Higher expression of the strawberry xyloglucan endotransglucosylase/hydrolase genes FvXTH9 and FvXTH6 accelerates fruit ripening.

Fruit softening in Fragaria (strawberry) is proposed to be associated with the modification of cell wall components such as xyloglucan by the action of cell wall-modifying enzymes. This study focuses on the in vitro and in vivo characterization of two recombinant xyloglucan endotransglucosylase/hydrolases (XTHs) from Fragaria vesca, FvXTH9 and FvXTH6. Mining of the publicly available F. vesca genome sequence yielded 28 putative XTH genes. FvXTH9 showed the highest expression level of all FvXTHs in a fruit transcriptome data set and was selected with the closely related FvXTH6 for further analysis. To investigate their role in fruit ripening in more detail, the coding sequences of FvXTH9 and FvXTH6 were cloned into the vector pYES2 and expressed in Saccharomyces cerevisiae. FvXTH9 and FvXTH6 displayed xyloglucan endotransglucosylase (XET) activity towards various acceptor substrates using xyloglucan as the donor substrate. Interestingly, FvXTH9 showed activity of mixed-linkage glucan:xyloglucan endotransglucosylase (MXE) and cellulose:xyloglucan endotransglucosylase (CXE). The optimum pH of both FvXTH9 and FvXTH6 was 6.5. The prediction of subcellular localization suggested localization to the secretory pathway, which was confirmed by localization studies in Nicotiana tabacum. Overexpression showed that Fragaria × ananassa fruits infiltrated with FvXTH9 and FvXTH6 ripened faster and showed decreased firmness compared with the empty vector control pBI121. Thus FvXTH9 and also FvXTH6 might promote strawberry fruit ripening by the modification of cell wall components.

Glucosylation of the phytoalexin N-feruloyl tyramine modulates the levels of pathogen-responsive metabolites in Nicotiana benthamiana.

Enzyme promiscuity, a common property of many uridine diphosphate sugar-dependent glycosyltransferases (UGTs) that convert small molecules, significantly hinders the identification of natural substrates and therefore the characterization of the physiological role of enzymes. In this paper we present a simple but effective strategy to identify endogenous substrates of plant UGTs using LC-MS-guided targeted glycoside analysis of transgenic plants. We successfully identified natural substrates of two promiscuous Nicotiana benthamiana UGTs (NbUGT73A24 and NbUGT73A25), orthologues of pathogen-induced tobacco UGT (TOGT) from Nicotiana tabacum, which is involved in the hypersensitive reaction. While in N. tabacum, TOGT glucosylated scopoletin after treatment with salicylate, fungal elicitors and the tobacco mosaic virus, NbUGT73A24 and NbUGT73A25 produced glucosides of phytoalexin N-feruloyl tyramine, which may strengthen cell walls to prevent the intrusion of pathogens, and flavonols after agroinfiltration of the corresponding genes in N. benthamiana. Enzymatic glucosylation of fractions of a physiological aglycone library confirmed the biological substrates of UGTs. In addition, overexpression of both genes in N. benthamiana produced clear lesions on the leaves and led to a significantly reduced content of pathogen-induced plant metabolites such as phenylalanine and tryptophan. Our results revealed some additional biological functions of TOGT enzymes and indicated a multifunctional role of UGTs in plant resistance.

Structural and Functional Analysis of UGT92G6 Suggests an Evolutionary Link Between Mono- and Disaccharide Glycoside-Forming Transferases.

Glycosylation mediated by UDP-dependent glycosyltransferase (UGT) is one of the most common reactions for the biosynthesis of small molecule glycosides. As glycosides have various biological roles, we characterized UGT genes from grapevine (Vitis vinifera). In silico analysis of VvUGT genes that were highly expressed in leaves identified UGT92G6 which showed sequence similarity to both monosaccharide and disaccharide glucoside-forming transferases. The recombinant UGT92G6 glucosylated phenolics, among them caffeic acid, carvacrol, eugenol and raspberry ketone, and also accepted geranyl glucoside and citronellyl glucoside. Thus, UGT92G6 formed mono- and diglucosides in vitro from distinct compounds. The enzyme specificity constant Vmax/Km ratios indicated that UGT92G6 exhibited the highest specificity towards caffeic acid, producing almost equal amounts of the 3- and 4-O-glucoside. Transient overexpression of UGT92G6 in Nicotiana benthamiana leaves confirmed the production of caffeoyl glucoside; however, the level of geranyl diglucoside was not elevated upon overexpression of UGT92G6, even after co-expression of genes encoding geraniol synthase and geraniol UGT to provide sufficient precursor. Comparative sequence and 3-D structure analysis identified a sequence motif characteristic for monoglucoside-forming UGTs in UGT92G6, suggesting an evolutionary link between mono- and disaccharide glycoside UGTs. Thus, UGT92G6 functions as a mono- and diglucosyltransferase in vitro, but acts as a caffeoyl glucoside UGT in N. benthamiana.

