Cloning and expression analysis of dihydroxyflavonol 4-reductase (DFR) in Ascocenda spp.

Dihydroflavonol 4-reductase (DFR) gene is a key gene of anthocyanins biosynthesis pathway, which represent an importance pathway for orchid flower. In this study, cloning and expression analysis of DFR gene in Ascocenda spp. were carried out. Nucleotide analysis revealed that the Ascocenda DFR gene was 1,056 bp in length, and encoded a protein of 351 amino acid residues. A typical translation initiation codon (ATG) and translation termination codon (TGA), the most frequently found codon in plant were identified, indicating a full-length coding sequence of the DFR gene. The calculated molecular mass of the deduced polypeptide was 39.8 kDa and the predicted isoelectric point was 5.58. Homology analysis revealed that the amino acid sequence of the Ascocenda DFR gene product was 80 to 87% identity to amino acid sequences of DFR gene products of other orchids such as Bromheadia, Dendrobium, Cymbidium and Oncidium. It also showed a high degree of identity to the DFR gene products of other flowers such as Lilium, Tilipa, Allium, Gentiana and Chrysanthenum. Southern blot analysis indicate that DFR is presented as a single copy in the Ascocenda spp. genome. The AscoDFR gene was highly expressed in the flower stages 2 and 3 of development as well as in the sepal and petal of the orchid flower.Keywords: Orchid, dihydroxyflavonol 4-reductase, anthocyanins, gene clonin

Fermentation of rice bran hydrolysate to ethanol using Zymomonas mobilis biofilm immobilization on DEAE-cellulose

Background: The major challenges associated with the fermentation of lignocellulosic hydrolysates are the reduction in the operating cost and minimizing the complexity of the process. Zymomonas mobilis biofilm has been emerged to resolve these complexities. Biofilm has been reported to tolerate to the toxic inhibitors and easily manipulated toward the cell recycle through the cell immobilization. Results: Z.mobilis ZM4 and TISTR 551 were able to develop biofilms on DEAE cellulose under the differences in the morphologies. Z. mobilis ZM4 developed homogeneous biofilm that brought DEAE fiber to be crosslinking, while Z. mobilis TISTR 551 developed heterogeneous biofilm in which crosslinking was not observed. Ethanol production under batch and repeated batch fermentation of rice bran hydrolysate containing toxic inhibitors were compared between these two biofilms. TISTR 551 biofilm produced the maximum yield (YP/S) of 0.43 \ub1 0.09 g ethanol/g glucose (83.89% theoretical yield). However the repeated batch could not be proceeded due to the bacterial detachment. Z. mobilis ZM4 biofilm produced the maximum yield (YP/S) of 0.177 \ub1 0.05 g ethanol/g glucose (34.74% theoretical yield) in the batch culture and the biofilm remained intact to proceed along the repeated batch. The highest ethanol yield (YP/S) in the repeated batch of Z. mobilis ZM4 was 0.354 \ub1 0.07 g ethanol/g glucose (69.51% theoretical yield). Conclusions: Homogeneous biofilm structure of Z. mobilis provided more recycle beneficial over the heterogeneous biofilm structure for the ethanol production from lignocellulosic hydrolysate

Utilization capability of sucrose, raffinose and inulin and its less-sensitiveness to glucose repression in thermotolerant yeast Kluyveromyces marxianus DMKU 3-1042

Kluyveromyces marxianus possesses a useful potential to assimilate a wide variety of substrates at a high temperature, but the negative effect by coexisting glucose is critical for utilization of biomass containing various sugars. Such a negative effect on the activity of inulinase, which is the sole enzyme to hydrolyze sucrose, raffinose and inulin, has been demonstrated in K. marxianus without analysis at the gene level. To clarify the utilization capability of sucrose, raffinose and inulin and the glucose effect on inulinase in K. marxianus DMKU 3-1042, its growth and metabolite profiles on these sugars were examined with or without glucose under a static condition, in which glucose repression evidently occurs. Consumption of sucrose was not influenced by glucose or 2-deoxyglucose. On the other hand, raffinose and inulin consumption was hampered by glucose at 30°C but hardly hampered at 45°C. Unlike Saccharomyces cerevisiae, increase in glucose concentration had no effect on sucrose utilization. These sugar-specific glucose effects were consistent with the level of inulinase activity but not with that of the KmINU1 transcript, which was repressed in the presence of glucose via KmMig1p. This inconsistency may be due to sufficient activity of inulinase even when glucose is present. Our results encourage us to apply K. marxianus DMKU 3-1042 to high-temperature ethanol fermentation with biomass containing these sugars with glucose

