Sorbitol-6-phosphate Dehydrogenase Research Articles

Crucial roles of sorbitol metabolism and energy status in the chilling tolerance of yellow peach

In this study, we compared sorbitol metabolism, energy metabolism, and CI development in yellow peach fruit at 1 °C (less susceptible to CI) and 8 °C (more susceptible to CI) storage to elucidate potential connections between them. The results indicated that storage at 1 °C effectively maintained the textural quality of yellow peach fruit and delayed the onset of CI by 12 days compared to 8 °C. This positive effect might be attributable to 1 °C storage maintaining higher sorbitol content throughout the storage duration, thus sustaining the higher adenosine triphosphate (ATP) level and energy charge. The regulation of sorbitol accumulation by 1 °C storage was closely linked to the metabolic activity of sorbitol, which stimulated sorbitol synthesis by enhancing sorbitol-6-phosphate dehydrogenase (S6PDH) activity after 12 days while suppressing sorbitol degradation via decreased sorbitol oxidase (SOX) and NAD+-sorbitol dehydrogenase (NAD+-SDH) activities before 24 days. In addition, the notable up-regulation in the NAD+-SDH activity in the late storage period promoted the conversion of sorbitol to fructose and glucose under 1 °C storage, thereby providing ample energy substrate for ATP generation. Moreover, sorbitol acts as a vital signaling molecule, and substantially up-regulated expressions of sorbitol transporters genes (PpeSOT3, PpeSOT5, and PpeSOT7) were observed in fruit stored at 1 °C, which might promote sorbitol transport and improve cold tolerance in peach fruit. Taken together, these findings suggested that 1 °C storage delayed CI by enhancing sorbitol metabolism and transporter activity, promoting sorbitol accumulation, and finally elevating the energy status in yellow peach fruit.

Genome-wide identification and comparative evolutionary analysis of sorbitol metabolism pathway genes in four Rosaceae species and three model plants

In contrast to most land plant species, sorbitol, instead of sucrose, is the major photosynthetic product in many Rosaceae species. It has been well illustrated that three key functional genes encoding sorbitol-6-phosphate dehydrogenase (S6PDH), sorbitol dehydrogenase (SDH), and sorbitol transporter (SOT), are mainly responsible for the synthesis, degradation and transportation of sorbitol. In this study, the genome-wide identification of S6PDH, SDH and SOT genes was conducted in four Rosaceae species, peach, mei, apple and pear, and showed the sorbitol bio-pathway to be dominant (named sorbitol present group, SPG); another three related species, including tomato, poplar and Arabidopsis, showed a non-sorbitol bio-pathway (named sorbitol absent group, SAG). To understand the evolutionary differences of the three important gene families between SAG and SPG, their corresponding gene duplication, evolutionary rate, codon bias and positive selection patterns have been analyzed and compared. The sorbitol pathway genes in SPG were found to be expanded through dispersed and tandem gene duplications. Branch-specific model analyses revealed SDH and S6PDH clade A were under stronger purifying selection in SPG. A higher frequency of optimal codons was found in S6PDH and SDH than that of SOT in SPG, confirming the purifying selection effect on them. In addition, branch-site model analyses revealed SOT genes were under positive selection in SPG. Expression analyses showed diverse expression patterns of sorbitol-related genes. Overall, these findings provide new insights in the evolutionary characteristics for the three key sorbitol metabolism-related gene families in Rosaceae and other non-sorbitol dominant pathway species.

Trehalose Regulates Starch, Sorbitol, and Energy Metabolism to Enhance Tolerance to Blue Mold of “Golden Delicious” Apple Fruit

The efficacy of trehalose on the lesion diameter of apples (cv. Golden Delicious) inoculated with Penicillium expansum was evaluated to screen the optimal concentration. The changes in gene expression and activity of the enzyme in starch, sorbitol, and energy metabolism were also investigated in apples after trehalose treatment. The results revealed that trehalose dipping reduced the lesion diameter of apples inoculated with P. expansum. Trehalose suppressed the activities and gene expressions of β-amylase, NAD-sorbitol dehydrogenase, and NADP-sorbitol dehydrogenase, whereas it decreased the sorbitol 6-phosphate dehydrogenase gene expression and amylose, amylopectin, total starch, and reducing sugar contents. Additionally, trehalose improved the gene expressions and activities of α-amylase, starch-branching enzymes, total amylase, H+-ATPase, and Ca2+-ATPase, as well as soluble sugar, adenosine triphosphate, and adenosine diphosphate contents and energy charge in apples. These findings imply that trehalose could induce tolerance to the blue mold of apple fruit by regulating starch, sorbitol, and energy metabolism.