Glucosylation of 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone, the Key Strawberry Flavor Compound in Strawberry Fruit.

Strawberries emit hundreds of different volatiles, but only a dozen, including the key compound HDMF [4-hydroxy-2,5-dimethyl-3(2H)-furanone] contribute to the flavor of the fruit. However, during ripening, a considerable amount of HDMF is metabolized to the flavorless HDMF ß-d-glucoside. Here, we functionally characterize nine ripening-related UGTs (UDP-glucosyltransferases) in Fragaria that function in the glucosylation of volatile metabolites by comprehensive biochemical analyses. Some UGTs showed a rather broad substrate tolerance and glucosylated a range of aroma compounds in vitro, whereas others had a more limited substrate spectrum. The allelic UGT71K3a and b proteins and to a lesser extent UGT73B24, UGT71W2, and UGT73B23 catalyzed the glucosylation of HDMF and its structural homolog 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone. Site-directed mutagenesis to introduce single K458R, D445E, D343E, and V383A mutations and a double G433A/I434V mutation led to enhanced HDMF glucosylation activity compared to the wild-type enzymes. In contrast, a single mutation in the center of the plant secondary product glycosyltransferase box (A389V) reduced the enzymatic activity. Down-regulation of UGT71K3 transcript expression in strawberry receptacles led to a significant reduction in the level of HDMF-glucoside and a smaller decline in HDMF-glucoside-malonate compared with the level in control fruits. These results provide the foundation for improvement of strawberry flavor and the biotechnological production of HDMF-glucoside.

Acylphloroglucinol Biosynthesis in Strawberry Fruit.

Phenolics have health-promoting properties and are a major group of metabolites in fruit crops. Through reverse genetic analysis of the functions of four ripening-related genes in the octoploid strawberry (Fragaria × ananassa), we discovered four acylphloroglucinol (APG)-glucosides as native Fragaria spp. fruit metabolites whose levels were differently regulated in the transgenic fruits. The biosynthesis of the APG aglycones was investigated by examination of the enzymatic properties of three recombinant Fragaria vesca chalcone synthase (FvCHS) proteins. CHS is involved in anthocyanin biosynthesis during ripening. The F. vesca enzymes readily catalyzed the condensation of two intermediates in branched-chain amino acid metabolism, isovaleryl-Coenzyme A (CoA) and isobutyryl-CoA, with three molecules of malonyl-CoA to form phlorisovalerophenone and phlorisobutyrophenone, respectively, and formed naringenin chalcone when 4-coumaroyl-CoA was used as starter molecule. Isovaleryl-CoA was the preferred starter substrate of FvCHS2-1. Suppression of CHS activity in both transient and stable CHS-silenced fruit resulted in a substantial decrease of APG glucosides and anthocyanins and enhanced levels of volatiles derived from branched-chain amino acids. The proposed APG pathway was confirmed by feeding isotopically labeled amino acids. Thus, Fragaria spp. plants have the capacity to synthesize pharmaceutically important APGs using dual functional CHS/(phloriso)valerophenone synthases that are expressed during fruit ripening. Duplication and adaptive evolution of CHS is the most probable scenario and might be generally applicable to other plants. The results highlight that important promiscuous gene function may be missed when annotation relies solely on in silico analysis.

Non-water miscible ionic liquid improves biocatalytic production of geranyl glucoside with Escherichia coli overexpressing a glucosyltransferase.

Whole cells of Escherichia coli overexpressing a glucosyltransferase from Vitis vinifera were used for the glucosylation of geraniol to geranyl glucoside. A high cell density cultivation process for the production of whole-cell biocatalysts was developed, gaining a dry cell mass concentration of up to 67.6 ± 1.2 g L(-1) and a glucosyltransferase concentration of up to 2.7 ± 0.1 g protein L(-1) within a process time of 48 h. Whole-cell batch biotransformations in milliliter-scale stirred-tank bioreactors showed highest conversion of geraniol at pH 7.0 although the pH optimum of the purified glucosyltransferase was at pH 8.5. The biocatalytic batch process performance was improved significantly by the addition of a water-immiscible ionic liquid (N-hexylpyridinium bis(trifluoromethylsulfonyl)imid) for in situ substrate supply. The so far highest final geranyl glucoside concentration (291 ± 9 mg L(-1)) and conversion (71 ± 2 %) reported for whole-cell biotransformations of geraniol were achieved with 5 % (v/v) of the ionic liquid.

