D-galacturonate Reductase Research Articles

Genome-Wide Analysis of Ascorbic Acid Metabolism Related Genes in Fragaria × ananassa and Its Expression Pattern Analysis in Strawberry Fruits.

Ascorbic acid (AsA) is an important antioxidant for scavenging reactive oxygen species and it is essential for human health. Strawberry (Fragaria × ananassa) fruits are rich in AsA. In recent years, strawberry has been regarded as a model for non-climacteric fruit ripening. However, in contrast to climacteric fruits, such as tomato, the regulatory mechanism of AsA accumulation in strawberry fruits remains largely unknown. In this study, we first identified 125 AsA metabolism-related genes from the cultivated strawberry “Camarosa” genome. The expression pattern analysis using an available RNA-seq data showed that the AsA biosynthetic-related genes in the D-mannose/L-galactose pathway were downregulated remarkably during fruit ripening which was opposite to the increasing AsA content in fruits. The D-galacturonate reductase gene (GalUR) in the D-Galacturonic acid pathway was extremely upregulated in strawberry receptacles during fruit ripening. The FaGalUR gene above belongs to the aldo-keto reductases (AKR) superfamily and has been proposed to participate in AsA biosynthesis in strawberry fruits. To explore whether there are other genes in the AKR superfamily involved in regulating AsA accumulation during strawberry fruit ripening, we further implemented a genome-wide analysis of the AKR superfamily using the octoploid strawberry genome. A total of 80 FaAKR genes were identified from the genome and divided into 20 subgroups based on phylogenetic analysis. These FaAKR genes were unevenly distributed on 23 chromosomes. Among them, nine genes showed increased expression in receptacles as the fruit ripened, and notably, FaAKR23 was the most dramatically upregulated FaAKR gene in receptacles. Compared with fruits at green stage, its expression level increased by 142-fold at red stage. The qRT-PCR results supported that the expression of FaAKR23 was increased significantly during fruit ripening. In particular, the FaAKR23 was the only FaAKR gene that was significantly upregulated by abscisic acid (ABA) and suppressed by nordihydroguaiaretic acid (NDGA, an ABA biosynthesis blocker), indicating FaAKR23 might play important roles in ABA-mediated strawberry fruit ripening. In a word, our study provides useful information on the AsA metabolism during strawberry fruit ripening and will help understand the mechanism of AsA accumulation in strawberry fruits.

Identification of a D-galacturonate reductase efficiently using NADH as a cofactor

D-galacturonate reductases are catalysing the reversible reduction of D-galacturonate to L-galactonate using NAD(P)H as a cofactor. The enzymes are part of two different pathways. One pathway is the fungal pathway for the catabolism of the main compound of pectin, D-galacturonate. The other pathway is a a pathway in plants for L-ascorbic acid synthesis. The previously described naturally occurring enzymes preferably use NADPH as a cofactor. Although certain D-galacturonate reductases, such as the reductases from Aspergillus niger or Euglena gracilis also accept NADH, their activity is significantly higher with NADPH. We identified in E. gracilis a gene, called gaa1, coding for a D-galacturonate reductase with similar activities with NADH and NADPH. It is potentially useful for the metabolic engineering of microbes to make use of pectin rich biomass.

Comparative transcriptome analysis reveals genes and pathways associated with anthocyanins in strawberry