Effects of zinc levels on synthesis and translocation of 13C-photoassimilates in leaves to fruit of apple during fruit expanding stage

To explore the effects of zinc levels on the synthesis and translocation of photosynthetic products from leaves to fruits, and to lay a theoretical foundation for improving fruit quality through zinc supplementation during the critical period of apple fruit development, a field experiment was carried out with a eight-year old 'Hanfu'/GM256/Malus baccata Borkh apple. We used the 13C tracer method to examine the effects of different zinc levels (ZnSO4·H2O 0, 0.1%, 0.2%, 0.3%, 0.4%, expressed by CK, Zn1, Zn2, Zn3, Zn4, respectively) on translocation of photosynthate to fruit during the stage of fruit expanding. The results showed that, with increasing zinc concentration, Rubisco enzyme activity, net photosynthetic rate, sorbitol and sucrose content, sorbitol 6-phosphate dehydrogenase, and sucrose phosphate synthase enzyme activities of leaves first increased and then decreased, with the highest values being observed in Zn3 treatment. Zn3 treatment significantly increased the 13C assimilation capability of leaves. Compared with other treatments, the 13C of self-retention (labeled leaves and labeled branches) was lowest in Zn3 treatment (61.2%) and the output of 13C photoassimilates was highest in Zn3 treatment (38.8%). 13C absorption of apple fruit showed a trend of Zn3 ＞ Zn2 ＞ Zn4 ＞ Zn1 ＞ CK. In summary, foliar zinc application under appropriate concentration (0.3% ZnSO4·H2O) enhanced photosynthesis, increased the assimilation capability of leaves, and promoted the directional transportation of photosynthate to fruit.

Effects of potassium levels on translocation of 13C-photoassimilates to fruit in 'Fuji' apple during fruit expanding period

A field experiment was carried out in a six-year old 'Fuji'3/M26/Malus hupehensis Rehd. apple with the 13C tracer method to examine the changes of chlorophyll fluorescence parameters, photosynthetic characteristics of leaf, sugar transporter gene expression, 13C assimilation capability and the characteristics of translocation and distribution of 13C-photoassimilates to fruit under different levels of potassium addition (K2O 0, 0.5%, 1.0%, 1.5%, 2.0%, expressed by CK, K1, K2, K3, K4, respectively). Potassium aqueous solution smear the leaves within 20 cm around the fruit at fruit enlargement stage. Compared with other treatments, K3 treatment significantly increased Rubisco enzyme activity, net photosynthetic rate, maximal photochemical efficiency of PSII, actual photochemical efficiency of PSII, coefficient of photochemical quenching, sorbitol and sucrose content, sorbitol 6-phosphate dehydrogenase (S6PDH) and sucrose phosphate synthase (SPS) enzyme activities and 13C assimilation capability of leaves. Furthermore, K3 treatment increased gene expression of sorbitol transporter MdSOT1 and MdSOT2 and sucrose transporter MdSUT4, and promoted the unloading of sugar in fruit. The 13C of self retention (self leaves and self branches) was the highest in CK (82.6%) and the lowest in K3 treatment (60.5%). With increasing potassium concentration, the 13C absorption of fruit first increased and then decreased, which was the highest in K3 treatment (1.31 mg·g-1) and the lowest in CK (0.57 mg·g-1). Our results indicated that foliage application of potassium solution improved PSII photochemical efficiency, activities of key enzymes related with carbon assimilation, synthesis ability, and outward transport ability of photosynthates in leaves, and consequently promoted the directional transportation of sugar to fruit. The amount of photoassimilates transported to fruit was the most under 1.5% K2O treatment (K3).