Expression and characterization of CYP52 genes involved in the biosynthesis of sophorolipid and alkane metabolism from Starmerella bombicola.

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C16 to C20 fatty acids preferentially. It converted oleic acid (C18:1) more efficiently than stearic acid (C18:0) and linoleic acid (C18:2) and much more effectively than α-linolenic acid (C18:3). No products were detected when C10 to C12 fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C14 to C20 saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.

Overexpression of hydroperoxide lyase, peroxygenase and epoxide hydrolase in tobacco for the biotechnological production of flavours and polymer precursors.

Plants produce short-chain aldehydes and hydroxy fatty acids, which are important industrial materials, through the lipoxygenase pathway. Based on the information that lipoxygenase activity is up-regulated in tobacco leaves upon infection with tobacco mosaic virus (TMV), we introduced a melon hydroperoxide lyase (CmHPL) gene, a tomato peroxygenase (SlPXG) gene and a potato epoxide hydrolase (StEH) into tobacco leaves using a TMV-based viral vector system to afford aldehyde and hydroxy fatty acid production. Ten days after infiltration, tobacco leaves infiltrated with CmHPL displayed high enzyme activities of 9-LOX and 9-HPL, which could efficiently transform linoleic acid into C(9) aldehydes. Protein extracts prepared from 1 g of CmHPL-infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of control vector-infiltrated tobacco leaves (as an additional 9-LOX source) produced 758 ± 75 µg total C(9) aldehydes in 30 min. The yield of C(9) aldehydes from linoleic acid was 60%. Besides, leaves infiltrated with SlPXG and StEH showed considerable enzyme activities of 9-LOX/PXG and 9-LOX/EH, respectively, enabling the production of 9,12,13-trihydroxy-10(E)-octadecenoic acid from linoleic acid. Protein extracts prepared from 1 g of SlPXG-infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of StEH-infiltrated tobacco leaves produced 1738 ± 27 µg total 9,12,13-trihydroxy-10(E)-octadecenoic acid isomers in 30 min. The yield of trihydroxyoctadecenoic acids from linoleic acid was 58%. C(9) aldehydes and trihydroxy fatty acids could likely be produced on a larger scale using this expression system with many advantages including easy handling, time-saving and low production cost.

Comparative Physicochemical and Biochemical Characterization of Small-Molecule Glucosides.

Glycosylation of small molecules can significantly improve their physicochemical and biological properties. Only recently, decisive improvements in the biotechnological production of small-molecule glucosides (SMGs) have resulted in a large number of these compounds now being commercially available. In this study, we have analyzed a number of physical, chemical, and biological parameters of 31 SMGs, including solubility, stability, melting and pyrolysis points, partition coefficient log P, minimum inhibitory concentration against Escherichia coli (MIC), and enzymatic degradability. The properties such as water solubility, pH stability, and MICs of the glycosides were strongly dependent on the structures of the respective aglycones, which is why the SMG clustered according to their aglycones in most cases. Phenolic and furanone glucosides were readily hydrolyzed by saliva and skin microflora, whereas monoterpenol glycosides were poorer substrates for the enzymes involved. The results of this comparative analysis of SMGs provide valuable information for elucidating the biological functions of SMGs and the future technological applications of these useful natural products.

Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamiana for biotechnological application.

BACKGROUND: Plant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system. RESULTS: A LOX gene which is expressed after treatment of the viral vectors was isolated from Nicotiana benthamiana. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as Nb-9-LOX. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml(-1) cell cultures) which can oxygenate linoleic acid resulting in high yields (18 µmol ml(-1) cell cultures) of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL) or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL) and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C9-aldehydes (3.3 µmol mg(-1) crude protein). The yield of C9-aldehydes from linoleic acid was 64%. CONCLUSION: The yeast expressed Nb-9-LOX can be used to produce C9-aldehydes on a large scale in combination with a HPL gene with 9-HPL function, or to effectively produce 9-hydroxy-10(E),12(Z)-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent.

An oxygenase inhibitor study in Solanum lycopersicum combined with metabolite profiling analysis revealed a potent peroxygenase inactivator.