BACKGROUND: Anthocyanins are not only one of the most important antioxidants in plants but also responsible for the coloration in strawberry (Fragaria×ananassa), it is suggested to be associated with ascorbic acid (AsA), sugars and plant hormones metabolism. OBJECTIVE: To elucidate the molecular differences of genes and pathways in the presence and absence of anthocyanins. METHODS: RNA sequencing (RNAseq) of red-fleshed strawberry ‘Benihoppe’ and its white-fleshed mutant ‘Xiaobai’ was performed. RESULTS: A total of 1,156 and 1,378 transcripts were differentially expressed (DE) in two cultivars at white stage and red stage respectively. Among them, 28, 49 and 72 DE transcripts were involved in AsA, sugars metabolism and plant hormones signaling respectively. Fruits of ‘Benihoppe’ with higher anthocyanins content contained higher AsA, the expression of D-galacturonate reductase (GalUR) in white-fleshed strawberry was down-regulated. Particularly, the expression of transcripts involved in sugars, jasmonic acid (JA) and abscisic acid (ABA) signaling was decreased, by contrast, the expression of transcripts involved in auxin and gibberellins (GAs) signaling was significantly increased. CONCLUSIONS: The lower AsA level in white-fleshed strawberry is possibly because of the lower flux of D-galacturonate biosynthesis pathway. Anthocyanins was associated with gene expression involved in AsA, sugars, and hormones signaling metabolisms.

Vitamin C in fleshy fruits: biosynthesis, recycling, genes, and enzymes

L-ascorbic acid (vitamin C) is a plant secondary metabolite that has a variety of functions both in plant tissues and in the human body. Plants are the main source of vitamin C in human nutrition, especially citrus, rose hip, tomato, strawberry, pepper, papaya, kiwi, and currant fruits. However, in spite of the biological significance of L-ascorbic acid, the pathways of its biosynthesis in plants were fully understood only in 2007 by the example of a model plant Arabidopsis thaliana. In the present review, the main biosynthetic pathways of vitamin C are described: the L-galactose pathway, L-gulose pathway, galacturonic and myo-inositol pathway. To date, the best studied is the L-galactose pathway (Smyrnoff–Wheeler pathway). Only for this pathway all the enzymes and the entire cascade of reactions have been described. For other pathways, only hypothetical metabolites are proposed and not all the catalyzing enzymes have been identified. The key genes participating in ascorbic acid biosynthesis and accumulation in fleshy fruits are highlighted. Among them are L-galactose pathway proteins (GDP-mannose phosphorylase (GMP, VTC1), GDP-D-mannose epimerase (GME), GDP-L-galactose phosphorylase (GGP, VTC2/VTC5), L-galactose-1-phosphate phosphatase (GPP/VTC4), L-galactose-1-dehydrogenase (GalDH), and L-galactono1,4-lactone dehydrogenase (GalLDH)); D-galacturonic pathway enzymes (NADPH-dependent D-galacturonate reductase (GalUR)); and proteins, controlling the recycling of ascorbic acid (dehydroascorbate reductase (DHAR1) and monodehydroascorbate reductase (MDHAR)). Until now, there is no clear and unequivocal evidence for the existence of one predominant pathway of vitamin C biosynthesis in fleshy fruits. For example, the L-galactose pathway is predominant in peach and kiwi fruits, whereas the D-galacturonic pathway seems to be the most essential in grape and strawberry fruits. However, in some plants, such as citrus and tomato fruits, there is a switch between different pathways during ripening. It is noted that the final ascorbic acid content in fruits depends not only on biosynthesis but also on the rate of its oxidation and recirculation.

L-ascorbic acid metabolism in an ascorbate-rich kiwifruit (Actinidia. Eriantha Benth.) cv. ‘White’ during postharvest

Kiwifruit (Actinidia eriantha Benth.) ‘White’, a novel cultivar with higher L-ascorbic acid (AsA) level, is registered in China. Changes in AsA, related metabolites, enzymatic activity, and gene expression associated with AsA biosynthesis and recycling process were investigated in this paper. The results indicated that AsA biosynthesis through L-galactose pathway supplemented by D-galacturonic acid pathway and AsA recycling collectively contributed to accumulating and remaining higher AsA level in kiwifruit cv. ‘White’ during postharvest. Moreover, L-galactose dehydrogenase (GalDH) activity and relative expressions of the genes encoding GDP-D-mannose pyrophosphorylase (GMP), L-galactose-1-P phosphatase (GPP), GDP-L-galactose phosphorylase (GGP), GalDH and D-galacturonate reductase (GalUR) were important for regulation of AsA biosynthesis, and the activity and expression of dehydroascorbate reductase (DHAR) were primarily responsible for regulation of AsA recycling in kiwifruit ‘White’ during postharvest.