Effects of nitrogen application levels on translocation and distribution of 13C-photosynthate and 15N to fruit from leaves of apple tree

A field experiment was carried out in a six-year old 'Fuji3'/M26/Malus hupehensis Rehd. apple with 15N and 13C labeled tracers, to understand 13C assimilation capability and the characteristics of translocation and distribution of 13C-photosynthate and 15N to fruit under different nitrogen application levels (urea 0%, 0.6%, 1.2%, 1.8%, 2.4%, CK, N1, N2, N3, N4, respectively) to smear the leaves within 20 cm around the fruit at late stage of fruit enlargement. The results showed that, with the increases of urea application, the chlorophyll content, nitrogen content, net photosynthetic rate, sorbitol and sucrose content, sorbitol 6-phosphate dehydrogenase (S6PDH) and sucrose phosphate synthase (SPS) activities, 13C assimilation capability of leaves were first increased and then decreased, with the highest value in 1.8% urea smear treatment and the lowest value with the treatment of clear water. The 13C of self retention (self leaves and self branches) was the highest in clear water (81.6%) and the lowest in 1.8% urea smear treatment (63.5%). The 13C was mainly allocated to fruit, followed by unlabeled perennial branch, and the lowest in unlabeled leaves. With the increases of urea application, the 13C absorption of fruit was first increased and then decreased, with the highest value in 1.8% urea smear treatment (1.21 mg·g-1) and the lowest value in clear water (0.51 mg·g-1). The 15N absorption of fruit was enhanced with the increases of urea application. These results indicated that foliage application of urea solution improved translocation and distribution of leaf photosynthate and nitrogen to fruit with varying degrees, which was the highest in 1.8% urea smear treatment and could avoid excessive intake of nitrogen to fruit.

Improved sorbitol production and growth in cyanobacteria using promiscuous haloacid dehalogenase-like hydrolase

Sorbitol can be produced photosynthetically in the model cyanobacterium Synechocystis sp. PCC 6803 harboring sorbitol-6-phosphate (S6P) dehydrogenase (S6PDH) from apple, representing a promising metabolic engineering strategy for environmentally friendly carbon-based compound production. However, no gene for S6P phosphatase has been reported to date. We previously found that members of the Escherichia coli haloacid dehalogenase-like hydrolase (HAD) superfamily, a group of promiscuous hydrolases, exert phosphatase activity to S6P in vitro. Here, we examined the effects of HADs on sorbitol production in cyanobacterial cells. Overexpression of E. coli HAD1 improved sorbitol production induced by toxic S6PDH, whereas it suppressed growth even without induction. Moreover, overexpression of HAD1 in combination with engineering of other pathways successfully allowed for the constitutive expression of toxic S6PDH. Consequently, the sorbitol production was highly improved, which in turn suppressed the growth suppression effect of promiscuous HAD1. These results provide a good example of a novel metabolic engineering strategy using a combination of a promiscuous enzyme with an abundant supply of one of its substrates.

Apple fruit superficial scald resistance mediated by ethylene inhibition is associated with diverse metabolic processes.

Fruits stored at low temperature can exhibit different types of chilling injury. In apple, one of the most serious physiological disorders is superficial scald, which is characterized by discoloration and brown necrotic patches on the fruit exocarp. Although this phenomenon is widely ascribed to the oxidation of α-farnesene, its physiology is not yet fully understood. To elucidate the mechanism of superficial scald development and possible means of prevention, we performed an integrated metabolite screen, including an analysis of volatiles, phenols and lipids, together with a large-scale transcriptome study. We also determined that prevention of superficial scald, through the use of an ethylene action inhibitor, is associated with the triggering of cold acclimation-related processes. Specifically, the inhibition of ethylene perception stimulated the production of antioxidant compounds to scavenge reactive oxygen species, the synthesis of fatty acids to stabilize plastid and vacuole membranes against cold temperature, and the accumulation of the sorbitol, which can act as a cryoprotectant. The pattern of sorbitol accumulation was consistent with the expression profile of a sorbitol 6-phosphate dehydrogenase, MdS6PDH, the overexpression of which in transgenic Arabidopsis thaliana plants confirmed its involvement in the cold acclimation and freezing tolerance.