Sorbitol required for cell growth and ethanol production by Zymomonas mobilis under heat, ethanol, and osmotic stresses

BACKGROUND: During ethanol fermentation, the ethanologenic bacterium, Zymomonas mobilis may encounter several environmental stresses such as heat, ethanol and osmotic stresses due to high sugar concentration. Although supplementation of the compatible solute sorbitol into culture medium enhances cell growth of Z. mobilis under osmotic stress, the protective function of this compound on cell growth and ethanol production by this organism under other stresses such as heat and ethanol has not been described yet. The formation of sorbitol in Z. mobilis was carried out by the action of the glucose-fructose oxidoreductase (GFOR) enzyme which is regulated by the gfo gene. Therefore, the gfo gene in Z. mobilis was disrupted by the fusion-PCR-based construction technique in the present study, and the protective function of sorbitol on cell growth, protein synthesis and ethanol production by Z. mobilis under heat, ethanol, and osmotic stresses was investigated. RESULTS: Based on the fusion-PCR-based construction technique, the gfo gene in Z. mobilis was disrupted. Disruption of the Z. mobilis gfo gene resulted in the reduction of cell growth and ethanol production not only under osmotic stress but also under heat and ethanol stresses. Under these stress conditions, the transcription level of pdc, adhA, and adhB genes involved in the pyruvate-to-ethanol (PE) pathway as well as the synthesis of proteins particularly in Z. mobilis disruptant strain were decreased compared to those of the parent. These findings suggest that sorbitol plays a crucial role not only on cell growth and ethanol production but also on the protection of cellular proteins from stress responses. CONCLUSION: We showed for the first time that supplementation of the compatible solute sorbitol not only promoted cell growth but also increased the ethanol fermentation capability of Z. mobilis under heat, ethanol, and osmotic stresses. Although the molecular mechanism involved in tolerance to stress conditions after sorbitol supplementation is still unclear, this research has provided useful information for the development of the effective ethanol fermentation process particularly under environmental conditions with high temperature or high ethanol and sugar concentration conditions

In Vitro Propagation of <i>Philodendron erubescens</i> ‘Pink Princess’ and Ex Vitro Acclimatization of the Plantlets

This study describes the in vitro propagation and ex vitro acclimatization of Philodendron erubescens pink princess, one of the most popular ornamental variegated foliage plants. For shoot proliferation, the protocorm-like bodies of the Philodendron pink princess were cultured on solid Murashige and Skoog (MS) media supplemented with 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) at different concentrations. The results revealed that supplementation with BAP alone at a concentration of 1.0 mg/L yielded the maximum number of shoots and leaves. Furthermore, the application of BAP at 1.0 mg/L significantly enhanced the shoot proliferation of Philodendron pink princess when grown in liquid MS medium, yielding 11.2 shoots/explant and 4.7 leaves/explant. When the established microshoots were subjected to root induction using solid MS media supplemented with different kinds and concentrations of auxins, indole-3-butyric acid (IBA) at 3 mg/L resulted in the highest number of roots (3.2 roots/explant) and longest root length (1.9 cm). Three supporting materials, i.e., peat moss, vermiculite, and perlite, were used as planting media for the ex vitro acclimatization of the Philodendron pink princess plantlets. The results demonstrated that the in vitro plantlets acclimatized and exhibited a relatively high survival frequency in all planting media without morphological abnormalities. Peat moss outperformed all other types of planting media in terms of sustaining the vegetative growth of the plantlets. In the future, the approach established in this study could be employed for the extensive production of Philodendron pink princess