Fermentative production of l-galactonate by using recombinant Saccharomyces cerevisiae containing the endogenous galacturonate reductase gene from Cryptococcus diffluens

The GAR1 gene, encoding d-galacturonate reductase in Cryptococcus diffluens, was isolated, and the GAR1-expression plasmid was constructed by insertion of GAR1 downstream of the yeast constitutive promoter in the yeast-integrating vector. Recombinant Saccharomyces cerevisiae expressing C.diffluensd-galacturonate reductase from a genome integrated copy of the gene was cultured for use the conversion of d-galacturonic acid to l-galactonic acid. The optimum conditions for l-galactonic acid production were determined in terms of the initial concentration of d-galacturonic acid, fermentation pH, and mixed sugars. The following conditions yielded high efficiency in the conversion of d-galacturonic acid to l-galactonic acid in large-scale cultures: 0.1% initial d-galacturonic acid concentration, pH 3.5, and glucose as additional sugar. The aerobic condition was necessary for the conversion of d-galacturonic acid. Subculture of that recombinant was not showing to decrease of the d-galacturonic acid conversion rate even though it was repeated in ten generations. Culturing in scale-up, the conversion rate of d-galacturonic acid to l-galactonic acid was increased.

Metabolic engineering of the fungal D-galacturonate pathway for L-ascorbic acid production.

BackgroundSynthetic L-ascorbic acid (vitamin C) is widely used as a preservative and nutrient in food and pharmaceutical industries. In the current production method, D-glucose is converted to L-ascorbic acid via several biochemical and chemical steps. The main source of L-ascorbic acid in human nutrition is plants. Several alternative metabolic pathways for L-ascorbic acid biosynthesis are known in plants. In one of them, D-galacturonic acid is the precursor. D-Galacturonic acid is also the main monomer in pectin, a plant cell wall polysaccharide. Pectin is abundant in biomass and is readily available from several waste streams from fruit and sugar processing industries.ResultsIn the present work, we engineered the filamentous fungus Aspergillus niger for the conversion of D-galacturonic acid to L-ascorbic acid. In the generated pathway, the native D-galacturonate reductase activity was utilized while the gene coding for the second enzyme in the fungal D-galacturonic acid pathway, an L-galactonate consuming dehydratase, was deleted. Two heterologous genes coding for enzymes from the plant L-ascorbic acid pathway – L-galactono-1,4-lactone lactonase from Euglena gracilis (EgALase) and L-galactono-1,4-lactone dehydrogenase from Malpighia glabra (MgGALDH) – were introduced into the A. niger strain. Alternatively, an unspecific L-gulono-1,4-lactone lactonase (smp30) from the animal L-ascorbic acid pathway was introduced in the fungal strain instead of the plant L-galactono-1,4-lactone lactonase. In addition, a strain with the production pathway inducible with D-galacturonic acid was generated by using a bidirectional and D-galacturonic acid inducible promoter from the fungus. Even though, the lactonase enzyme activity was not observed in the resulting strains, they were capable of producing L-ascorbic acid from pure D-galacturonic acid or pectin-rich biomass in a consolidated bioprocess. Product titers up to 170 mg/l were achieved.ConclusionsIn the current study, an L-ascorbic acid pathway using D-galacturonic acid as a precursor was introduced to a microorganism for the first time. This is also the first report on an engineered filamentous fungus for L-ascorbic acid production and a proof-of-concept of consolidated bioprocess for the production.

Increased antioxidant capacity in tomato by ectopic expression of the strawberry D-galacturonate reductase gene.