TRANSCRIPTOMIC ANALYSIS OF LEAVES OF TRANSGENIC APPLE SILENCED FOR SORBITOL-6-PHOSPHATE DEHYDROGENASE GENE

Plants in the Rosaceae synthesize both sorbitol and sucrose as major end products of photosynthesis. Sorbitol helps determine fruit and vegetative quality. To clarify the mechanism through which sorbitol acts as a signal molecule, we conducted comprehensive analyses of genes regulated by sorbitol in mature leaves of transgenic apple (Malus × domestica Borkh. ‘Greensleeves’) that were silenced for the sorbitol-6-phosphate dehydrogenase (S6PDH) gene. The microarray was designed using 23,731 apple unigenes opened by NCBI (http://www.ncbi.nlm. nih.gov/). Trees of sense clone GSS78 and antisense clone GSA04 had reduced S6PDH transcript compared to an untransformed control. In GSA04, 138 down-regulated ( 2-fold) were identified. In GSS78, 2,687 down-regulated genes ( 2-fold) were identified, 70 of which were also up-regulated in GSA04. Expression of these genes may be directly or indirectly regulated by sorbitol. Analysis of GO terms revealed that, among down-regulated genes in GSA04 involved in biological processes, the represented categories included “oxidation reduction”, “response to stress”, “primary metabolic process”, “response to hormone stimulus”, “regulation of transcription, DNA-dependent”, “cellular macromolecule metabolic process”, and “regulation of cellular process”. The functions of the drastically up-regulated genes are unknown except for sorbitol metabolism. These results are a first step toward functional genomics research for understanding gene functions and regulatory networks upon which sorbitol act as a signal molecule, affecting fruit quality and growth.

Identification and functional characterization of sorbitol-6-phosphate dehydrogenase protein from rice and structural elucidation by in silico approach

The sorbitol-6-phosphate dehydrogenase (S6PDH) is a key enzyme for sorbitol synthesis and plays an important role in the alleviation of salinity stress in plants. Despite the huge significance, the structure and the mode of action of this enzyme are still not known. In the present study, sequence analysis, cloning, expression, activity assays and enzyme kinetics using various substrates (glucose-6-phosphate, sorbitol-6-phosphate and mannose-6-phosphate) were performed to establish the functional role of S6PDH protein from rice (Oryza sativa). For the structural analysis of the protein, a comparative homology model was prepared on the basis of percentage sequence identity and substrate similarity using the crystal structure of human aldose reductase in complex with glucose-6-phosphate and NADP(+) (PDB ID: 2ACQ) as a template. Molecular docking was performed for studying the structural details of substrate binding and possible enzyme mechanism. The cloned sequence resulted into an active recombinant protein when expressed into a bacterial expression system. The purified recombinant protein was found to be active with glucose-6-phosphate and sorbitol-6-phosphate; however, activity against mannose-6-phosphate was not found. The K m values for glucose-6-phosphate and sorbitol-6-phosphate were found to be 15.9 ± 0.2 and 7.21 ± 0.5 mM, respectively. A molecular-level analysis of the active site of OsS6PDH provides valuable information about the enzyme mechanism and requisite enantioselectivity for its physiological substrates. Thus, the fundamental studies of structure and function of OsS6PDH could serve as the basis for the future studies of bio-catalytic applications of this enzyme.

Mining and comparison of the genes encoding the key enzymes involved in sugar biosynthesis in apple, grape, and sweet orange

Fruit taste depends on the contents and types of soluble sugars and organic acids. The large-scale identification and comparison of genes involved in sugar biosynthesis in apple, grape, and sweet orange based on the genome and expressed sequence tag information have not been reported. In this study, we found that sorbitol-6-phosphate dehydrogenase (S6PDH) was one of the most important genes in sorbitol biosynthesis in apple leaf, and was decomposed by NAD+-dependent sorbitol dehydrogenase (NAD+SDH) in apple fruit. The high expression level of soluble acidic invertase (AIV) gene possibly caused the high soluble sugar content (i.e., glucose and fructose) in grape. The expression level of sucrose synthase (SUS) in grape and sweet orange was also significantly higher than that in apple. The results show that SUS was involved in the resynthesis of fructose and UDP-glucose into sucrose in grape and sweet orange. By contrast, sucrose was transported into a vacuole and decomposed into glucose and fructose by AIV. Thus, ADP-glucose pyrophosphorylase (AGP), granule-bound starch synthase (GBSS), isoamylase 1 (ISA1), and isoamylase 2 (ISA2) could be the key genes in starch biosynthesis, whereas α-amylase (AMY3), isoamylse 3 (ISA3), and pullulanase (PUL) could be the key genes in starch degradation in apple. This study provides new insight into the isolation and comparison of candidate genes involved in the sugar biosynthetic pathway among multiple plants.

