity. This invertase could be suitable for producing inverted sugar used in the food industry.

Gene expression patterns of invertase gene families and modulation of the inhibitor gene in tomato sucrose metabolism.

Patterns of gene expression in the different types of sucrose metabolism in the tomato are highly variable and heritable. This genetic variation causes considerable functional differences. We examined the patterns of expression of invertase (Inv) gene families and an invertase inhibitor (INH) gene involved in elongating roots, hypocotyls, and fruit of the tomato (Lycopersicon esculentum cv. Micro-Tom and L. chmielewskii) through a real-time quantitative PCR analysis. We found that the Lin6 gene plays an important role in the vegetative growth stage. Lin5 and Lin7 did not express in Micro-Tom, but did express in L. chmielewskii. Overall relative expression levels of sucrose Inv gene families were significantly lower in L. chmielewskii during the reproductive growth stage than in Micro-Tom, being up to hundreds of times lower. It was not expressed in the dissepiment in L. chmielewskii. We suggest that differences in sucrose accumulation in tomato fruit is mainly due to differentially expressed invertase gene families at the later fruit growth stages.

Constitutive high-level expression of a codon-optimized ß-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris.

Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable ß-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.

Short-term proteomic dynamics reveal metabolic factory for active extrafloral nectar secretion by Acacia cornigera ant-plants.

Despite the ecological and evolutionary importance of nectar, mechanisms controlling its synthesis and secretion remain largely unknown. It is widely believed that nectar is 'secreted phloem sap', but current research reveals a biochemical complexity that is unlikely to stem directly from the phloem. We used the short daily peak in production of extrafloral nectar by Acacia cornigera to investigate metabolic and proteomic dynamics before, during and after 2 h of diurnal secretion. Neither hexoses nor dominating nectar proteins (nectarins) were detected in the phloem before or during nectar secretion, excluding the phloem as the direct source of major nectar components. Enzymes involved in the anabolism of sugars, amino acids, proteins, and nectarins, such as invertase, ß-1,3-glucanase and thaumatin-like protein, accumulated in the nectary directly before secretion and diminished quantitatively after the daily secretion process. The corresponding genes were expressed almost exclusively in nectaries. By contrast, protein catabolic enzymes were mainly present and active after the secretion peak, and may function in termination of the secretion process. Thus the metabolic machinery for extrafloral nectar production is synthesized and active during secretion and degraded thereafter. Knowing the key enzymes involved and the spatio-temporal patterns in their expression will allow elucidation of mechanisms by which plants control nectar quality and quantity.

A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis.

Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle.

The genome sequence of Propionibacterium acidipropionici provides insights into its biotechnological and industrial potential.

BACKGROUND: Synthetic biology allows the development of new biochemical pathways for the production of chemicals from renewable sources. One major challenge is the identification of suitable microorganisms to hold these pathways with sufficient robustness and high yield. In this work we analyzed the genome of the propionic acid producer Actinobacteria Propionibacterium acidipropionici (ATCC 4875). RESULTS: The assembled P. acidipropionici genome has 3,656,170 base pairs (bp) with 68.8% G + C content and a low-copy plasmid of 6,868 bp. We identified 3,336 protein coding genes, approximately 1000 more than P. freudenreichii and P. acnes, with an increase in the number of genes putatively involved in maintenance of genome integrity, as well as the presence of an invertase and genes putatively involved in carbon catabolite repression. In addition, we made an experimental confirmation of the ability of P. acidipropionici to fix CO2, but no phosphoenolpyruvate carboxylase coding gene was found in the genome. Instead, we identified the pyruvate carboxylase gene and confirmed the presence of the corresponding enzyme in proteome analysis as a potential candidate for this activity. Similarly, the phosphate acetyltransferase and acetate kinase genes, which are considered responsible for acetate formation, were not present in the genome. In P. acidipropionici, a similar function seems to be performed by an ADP forming acetate-CoA ligase gene and its corresponding enzyme was confirmed in the proteome analysis. CONCLUSIONS: Our data shows that P. acidipropionici has several of the desired features that are required to become a platform for the production of chemical commodities: multiple pathways for efficient feedstock utilization, ability to fix CO2, robustness, and efficient production of propionic acid, a potential precursor for valuable 3-carbon compounds.

Molecular and functional characterization of novel fructosyltransferases and invertases from Agave tequilana.

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants.

Changes in stomatal function and water use efficiency in potato plants with altered sucrolytic activity.

As water availability for agriculture decreases, breeding or engineering of crops with improved water use efficiency (WUE) will be necessary. As stomata are responsible for controlling gas exchange across the plant epidermis, metabolic processes influencing solute accumulation in guard cells are potential targets for engineering. In addition to its role as an osmoticum, sucrose breakdown may be required for synthesis of other osmotica or generation of the ATP needed for solute uptake. Thus, alterations in partitioning of sucrose between storage and breakdown may affect stomatal function. In agreement with this hypothesis, potato (Solanum tuberosum) plants expressing an antisense construct targeted against sucrose synthase 3 (SuSy3) exhibited decreased stomatal conductance, a slight reduction in CO(2) fixation and increased WUE. Conversely, plants with increased guard cell acid invertase activity caused by the introduction of the SUC2 gene from yeast had increased stomatal conductance, increased CO(2) fixation and decreased WUE. (14)CO(2) feeding experiments indicated that these effects cannot be attributed to alterations in photosynthetic capacity, and most likely reflect alterations in stomatal function. These results highlight the important role that sucrose breakdown may play in guard cell function and indicate the feasibility of manipulating plant WUE through engineering of guard cell sucrose metabolism.