Increasing L-ascorbic acid (AsA, vitamin C) content in fruits is a common goal in current breeding programs due to its beneficial effect on human health. Attempts to increase AsA content by genetic engineering have resulted in variable success likely due to AsA's complex regulation. Here, we report the effect of ectopically expressing in tomato the D-galacturonate reductase (FaGalUR) gene from strawberry, involved in AsA biosynthesis, either under the control of the constitutive 35S or the tomato fruit-specific polygalucturonase (PG) promoters. Although transgenic lines showed a moderate increase on AsA content, complex changes in metabolites were found in transgenic fruits. Metabolomic analyses of ripe fruits identified a decrease in citrate, glutamate, asparagine, glucose, and fructose, accompanied by an increase of sucrose, galactinol, and chlorogenic acid. Significant metabolic changes also occurred in leaves of 35S-FaGalUR lines, which showed higher non-photochemical fluorescence quenching (NPQ), indicative of a higher constitutive photo-protective capacity. Overall, overexpression of FaGalUR increased total antioxidant capacity in fruits and the results suggest a tight control of AsA content, probably linked to a complex regulation of cellular redox state and metabolic adjustment.

Identification and characterization of a galacturonic acid transporter from Neurospora crassa and its application for Saccharomyces cerevisiae fermentation processes

BackgroundPectin-rich agricultural wastes potentially represent favorable feedstocks for the sustainable production of alternative energy and bio-products. Their efficient utilization requires the conversion of all major constituent sugars. The current inability of the popular fermentation host Saccharomyces cerevisiae to metabolize the major pectic monosaccharide D-galacturonic acid (D-GalA) significantly hampers these efforts. While it has been reasoned that the optimization of cellular D-GalA uptake will be critical for the engineering of D-GalA utilization in yeast, no dedicated eukaryotic transport protein has been biochemically described. Here we report for the first time such a eukaryotic D-GalA transporter and characterize its functionality in S. cerevisiae.ResultsWe identified and characterized the D-GalA transporter GAT-1 out of a group of candidate genes obtained from co-expression analysis in N. crassa. The N. crassa Δgat-1 deletion strain is substantially affected in growth on pectic substrates, unable to take up D-GalA, and impaired in D-GalA-mediated signaling events. Moreover, expression of a gat-1 construct in yeast conferred the ability for strong high-affinity D-GalA accumulation rates, providing evidence for GAT-1 being a bona fide D-GalA transport protein. By recombinantly co-expressing D-galacturonate reductase or uronate dehydrogenase in yeast we furthermore demonstrated a transporter-dependent conversion of D-GalA towards more reduced (L-galactonate) or oxidized (meso-galactaric acid) downstream products, respectively, over a broad concentration range.ConclusionsBy utilizing the novel D-GalA transporter GAT-1 in S. cerevisiae we successfully generated a transporter-dependent uptake and catalysis system for D-GalA into two products with high potential for utilization as platform chemicals. Our data thereby provide a considerable first step towards a more complete utilization of biomass for biofuel and value-added chemicals production.

Accumulation of Vitamin C and Enhanced Tolerance to Iron Deficiency Stress in Transgenic Tomato with GalUR Gene

The genes dominating vitamin C (AsA) synthesis in strawberry fruits may be used in increasing levels of AsA and tolerance to environmental stress in other plants by transgenic method. D-galacturonate reductase (GalUR) plays a prominent role in the regulation of the AsA biosynthetic pathway. In this study, transgenic tomato plants (Tg1, Tg2) overexpressing strawberry GalUR gene have been developed and resulted in 2.6-fold increase of AsA in fruits and 1.6-fold increase of AsA in leaves over non-transformed tomato (WT) and the levels of AsA were positively correlated with increased GalUR activity. Moreover, Tg1, Tg2 showed enhanced tolerance to iron deficiency as compared to WT. Under Fe (II) deficiency condition the plant height of Tg1, Tg2 was 1.2-1.7 times, AsA content of Tg1 was 1.8-2.8 times and Fe (II) content of Tg1 was 1.1-1.4 times as much as WT. Otherwise, AsA content of transgenic FRO2 plant (TFRO2) was more than WT but less than Tg1 and its Fe (II) content was more than WT and Tg1. The study demonstrated that the over-expression of GalUR gene enhanced the level of AsA and Fe (II) of transgenic tomato plants which had better growth than WT under iron stresses. Therefore, GalUR and FRO should be interrelate and stimulative each other.