High Temperature Alters Sorbitol Metabolism in Pyrus pyrifolia Leaves and Fruit Flesh during Late Stages of Fruit Enlargement

Sorbitol is the main photosynthetic product and primary translocated carbohydrate in the Rosaceae and plays fundamental roles in plant growth, fruit quality, and osmotic stress adaptation. To investigate the effect of frequent high temperature during advanced fruit development on fruit quality of chinese sand pear [Pyrus pyrifolia (Burm. f.) Nakai], we analyzed sorbitol metabolism in mature leaves and fruit flesh of potted ‘Wonhwang’ pear trees. In mature leaves, sorbitol synthesis catalyzed by NADP+-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) was repressed, while sorbitol utilization mainly catalyzed by NAD+-dependent sorbitol dehydrogenase (NAD+-SDH) and NADP+-dependent sorbitol dehydrogenase (NADP+-SDH) was higher than that before high-temperature treatment, which resulted in decreased sorbitol accumulation. In contrast, sucrose accumulation in mature leaves was significantly enhanced in response to high temperatures. In fruit flesh, accumulation of sorbitol and sucrose was increased at the time of harvest under high temperatures. Among sorbitol metabolic enzymes, only NAD+-SDH was sensitive to high temperature in fruit flesh, and significant decrease of NAD+-SDH activity indicated that the fruit sorbitol-uptake capacity was undermined under high temperatures. Transcription analysis revealed tissue-specific responses of NAD+-SDH genes (PpSDH1, PpSDH2, and PpSDH3) to high-temperature treatment. The NAD+-SDH activity and regulation of PpSDH1 and PpSDH3 were positively correlated in mature leaves. However, the downregulation of PpSDH1 and PpSDH2 was consistent with decreased enzyme activity in the fruit flesh. With regard to sorbitol transport, two sorbitol transporter genes (PpSOT1 and PpSOT2) were isolated, and downregulation of PpSOT2 expression in mature leaves indicated that the sorbitol-loading capability decreased under high-temperature conditions because of the limited sorbitol supply. These findings suggested that sorbitol metabolism responded differently in mature leaves and fruit flesh under high temperature, and that these dissimilar responses influenced fruit quality and may play important roles in adaptation to high temperatures.

CDNA-AFLP analysis of gene expression differences between the flower bud and sprout-shoot apical meristem of Angelica sinensis (Oliv.) Diels

Angelica sinensis (Oliv.) Diels (Umbelliferae) is a well-known medicinal plant mainly distributed in Gansu Province of China. Its local and global demand is significant because of its food and medicinal applications. However, the early bolting rate of Angelica sinensis (Oliv.) Diels reaches 20%–60%, which seriously affects its food and medicinal qualities. Thus, differences in gene expression between the flower bud and sprout-shoot apical meristem underwent analysis, by means of cDNA-amplified restriction fragment length polymorphism, to better understand the flowering mechanism. 64 primer sets, each of which amplified to 60 transcript-derived fragments (TDFs), were used. Among these TDFs, 26 were expressed specifically in the flower bud. After cloning and sequencing, 32 distinct sequences were obtained from these 26 TDFs, and 25 were found with homologous sequences in databases. Confirmation of differential expression of 13 sequences was obtained by semi-quantitative RT-PCR, their showing higher expression levels in flower buds. These homologous sequences encode transposable elements, pentatricopeptide repeat-containing proteins, DNA-binding transcription factors, zinc finger (B-box type) family proteins, NADP-dependent sorbitol 6-phosphate dehydrogenase (S6PDH), amongst others.

Quantitative real-time PCR to determine allele number for the astringency locus by analysis of a linked marker in Diospyros kaki Thunb

Persimmon (Diospyros kaki Thunb.) is a polyploidy fruit tree species of economic importance to East Asia. Natural astringency loss is an important trait in persimmon breeding programs. Quantitative real-time PCR was used to determine the number of AST/ast alleles for fruit astringency in persimmon (D. kaki Thunb.). To this end, the cultivar Jiro was transformed with one or two copies of a gene encoding NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH), which was used as a standard for measuring the allele number of a sequenced marker tightly linked to the recessive ast locus for nonastringency. Primers for markers linked to the AST or ast allele were then used to measure the AST to ast ratio directly in the progeny of a full-sib cross. From determination of the AST to ast ratio and the results of the S6PDH copy number, the number of AST and ast alleles at the AST/ast locus was estimated. This research supported the hypothesis that D. kaki is a hexaploid with six AST and/or ast alleles. In addition to the determination of the allelic status of the AST locus, the application of real-time PCR for confirmation of the ploidy level and allelic composition of target genes in autopolyploids or allopolyploids was demonstrated.