Plant genomes contain a vast number of oxygenase genes, but only very few have been functionally characterized. To devise an alternative method for the detection of novel oxygenase-catalysed reactions the effects of the cytochrome P450 oxygenase inhibitors 1-aminobenzotriazole (ABT) and tetcyclacis (TET) have been examined by metabolite profiling analysis in tomato fruit (Solanum lycopersicum). Treatment with TET resulted in significant increases in the levels of certain flavonoids, whereas ABT strongly inhibited their formation during fruit ripening. Injections of buffered solutions of ABT into tomato fruits led rather to an accumulation of 9,12,13-trihydroxy-10(E)-octadecenoic acid probably due to retarded metabolism of the hydroxylated acid, while TET completely repressed its formation. Peroxygenase, a hydroperoxide-dependent hydroxylase involved in the formation of the trihydroxy fatty acid, is strongly inhibited by TET (IC(50) 2.6 µM) as was demonstrated by studies with the recombinant tomato enzyme expressed in yeast. The data show that ABT and TET affect oxygenases differently in tomato fruit and reveal that these enzymes catalyse distinct reactions in different metabolic pathways, among which C(18)-trihydroxy fatty acid and flavonoid metabolism involve novel oxygenase-catalysed reactions. The method is suitable to identify potential substrates and products of ripening-related, putative oxygenases and can support functional analyses of recombinant enzymes.

Overexpression of hydroperoxide lyase gene in Nicotiana benthamiana using a viral vector system.

13-Lipoxygenase (13-LOX) and 13-hydroperoxide lyases (13-HPL) are the key enzymes for the production of the 'green note' compounds hexanal, (3Z)- and (2E)-hexenal in plant tissues. To produce high levels of 13-LOX and 13-HPL enzymatic activities for a biocatalytic process to generate C(6)-aldehydes on a large scale, soya bean 13-LOX (GmVLXC) and watermelon 13-HPL (ClHPL) genes were expressed in Nicotiana benthamiana using a viral vector system mediated by agroinfiltration. The N. benthamiana leaves produced high activity of watermelon HPL, but not GmVLXC 13-LOX. In addition, all leaves treated with bacterial suspension displayed a high activity of 9-LOX, indicating that the internal tobacco 9-LOX gene was highly induced through agroinfiltration because of wounding. GmVLXC and ClHPL transcripts could be detected in the corresponding transformed tobacco leaves by real-time RT-PCR analysis but the expression level of ClHPL was 24-fold higher than that of GmVLXC. Western blot analysis showed that LOX was present in all tobacco leaves which were treated with bacterial suspensions, but not in the untreated wild-type control. This result confirms that internal 9-LOX was highly induced by agroinfiltration. The highest levels of ClHPL activity under optimal infiltration conditions were 80 times the HPL activity of wild-type plants or plants transformed with control vector. A large amount of hexanal was formed when linoleic acid was incubated with extracts from N. benthamiana leaves over-expressing ClHPL in combination with GmVLXC-expressing yeast extracts. One gram of ClHPL-expressing N. benthamiana leaves (fresh weight) could produce 17 +/- 0.4 mg hexanal from 50 mg linoleic acid after 30 min.

Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes.

Although a number of plant carotenoid cleavage dioxygenase (CCD) genes have been functionally characterized in different plant species, little is known about the biochemical role and enzymatic activities of members of the subclass 4 (CCD4). To gain insight into their biological function, CCD4 genes were isolated from apple (Malus x domestica, MdCCD4), chrysanthemum (Chrysanthemum x morifolium, CmCCD4a), rose (Rosa x damascena, RdCCD4), and osmanthus (Osmanthus fragrans, OfCCD4), and were expressed, together with AtCCD4, in Escherichia coli. In vivo assays showed that CmCCD4a and MdCCD4 cleaved beta-carotene well to yield beta-ionone, while OfCCD4, RdCCD4, and AtCCD4 were almost inactive towards this substrate. No cleavage products were found for any of the five CCD4 genes when they were co-expressed in E. coli strains that accumulated cis-zeta-carotene and lycopene. In vitro assays, however, demonstrated the breakdown of 8'-apo-beta-caroten-8'-al by AtCCD4 and RdCCD4 to beta-ionone, while this apocarotenal was almost not degraded by OfCCD4, CmCCD4a, and MdCCD4. Sequence analysis of genomic clones of CCD4 genes revealed that RdCCD4, like AtCCD4, contains no intron, while MdCCD, OfCCD4, and CmCCD4a contain introns. These results indicate that plants produce at least two different forms of CCD4 proteins. Although CCD4 enzymes cleave their substrates at the same position (9,10 and 9',10'), they might have different biochemical functions as they accept different (apo)-carotenoid substrates, show various expression patterns, and are genomically differently organized.