Expression profiling of genes involved in ascorbate biosynthesis and recycling during fleshy root development in radish

Ascorbate is a primary antioxidant and an essential enzyme cofactor in plants, which has an important effect on the development of plant root system. To investigate the molecular mechanisms of ascorbate accumulation during root development and reveal the key genes of the ascorbate biosynthesis and recycling pathways, the expression of 16 related genes together with ascorbate abundance were analyzed in the flesh and skin of radish (Raphanus sativus L.) fleshy root. The content of ascorbate decreased with root growth in both the flesh and skin. Expression of GDP-d-mannose pyrophosphorylase, GDP-d-mannose-3',5'-epimerase and d-galacturonate reductase were also decreased and correlated with ascorbate levels in the flesh. In the skin, the expression of GDP-d-mannose pyrophosphorylase and l-galactose dehydrogenase was correlated with ascorbate levels. These results suggested that ascorbate accumulation is affected mainly by biosynthesis rather than recycling in radish root, and the l-galactose pathway may be the major biosynthetic route of ascorbate, and moreover, the salvage pathway may also contribute to ascorbate accumulation. The data suggested that GDP-d-mannose pyrophosphorylase could play an important role in the regulation of ascorbate accumulation during radish fleshy taproot development.

Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway

The D-mannose/L-galactose pathway for the biosynthesis of vitamin C (L-ascorbic acid; AsA) has greatly improved the understanding of this indispensable compound in plants, where it plays multifunctional roles. However, it is yet to be proven whether the same pathway holds for all the different organs of plants, especially the fruit-bearing plants, at different stages of development. Micro-Tom was used here to elucidate the mechanisms of AsA accumulation and regulation in tomato fruits. The mRNA expression of the genes in the D-mannose/L-galactose pathway were inversely correlated with increasing AsA content of Micro-Tom fruits during ripening. Feeding L-[6-14C]AsA to Micro-Tom plants revealed that the bulk of the label from AsA accumulated in the source leaf was transported to the immature green fruits, and the rate of translocation decreased as ripening progressed. L-Galactose feeding, but neither D-galacturonate nor L-gulono-1,4-lactone, enhanced the content of AsA in immature green fruit. On the other hand, L-galactose and D-galacturonate, but not L-gulono-1,4-lactone, resulted in an increase in the AsA content of red ripened fruits. Crude extract prepared from insoluble fractions of green and red fruits showed D-galacturonate reductase- and aldonolactonase-specific activities, the antepenultimate and penultimate enzymes, respectively, in the D-galacturonate pathway, in both fruits. Taken together, the present findings demonstrated that tomato fruits could switch between different sources for AsA supply depending on their ripening stages. The translocation from source leaves and biosynthesis via the D-mannose/L-galactose pathway are dominant sources in immature fruits, while the alternative D-galacturonate pathway contributes to AsA accumulation in ripened Micro-Tom fruits.