Isolation and characterization of genes associated with shade-induced apple abscission

Apple trees generally produce excessive number of flowers and young fruitlets, which negatively affects commercial value. Thus, thinning is an important practice by which excessive flowers and fruitlets abort. Fruit abscission is difficult to predict or control, as many factors including shading can cause fruitlets to abort. In order to understand the molecular basis of shade-induced fruit abscission, two cDNA libraries were constructed using the suppression-subtractive hybridization (SSH) method and 347 expressed sequence tags (ESTs) were obtained. 168 ESTs represent transcripts that are preferentially expressed after 24 h of shading, and the other 179 are derived from RNAs of small apple fruits that were shaded for 72 h. Sequence analyses revealed that these clones represent 68 (24 h) and 44 (72 h) unique genes; these genes belong in eight functional categories. The largest set of genes is related to carbohydrate metabolism including the sorbitol 6-phosphate dehydrogenase (S6PDH) gene that was generally believed not to be expressed in young fruits, while the second largest group contains unclassified or unknown genes. RNA gel blot analysis confirmed that at least 26 genes are up-regulated after shade treatment. Some of these known genes may serve as molecular markers for apple monitoring the induction of fruitlet abscission. Improved genetic understanding is critical to the development of targeted abscission agents to better control apple crop loads and optimize apple production.

EFFECTS OF ENVIRONMENTAL STRESSES AND ABSCISIC ACID ON SORBITOL-6-PHOSPHATE DEHYDROGENASE EXPRESSION IN ROSACEAE FRUIT TREES

Sorbitol plays an important role in carbohydrate translocation in Rosaceae fruit trees such as apple, pear, and peach and may also contribute to the stress response as a compatible solute. Leaf disc experiments showed enhanced sorbitol accumulation under salt, osmotic, and low-temperature stresses. Northern blot analysis indicated a higher level of mRNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH), a key enzyme in sorbitol biosynthesis, under stress conditions. Thus, sorbitol accumulation under stress may reflect an increase in sorbitol biosynthesis related to expression of the S6PDH gene. Abscisic acid (ABA) also increased in apple leaves under low-temperature conditions. Further investigation revealed that the addition of ABA enhanced S6PDH expression in apple leaves, indicating that S6PDH is an ABA-inducible gene. These results suggest that ABA-mediated S6PDH plays a role in sorbitol biosynthesis in various stress responses and is involved in photo-synthate translocation.

Expression and cloning of the full-length cDNA for sorbitol-6-phosphate dehydrogenase and NAD-dependent sorbitol dehydrogenase from pear ( Pyrus pyrifolia N.)

Sorbitol is the primary product of photosynthesis and the major translocation form of photosynthate in the Rosaceae family, which includes pear plants. Therefore, the biosynthesis and breakdown of sorbitol is an important step in the carbohydrate metabolism of pear. In sorbitol metabolism, sorbitol-6-phosphate dehydrogenase (S6PDH) and NAD-sorbitol dehydrogenase (NAD-SDH) play key roles in biosynthesis and breakdown of sorbitol, respectively. We isolated full-length cDNAs encoding S6PDH and NAD-SDH from pear ( Pyrus pyrifolia N. cv Niitaka). PyS6PDH cDNA encodes a polypeptide consisting of 311 amino acid residues. PyNAD-SDH cDNA encodes a 371 amino acid polypeptide. The PyS6PDH gene was expressed at high levels in leaves at 30 day after full bloom (DAFB), but its expression was decreased later. Conversely, the transcripts of the PyNAD- SDH gene were detected at low levels in young leaves, but increased with age. These genes may be regulated by the transcriptional level.