Substrate promiscuity of RdCCD1, a carotenoid cleavage oxygenase from Rosa damascena.

Several of the key flavor compounds in rose essential oil are C(13)-norisoprenoids, such as beta-damascenone, beta-damascone, and beta-ionone which are derived from carotenoid degradation. To search for genes putatively responsible for the cleavage of carotenoids, cloning of carotenoid cleavage (di-)oxygenase (CCD) genes from Rosa damascena was carried out by a degenerate primer approach and yielded a full-length cDNA (RdCCD1). The RdCCD1 gene was expressed in Escherichia coli and recombinant protein was assayed for its cleavage activity with a multitude of carotenoid substrates. The RdCCD1 protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' positions to produce a C(14) dialdehyde and two C(13) products, which vary depending on the carotenoid substrates. RdCCD1 could also cleave lycopene at the 5-6 and 5'-6' positions to produce 6-methyl-5-hepten-2-one. Expression of RdCCD1 was studied by real-time PCR in different tissues of rose. The RdCCD1 transcript was present predominantly in rose flower, where high levels of volatile C(13)-norisoprenoids are produced. Thus, the accumulation of C(13)-norisoprenoids in rose flower is correlated to the expression of RdCCD1.

Generation of marker-free plastid transformants using a transiently cointegrated selection gene.

Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops, the possibility of marker transfer to wild relatives or microorganisms cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.

Glucosylation of Smoke-Derived Volatiles in Grapevine (Vitis vinifera) is Catalyzed by a Promiscuous Resveratrol/Guaiacol Glucosyltransferase.

Vinification of grapes (Vitis vinifera) exposed to forest fire smoke can yield unpalatable wine due to the presence of taint compounds from smoke and the release of smoke derived volatiles from their respective glycosides during the fermentation process or in-mouth during consumption. To identify glycosyltransferases (GTs) involved in the formation of glycosidically bound smoke-derived volatiles we performed gene expression analysis of candidate GTs in different grapevine tissues. Second, substrates derived from bushfire smoke or naturally occurring in grapes were screened with the candidate recombinant GTs. A resveratrol GT (UGT72B27) gene, highly expressed in grapevine leaves and berries was identified to be responsible for the production of the phenolic glucosides. UGT72B27 converted the stilbene trans-resveratrol mainly to the 3-O-glucoside. Kinetic analyses yielded specificity constants (kcat/KM) of 114, 17, 9, 8, and 2 mM-1 s-1 for guaiacol, trans-resveratrol, syringol, methylsyringol, and methylguaiacol, respectively. This knowledge will help to design strategies for managing the risk of producing smoke-affected wines.

Genomic organization of a diverse ACC synthase gene family in banana and expression characteristics of the gene member involved in ripening of banana fruits.

The banana is one of the typical climacteric fruits with great economic importance in agriculture. To understand the basic mechanism underlying banana ripening, gene clones for banana ACC synthase (EC 4.4.1.14), a key regulatory enzyme in the ethylene biosynthetic pathway, were characterized. Genomic clones were analyzed by restriction mapping, and the data in conjunction with sequence comparisons with the previously isolated PCR fragments indicated that at least nine ACC synthase genes (MACS1-9) exist in the banana genome. Southern blot analysis showed they are located in different regions of the banana genome. Three lambda genomic clones (GMACS-1, -9, and -12) were completely sequenced, and gene structures of MACS1 (corresponding to alleles of GMACS-9 and -12) and MACS2 (corresponding to GMACS-1) were elucidated. The coding regions of these three genes were all interrupted by three introns. The size and location of introns are similar to the ACC synthase genes from tomato and Arabidopsis. Northern analysis showed that only MACS1 expressed during fruit ripening and was inducible by exogenous ethylene treatment, which indicates MACS1 is a significant member of the ACC synthase gene family related to ripening in banana fruit. The transcription initiation site of GMACS-12 containing MACS1 was defined. There is a TATTAAT sequence located at position -31 to -25 that qualifies as a TATA box. The delineation of transcription unit in MACS1 will facilitate the promoter studies for this gene and allow its specific functions involved in fruit ripening to be determined.

Enhanced production of ß-glucosides by in-situ UDP-glucose regeneration.

