Sudarat Thanonkeo

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

BackgroundDuring 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.ResultsBased 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.ConclusionWe 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.

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.

Ethanol production from Jerusalem artichoke ( Helianthus tuberosus L.) by Zymomonas mobilis TISTR548

The selection and characterization of Zymomonas mobilis for ethanol production from Jerusalem artichoke ( Helianthus tuberosus L.) juice was investigated. Growth and ethanol production of four Z. mobilis strains isolated in Thailand, that is, TISTR 405, TISTR 548, TISTR 550 and TISTR 551, were compared with those of the type strain Z. mobilis ZM4 (NRRL B-14023) at different temperatures. Among the strains tested, TISTR 548 gave the highest ethanol concentration at 30 to 35°C, as compared to the others. Therefore, this strain was chosen for ethanol production from Jerusalem artichoke juice after acid hydrolysis. The influence of some fermentation factors such as sugar concentration, pH of the fermentation medium, inoculation size and nitrogen source on ethanol production from Jerusalem artichoke juice was determined. The results show that the maximum ethanol concentration (95.9 g/L) with 98% of the theoretical ethanol yield was obtained when the fermentation was carried out in a medium containing 250 g/L total sugars, pH 5.0, inoculation size at 10% and using 0.5 g/L diammonium phosphate as nitrogen source. The maximum theoretical ethanol yield obtained in this study was higher than those previously reported. Key words : Ethanol production, Zymomonas mobilis, thermotolerant microorganism, Jerusalem artichoke.

Induced accumulation of 20-hydroxyecdysone in cell suspension cultures of Vitex glabrata R.Br.

This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata , an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production were not significantly different when cells were cultivated on B5 or half-strength MS medium. However, cultivation of V. glabrata cell cultures at 25°C yielded 1.06- and 1.09-fold higher values of cell growth and 20-hydroxyecdysone content, respectively than those at 30°C. Sucrose at 30 and 40 g/L favors the production of 20-hydroxyecdysone in suspension cultures of V. glabrata . Feeding of cholesterol at 5 mg/L, as precursor for biosynthesis of 20-hydroxyecdysone, yielded 1.11-fold higher accumulation of 20-hydroxyecdysone than the control cells. Increasing of cholesterol to 10 mg/L resulted in decreased production of 20-hydroxyecdysone. Key words : Vitex glabrata, 20-hydroxyecdysone, suspension culture, cholesterol.

Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.

The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production

High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160 g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85 g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51 g/L and 33.84 g/L at 37 °C and 40 °C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42 °C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.

Enhance production of 20-hydroxyecdysone in cell suspension cultures of Vitex glabrata R.Br. by elicitor feeding

The effects of chitosan and methyl jasmonate on growth and 20-hydroxyecdysone production in cell suspension cultures of Vitex glabrata, a medicinal plant in Thailand, were investigated. Elicitation with chitosan at 50 mg/L resulted in 17.16 g/L biomass and 377.09 mg/100 g dry weight (DW) 20hydroxyecdysone, which were 1.62 and 8.33 times higher than the control cultures, respectively. Likewise, addition of methyl jasmonate at 100 μM also enhanced growth and production of 20hydroxyecdysone. The highest growth and 20-hydroxyecdysone production reached 14.44 g/L and 621.76 mg/100 g DW, which were 1.35 and 14.54 times higher in comparison to the control cultures, respectively. This is the first report to indicate that elicitation with chitosan and methyl jasmonate enhanced the production of 20-hydroxyecdysone in cell suspension cultures of V. glabrata.

Ethanol production from sweet sorghum by Saccharomyces cerevisiae DBKKUY-53 immobilized on alginate-loofah matrices

Ethanol production from sweet sorghum juice (SSJ) using the thermotolerant Saccharomyces cerevisiae strain DBKKUY-53 immobilized in an alginate-loofah matrix (ALM) was successfully developed. As found in this study, an ALM with dimensions of 20 × 20 × 5 mm3 is effective for cell immobilization due to its compact structure and long-term stability. The ALM-immobilized cell system exhibited greater ethanol production efficiency than the freely suspended cell system. By using a central composite design (CCD), the optimum conditions for ethanol production from SSJ by ALM-immobilized cells were determined. The maximum ethanol concentration and volumetric ethanol productivity obtained using ALM-immobilized cells under the optimal conditions were 97.54 g/L and 1.36 g/L h, respectively. The use of the ALM-immobilized cells was successful for at least six consecutive batches (360 h) without any loss of ethanol production efficiency, suggesting their potential application in industrial ethanol production.

Agrobacterium-mediated transformation of Dendrobium orchid with the flavanone 3-hydroxylase gene

The Ascocenda flavanone 3-hydroxylase (AcF3H) gene was successfully transformed into Dendrobium 5N white orchid plants using Agrobacterium-mediated gene transformation. In this study, for the first time, we report the construction of a plant expression vector harboring the AcF3H gene using the Gateway cloning system. Protocorm-like bodies (PLBs) were cocultivated with the Agrobacterium tumefaciens strain AGL1 harboring the plant expression vector pGWB5-AcF3H, which contained the hygromycin phosphotransferase (hpt) gene as a selectable marker. The highest transformation efficiency (10.13%) was achieved when PLBs were cocultivated with Agrobacterium cells for 15 min. Three months after the transformation, the plantlets were regenerated, and the transgenic plants were confirmed by polymerase chain reaction (PCR) analysis using specific primers for the hpt gene and 35S promoter region. PCR products of approximately 400 and 500 bp, corresponding to the hpt gene and the 35S promoter, respectively, were detected in the transgenic plants, while no such product was observed in nontransgenic plants, indicating that the AcF3H gene was integrated into the genome of Dendrobium 5N white orchid plants. The transient expression of the AcF3H gene in Dendrobium 5N white and Dendrobium Anna petals was performed using the agroinfiltration technique, and the results demonstrated that no cyanidin content was detected in the Dendrobium 5N white petals after AcF3H infiltration. In contrast, the cyanidin content was increased by approximately 6% in the Dendrobium Anna petals, suggesting that the AcF3H gene was transiently expressed in this orchid.

Genetic diversity and population structure of blue-crested lizard, Calotes mystaceus Duméril & Bibron, 1837 (Squamata: Agamidae) in Thailand

The blue-crested lizard, Calotes mystaceus Duméril & Bibron, 1837, is listed as a protected wild animal in Thailand. Its population is likely to be dramatically reduced due to massive hunting in several areas in this country. Basic information on its population genetics is therefore needed to facilitate its conservation. Thus, in this study we investigated the mitochondrial cytochrome c oxidase subunit 1 (CO1) sequence variation of 238 individual C. mystaceus from 42 different geographical localities of the north, west, central, east and northeast regions of Thailand. High genetic diversity and genetic differentiation at the intrapopulation and interpopulation levels was observed. We detected 63 unique haplotypes and 12 common/shared haplotypes. The phylogenetic analysis reveals two major lineages, I and II. These two lineages are separated by mountain ranges, which play an important role as natural barriers blocking gene flow. Our finding reveal at least two cryptic lineages represented in C. mystaceus populations in Thailand. However, a comprehensive investigation of the morphology, biology, ecology and genetic diversity of C. mystaceus in other regions within its area of distribution is needed.