High-Level Production of the Low-Calorie Sugar Sorbitol by Lactobacillus plantarum through Metabolic Engineering

Sorbitol is a low-calorie sugar alcohol that is largely used as an ingredient in the food industry, based on its sweetness and its high solubility. Here, we investigated the capacity of Lactobacillus plantarum, a lactic acid bacterium found in many fermented food products and in the gastrointestinal tract of mammals, to produce sorbitol from fructose-6-phosphate by reverting the sorbitol catabolic pathway in a mutant strain deficient for both l- and d-lactate dehydrogenase activities. The two sorbitol-6-phosphate dehydrogenase (Stl6PDH) genes (srlD1 and srlD2) identified in the genome sequence were constitutively expressed at a high level in this mutant strain. Both Stl6PDH enzymes were shown to be active, and high specific activity could be detected in the overexpressing strains. Using resting cells under pH control with glucose as a substrate, both Stl6PDHs were capable of rerouting the glycolytic flux from fructose-6-phosphate toward sorbitol production with a remarkably high efficiency (61 to 65% glucose conversion), which is close to the maximal theoretical value of 67%. Mannitol production was also detected, albeit at a lower level than the control strain (9 to 13% glucose conversion), indicating competition for fructose-6-phosphate rerouting by natively expressed mannitol-1-phosphate dehydrogenase. By analogy, low levels of this enzyme were detected in both the wild-type and the lactate dehydrogenase-deficient strain backgrounds. After optimization, 25% of sugar conversion into sorbitol was achieved with cells grown under pH control. The role of intracellular NADH pools in the determination of the maximal sorbitol production is discussed.

Presence and expression of NAD+-dependent sorbitol dehydrogenase and sorbitol-6-phosphate dehydrogenase genes in strawberry

SummaryThe sorbitol metabolic pathway in strawberry (Fragaria ananassa Duch. var. ‘Nyoho’) is unclear, despite strawberry belonging to the family Rosaceae. Therefore, the presence or absence of genes for NAD+-dependent sorbitol dehydrogenase (NAD-SDH) and sorbitol-6-phosphate dehydrogenase (S6PDH) in strawberry, and their levels of expression related to growth were investigated. A full-length cDNA encoding the NAD-SDH gene in strawberry (FaSDH) was obtained using rapid amplification of cDNA ends (RACE). The full-length clone consisted of 1,399 bp and contained a 1,083 bp open reading frame encoding 361 amino acids with a calculated molecular mass of 38.8 kDa, and a predicted isoelectric point (pI) of 6.08. This nucleotide sequence showed 79 – 85% similarity with the NAD-SDH genes of other plants. Its amino acid sequence contained both zinc- and NAD-binding sites, confirming that this sequence belonged to the NAD-SDH gene family. Southern blotting suggested that the genomic copy number of FaSDH was less than in apple. The expression of FaSDH transcripts in strawberry fruit was higher at the young and mature stages than at other stages. The transcript level in mature leaves was higher than in the shoot tip, and in folded and young leaves. NAD-SDH activity was detected in strawberry fruits and leaves and the activity in fruit was comparable to that in other fruits. Reverse transcriptase (RT)-PCR using specific primers designed based on the S6PDH gene of Fragaria vesca, showed a clear single band in leaves and fruits of strawberry. Sequence analysis showed this RT-PCR fragment, named FaS6PDH, to have high homology to the S6PDH gene sequence of other plants, suggesting that the S6PDH gene exists in the strawberry genome and that its mRNA is expressed in leaves and fruits. However, neither the protein nor its enzyme activity were detected in leaves or fruit.

An engineered sorbitol cycle alters sugar composition, not growth, in transformed tobacco

Many efforts have been made to engineer stress tolerance by accumulating polyols. Transformants that accumulate polyols often show growth inhibition, because polyols are synthesized as a dead-end product in plants that do not naturally accumulate polyols. Here, we show a novel strategy in which a sorbitol cycle was engineered by introducing apple cDNA encoding NAD-dependent sorbitol dehydrogenase (SDH) in addition to sorbitol-6-phosphate dehydrogenase (S6PDH). Tobacco plants transformed only with S6PDH showed growth inhibition, and very few transformants were obtained. In contrast, many transgenic plants with both S6PDH and SDH were easily obtained, and their growth was normal despite their accumulation of sorbitol. Interestingly, the engineered sorbitol cycle enhanced the accumulation of sucrose instead of fructose that was expected to be increased. Sucrose, rather than fructose, was also increased in the immature fruit of tomato plants transformed with an antisense fructokinase gene in which the phosphorylation of fructose was inhibited. A common phenomenon was observed in the metabolic engineering of two different pathways, showing the presence of homeostatic regulation of fructose levels.