Ascorbic Acid Accumulation and Expression of Genes Involved in Its Biosynthesis and Recycling in Developing Apple Fruit

The objective of this study was to investigate ascorbic acid (AsA) accumulation, mRNA expression of genes involved in AsA biosynthesis as well as recycling, activity of key enzymes, and the relationship of them to AsA levels during the development of apple fruit (Malus ×domestica cv. Gala). AsA concentration, which mainly depends on biosynthesis, was the highest in young fruit post-anthesis and then decreased steadily toward maturation. However, AsA continued to accumulate over time because of the increase in fruit mass. Transcript levels of guanosine diphosphate (GDP)-L-galactose phosphorylase, GDP-mannose pyrophosphorylase, D-galacturonate reductase, and the post-transcriptionally regulated L-galactono-1,4-lactone dehydrogenase were not correlated with AsA accumulation in apple. In contrast, patterns of expression for L-galactose dehydrogenase, L-galactose-1-phosphate phosphatase, and GDP-mannose-3′,5′-epimerase showed a pattern of change similar to that of AsA accumulation. Although activities and expression levels of monodehydroascorbate reductase and dehydroascorbate reductase in fruit, which had less capacity for AsA recycling, were much lower than in leaves, they were not clearly correlated with AsA level during fruit development.

The d-galacturonic acid catabolic pathway in Botrytis cinerea

d-galacturonic acid is the most abundant component of pectin, one of the major polysaccharide constituents of plant cell walls. Galacturonic acid potentially is an important carbon source for microorganisms living on (decaying) plant material. A catabolic pathway was proposed in filamentous fungi, comprising three enzymatic steps, involving d-galacturonate reductase, l-galactonate dehydratase, and 2-keto-3-deoxy-l-galactonate aldolase. We describe the functional, biochemical and genetic characterization of the entire d-galacturonate-specific catabolic pathway in the plant pathogenic fungus Botrytis cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-l-galactonate aldolase gene (Bclga1). Their expression levels were highly induced in cultures containing GalA, pectate, or pectin as the sole carbon source. The four proteins were expressed in Escherichia coli and their enzymatic activity was characterized. Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on d-galacturonic acid, pectate, or pectin as the sole carbon source. In Aspergillus nidulans and A. niger, the first catabolic conversion only involves the Bcgar2 ortholog, while in Hypocrea jecorina, it only involves the Bcgar1 ortholog. In B. cinerea, however, BcGAR1 and BcGAR2 jointly contribute to the first step of the catabolic pathway, albeit to different extent. The virulence of all B. cinerea mutants in the d-galacturonic acid catabolic pathway on tomato leaves, apple fruit and bell peppers was unaltered.

Regulation of L-ascorbic acid content in strawberry fruits.

Plants have several L-ascorbic acid (AsA) biosynthetic pathways, but the contribution of each one to the synthesis of AsA varyies between different species, organs, and developmental stages. Strawberry (Fragaria×ananassa) fruits are rich in AsA. The pathway that uses D-galacturonate as the initial substrate is functional in ripe fruits, but the contribution of other pathways to AsA biosynthesis has not been studied. The transcription of genes encoding biosynthetic enzymes such as D-galacturonate reductase (FaGalUR) and myo-inositol oxygenase (FaMIOX), and the AsA recycling enzyme monodehydroascorbate reductase (FaMDHAR) were positively correlated with the increase in AsA during fruit ripening. Fruit storage for 72 h in a cold room reduced the AsA content by 30%. Under an ozone atmosphere, this reduction was 15%. Ozone treatment increased the expression of the FaGalUR, FaMIOX, and L-galactose-1-phosphate phosphatase (FaGIPP) genes, and transcription of the L-galactono-1,4-lactone dehydrogenase (FaGLDH) and FAMDHAR genes was higher in the ozone-stored than in the air-stored fruits. Analysis of AsA content in a segregating population from two strawberry cultivars showed high variability, which did not correlate with the transcription of any of the genes studied. Study of GalUR protein in diverse cultivars of strawberry and different Fragaria species showed that a correlation between GalUR and AsA content was apparent in most cases, but it was not general. Three alleles were identified in strawberry, but any sequence effect on the AsA variability was eliminated by analysis of the allele-specific expression. Taken together, these results indicate that FaGalUR shares the control of AsA levels with other enzymes and regulatory elements in strawberry fruit.

