The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of ¹⁴C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.     

Cloning, sequence, and disruption of the Saccharomyces diastaticus DAR1 gene encoding a glycerol-3-phosphate dehydrogenase.

The Saccharomyces diastaticus DAR1 gene was cloned by complementation in an Escherichia coli strain auxogrophic for glycerol-3-phosphate. DAR1 encodes an NADH-dependent dihydroxyacetone phosphate reductase (sn-glycerol-3-phosphate dehydrogenase [G3PDase; EC 1.1.1.8]) homologous to several other eukaryotic G3PDases. DAR1 is distinct from GUT2, which encodes a glucose-repressed mitochondrial G3PDase, but is identical to GPD1 from S. cerevisiae, a close relative of S. diastaticus. The level of DAR1-encoded G3PDase was increased about threefold in a medium of high osmolarity. Disruption of DAR1 in a haploid S. cerevisiae was not lethal but led to a decrease in cytoplasmic NADH-dependent G3PDase activity, an increase in osmotic sensitivity, and a 25% reduction in glycerol secretion from cells grown anaerobically on glucose.     

Glycerol Overproduction by Engineered Saccharomyces cerevisiae Wine Yeast Strains Leads to Substantial Changes in By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase

Six commercial wine yeast strains and three nonindustrial strains (two laboratory strains and one haploid strain derived from a wine yeast strain) were engineered to produce large amounts of glycerol with a lower ethanol yield. Overexpression of the GPD1 gene, encoding a glycerol-3-phosphate dehydrogenase, resulted in a 1.5- to 2.5-fold increase in glycerol production and a slight decrease in ethanol formation under conditions simulating wine fermentation. All the strains overexpressing GPD1 produced a larger amount of succinate and acetate, with marked differences in the level of these compounds between industrial and nonindustrial engineered strains. Acetoin and 2,3-butanediol formation was enhanced with significant variation between strains and in relation to the level of glycerol produced. Wine strains overproducing glycerol at moderate levels (12 to 18 g/liter) reduced acetoin almost completely to 2,3-butanediol. A lower biomass concentration was attained by GPD1-overexpressing strains, probably due to high acetaldehyde production during the growth phase. Despite the reduction in cell numbers, complete sugar exhaustion was achieved during fermentation in a sugar-rich medium. Surprisingly, the engineered wine yeast strains exhibited a significant increase in the fermentation rate in the stationary phase, which reduced the time of fermentation.     

Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol

Many microalgae and plants have the ability to synthesize large amounts of triacylglycerol (TAG) that can be used to produce biofuels. Presently, TAG-based biofuel production is limited by the feedstock supply. Metabolic engineering of lipid synthesis pathways to overproduce TAGs in oleaginous microalgae and oil crop plants has achieved only modest success. We demonstrate that inactivation of ADP-glucose pyrophosphorylase in a Chlamydomonas starchless mutant led to a 10-fold increase in TAG, suggesting that shunting of photosynthetic carbon partitioning from starch to TAG synthesis may represent a more effective strategy than direct manipulation of the lipid synthesis pathway to overproduce TAG.     

Engineered tobacco and microalgae secreting the fungal laccase POXA1b reduce phenol content in olive oil mill wastewater

Olive oil mill wastewaters (OMWs) are characterised by low pH and a high content of mono- and polyaromatic compounds that exert microbial and phytotoxic activity. The laccase cDNA of the poxA1b gene from Pleurotus ostreatus, carrying a signal peptide sequence for enzyme secretion and driven by the CaMV 35S promoter, was cloned into a plant expression vector. Nuclear genetic transformation was carried out by co-cultivation of Agrobacterium tumefaciens with tobacco cv Samsun NN leaves and cells of five different microalgae accessions belonging to the genera Chlamydomonas, Chlorella and Ankistrodesmus. Transgenic plants and microalgae were able to express and secrete the recombinant laccase in the root exudates and the culture medium, respectively. In comparison to untransformed controls, the ability to reduce phenol content in OMW solution was enhanced up to 2.8-fold in transgenic tobacco lines and by up to about 40% in two microalgae accessions. The present work provides new evidence for metabolic improvement of green organisms through the transgenic approach to remediation.     

Bacterial diketopiperazines stimulate diatom growth and lipid accumulation

Diatoms are photosynthetic microalgae that fix a significant fraction of the world’s carbon. Because of their photosynthetic efficiency and high-lipid content, diatoms are priority candidates for biofuel production. Here, we report that sporulating Bacillus thuringiensis and other members of the Bacillus cereus group, when in co-culture with the marine diatom Phaeodactylum tricornutum, significantly increase diatom cell count. Bioassay-guided purification of the mother cell lysate of B. thuringiensis led to the identification of two diketopiperazines (DKPs) that stimulate both P. tricornutum growth and increase its lipid content. These findings may be exploited to enhance P. tricornutum growth and microalgae-based biofuel production. As increasing numbers of DKPs are isolated from marine microbes, the work gives potential clues to bacterial-produced growth factors for marine microalgae.     