Glycosyltransferase (GT)-mediated methodology is recognized as one of the most practical approaches for large-scale production of glycosides. However, GT enzymes require a sugar nucleotide as donor substrate that must be generated in situ for preparative applications by recycling of the nucleotide moiety, e.g. by sucrose synthase (SUS). Three plant GT genes CaUGT2, VvGT14a, and VvGT15c and the fungal SbUGTA1 were successfully co-expressed with GmSUS from soybean in Escherichia coli BL21 and W cells. In vitro, the crude protein extracts prepared from four GT genes and GmSUS co-expressing cells were able to convert several small molecules to the corresponding glucosides, when sucrose and UDP were supplied. In addition, GmSUS was able to enhance the glucosylation efficiency and reduced the amount of supplying UDP-glucose. In the biotransformation system, co-expression of VvGT15c with GmSUS also improved the glucosylation of geraniol and enhanced the resistance of the cells against the toxic terpenol. GT-EcW and GTSUS-EcW cells tolerated up to 2mM geraniol and converted more than 99% of the substrate into the glucoside at production rates exceeding 40µgml(-1)h(-1). The results confirm that co-expression of SUS allows in situ regeneration of UDP-sugars and avoids product inhibition by UDP.

Glucosylation of aroma chemicals and hydroxy fatty acids.

To explore the utility of glycosyltransferases as novel biocatalysts, we isolated the glycosyltransferase genes CaUGT2 and SbUGTA1 from Catharanthus roseus and Starmerella bombicola, respectively and heterologously expressed them in Escherichia coli. The purified recombinant proteins were assayed with a variety of small molecule substrates. Carvacrol and its phenol isomer thymol are efficiently glucosylated by CaUGT2. The Vmax/Km ratios show that CaUGT2 exhibits the highest specificity towards carvacrol, followed by thymol, geraniol, eugenol, vanillin, menthol, and tyrosol. In contrast, SbUGTA1 accepts ω-hydroxy fatty acids and 1-alkanols as substrates. The Vmax/Km ratios indicate that SbUGTA1 exhibits the highest specificity towards 16-hydroxy palmitic acid, followed by octanol, decanol, and hexadecanol. In biotransformation experiments 23, 88 and 99% of octanol, 16-hydroxy palmitic acid, and decanol, respectively is converted into the corresponding ß-glucosides by E. coli cells expressing SbUGTA1 whereas those cells expressing CaUGT2 glucosylate 18, 61, 77 and 97% of applied eugenol, thymol, vanillin, and carvacrol, respectively. To optimize the biotransformation rate, the effects of the concentration of IPTG, glucose, and substrate on the production of glucosides were tested. Taken together, this procedure is a simple operation, environmentally friendly, and is useful for the preparation of glycosides as additives for food and cosmetics.

FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.).

The strawberry (Fragaria × ananassa) is one of the most preferred fresh fruit worldwide, accumulates numerous flavonoids but has limited shelf life due to excessive tissue softening caused by cell wall degradation. Since lignin is one of the polymers that strengthen plant cell walls and might contribute to some extent to fruit firmness monolignol biosynthesis was studied in strawberry fruit. Cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), and a peroxidase (POD27) gene were strongly expressed in red, ripe fruit whereas a second POD gene was primarily expressed in green, immature fruit. Moreover, FaPOD27 transcripts were strongly and constitutively induced in fruits exposed to Agrobacterium infection. Gene expression levels and enzymatic activities of FaCCR and FaCAD were efficiently suppressed through RNAi in FaCCR- and FaCAD-silenced strawberries. Besides, significantly elevated FaPOD transcript levels were detected after agroinfiltration of pBI-FaPOD constructs in fruits. At the same time, levels of G-monomers were considerably reduced in FaCCR-silenced fruits whereas the proportion of both G- and S-monomers decisively decreased in FaCAD-silenced and pBI-FaPOD fruits. Development, firmness, and lignin level of the treated fruits were similar to pBI-intron control fruits, presumably attributed to increased expression levels of FaPOD27 upon agroinfiltration. Additionally, enhanced firmness, accompanied with elevated lignin levels, was revealed in chalcone synthase-deficient fruits (CHS(-)), independent of down- or up-regulation of individual and combined FaCCR. FaCAD, and FaPOD genes by agroinfiltration, when compared to CHS(-)/pBI-intron control fruits. These approaches provide further insight into the genetic control of flavonoid and lignin synthesis in strawberries. The results suggest that FaPOD27 is a key gene for lignin biosynthesis in strawberry fruit and thus to improving the firmness of strawberries.