Characterization of d-galacturonate reductase purified from the psychrophilic yeast species Cryptococcus diffluens

D-Galacturonic acid reductase was purified from a psychrophilic yeast strain of Cryptococcus diffluens, which was isolated from Satho, a traditional alcohol drink in Thailand. This enzyme, named Cd-GalUAR, assimilates D-galacturonic acid and requires NADPH as a cofactor. Cd-GalUAR is about 45 kDa and stable from pH 6.5 to 7.5 and up to 35°C. Its optimum pH and temperature are pH 7.0 and 40°C, respectively. However, 80% of its maximum activity remained at 4°C. The reaction of Cd-GalUAR from D-galacturonic acid produces L-galactonic acid, which was identified by (13)C NMR and LC-MS. Three amino acid sequences were determined from trypsin-digested peptides of Cd-GalUAR. Similar sequences are found in many NAD or NADP oxidoreductases, including some D-galacturonate reductases. Our results suggest that Cd-GalUAR is the first D-galacturonate reductase identified in yeast.

Analysis of genes involved in l-ascorbic acid biosynthesis during growth and ripening of grape berries

Recent data indicate the existence of at least three l -ascorbic acid (AsA) biosynthetic pathways in plant cells. Studying their occurrence in different plant organs and species may help to decipher the precise role(s) of AsA in plant cell physiology. In grape berries, AsA is of particular importance since it is known to be the precursor of tartaric acid, an essential component of the grape fruit. The concentration of AsA increases during development of the fruit to reach a maximum at the full ripe stage. We followed the expression of genes related to the various AsA biosynthetic pathways in this plant organ during fruit ontogeny by real time RT-PCR. Among them, a gene ( VvGalUR), showing high homology to one from strawberry encoding a d-galacturonate reductase, was up-regulated during fruit ripening in parallel to the AsA content increase. Cloning of the corresponding full length cDNA showed highest similarity to the strawberry gene ( FaGalUR). Moreover, VvGalUR gene expression in grape was also up-regulated by high light, a condition that increased AsA content in grape fruits, while none of the genes involved in the other possible biosynthetic pathways analyzed increased their transcript levels. The results are discussed in relation to the presence of several AsA biosynthetic pathways in grape fruits.

The Missing Link in the Fungal D-Galacturonate Pathway: IDENTIFICATION OF THE L-THREO-3-DEOXY-HEXULOSONATE ALDOLASE

The fungal path for the catabolism of D-galacturonate is only partially known. It is however distinctly different to the well-known bacterial path. The known elements of the fungal path are D-galacturonate reductase converting D-galacturonate to L-galactonate and L-galactonate dehydratase converting L-galactonate to L-threo-3-deoxy-hexulosonate (2-keto-3-deoxy-L-galactonate). Here we describe the missing link in this pathway, an aldolase converting L-threo-3-deoxy-hexulosonate to pyruvate and L-glyceraldehyde. Fungal enzymes converting L-glyceraldehyde to glycerol have been described previously. The L-threo-3-deoxy-hexulosonate aldolase activity was induced in the mold Hypocrea jecorina (Trichoderma reesei) during growth on D-galacturonate. The enzyme was purified from this mold and a partial amino acid sequence obtained. This sequence was then used to identify the corresponding gene from the H. jecorina genome. The deletion of the gene resulted in a strain unable to grow on d-galacturonate and accumulating L-threo-3-deoxy-hexulosonate. The open reading frame was cloned from cDNA and functionally expressed in the yeast Saccharomyces cerevisiae. A histidine-tagged protein was expressed, purified, and characterized. The enzyme catalyzed reaction was reversible. With L-threo-3-deoxy-hexulosonate as substrate the K(m) was 3.5 mM and with pyruvate and L-glyceraldehyde the K(m) were 0.5 and 1.2 mM, respectively.