Lipid metabolism and potentials of biofuel and high added-value oil production in red algae

Biomass production is currently explored in microalgae, macroalgae and land plants. Microalgal biofuel development has been performed mostly in green algae. In the Japanese tradition, macrophytic red algae such as Pyropia yezoensis and Gelidium crinale have been utilized as food and industrial materials. Researches on the utilization of unicellular red microalgae such as Cyanidioschyzon merolae and Porphyridium purpureum started only quite recently. Red algae have relatively large plastid genomes harboring more than 200 protein-coding genes that support the biosynthetic capacity of the plastid. Engineering the plastid genome is a unique potential of red microalgae. In addition, large-scale growth facilities of P. purpureum have been developed for industrial production of biofuels. C. merolae has been studied as a model alga for cell and molecular biological analyses with its completely determined genomes and transformation techniques. Its acidic and warm habitat makes it easy to grow this alga axenically in large scales. Its potential as a biofuel producer is recently documented under nitrogen-limited conditions. Metabolic pathways of the accumulation of starch and triacylglycerol and the enzymes involved therein are being elucidated. Engineering these regulatory mechanisms will open a possibility of exploiting the full capability of production of biofuel and high added-value oil. In the present review, we will describe the characteristics and potential of these algae as biotechnological seeds.     

CCAAT/enhancer binding protein-beta negatively regulates the expression of glycerol-3-phosphate dehydrogenase 1 in pig PK-15 cells

Soluble glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays important roles in the synthesis of triacylglycerol and in the glycerol-3-phosphate shutter. Though GPD1 is expressed in most adult tissues, little is known about the regulation of its expression. In this study, we analyzed the characters, organization and core region of the promoter of pig GPD1 gene by in silico analysis and activity detection of deletion mutants. We also identified and testified the negative regulation effect of C/EBP β on pig GPD1 gene by Chromatin immunoprecipitation (ChIP) assay and over-expression experiments in cultured pig kidney cells. Compared to that of human, pig GPD1 gene promoter has three conserved regions and one deletion region. In silico analysis indicated that pig GPD1 promoter was TATA-less with at least 3 CpG islands of over 200 bp in length and over 60% in GC content. The activity detection of deletion mutants suggested that the essential elements required for the optimal promoter activity scatter in the promoter region, while the core promoter region was from -422 bp to -1 bp. Chromatin immunoprecipitation (ChIP) assay results indicated that C/EBP β had plenty of binding sites in pig GPD1 promoter with the common cis-element (5’- TKNNGCAAK -3’). The over-expression examination of C/EBP β showed that the expression of GPD1 was negatively regulated by C/EBP β in pig kidney cells. Overall, our study revealed that the pig GPD1 promoter is a TATA-less promoter, and in promoter region, the binding sites of C/EBP β share common motif of (5’-TKNNGCAAK -3’). We also showed that pig GPD1 gene is regulated negatively by C/EBP β in cultured kidney cells.     

Lipid productivity and fatty acid composition-guided selection of Chlorella strains isolated from Malaysia for biodiesel production

The need to develop biomass-based domestic production of high-energy liquid fuels (biodiesel) for transportation can potentially be addressed by exploring microalgae with high lipid content. Selecting the strains with adequate oil yield and quality is of fundamental importance for a cost-efficient biofuel feedstock production based on microalgae. This work evaluated 29 strains of Chlorella isolated from Malaysia as feedstock for biodiesel based on volumetric lipid productivity and fatty acid profiles. Phylogenetic studies based on 18S rRNA gene revealed that majority of the strains belong to true Chlorella followed by Parachlorella. The strains were similarly separated into two groups based on fatty acid composition. Of the 18 true Chlorella strains, Chlorella UMACC187 had the highest palmitic acid (C16:0) content (71.3?±?4.2 % total fatty acids, TFA) followed by UMACC84 (70.1?±?0.7 %TFA), UMACC283 (63.8?±?0.7 %TFA), and UMACC001 (60.3?±?4.0 %TFA). Lipid productivity of the strains at exponential phase ranged from 34.53 to 230.38 mg L^?1 day^?1, with Chlorella UMACC050 attaining the highest lipid productivity. This study demonstrated that Chlorella UMACC050 is a promising candidate for biodiesel feedstock production.     

Effects of GPD1 Overexpression in Saccharomyces cerevisiae Commercial Wine Yeast Strains Lacking ALD6 Genes

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP⁺-dependent Mg²⁺-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.