Reduced Glycerol Production Research Articles

Hepatic glycerol shunt and glycerol-3-phosphate phosphatase control liver metabolism and glucodetoxification under hyperglycemia

ObjectiveGlycerol-3-phosphate (Gro3P) phosphatase (G3PP) hydrolyzes Gro3P to glycerol that exits the cell, thereby operating a “glycerol shunt”, a metabolic pathway that we identified recently in mammalian cells. We have investigated the role of G3PP and the glycerol shunt in the regulation of glucose metabolism and lipogenesis in mouse liver. MethodsWe generated hepatocyte-specific G3PP-KO mice (LKO), by injecting AAV8-TBG-iCre to male G3PPfl/fl mice. Controls received AAV8-TBG-eGFP. Both groups were fed chow diet for 10 weeks. Hyperglycemia (16–20 mM) was induced by glucose infusion for 55 h. Hepatocytes were isolated from normoglycemic mice for ex vivo studies and targeted metabolomics were measured in mice liver after glucose infusion. ResultsLKO mice showed no change in body weight, food intake, fed and fasted glycemia but had increased fed plasma triglycerides. Hepatic glucose production from glycerol was increased in fasted LKO mice. LKO mouse hepatocytes displayed reduced glycerol production, elevated triglyceride and lactate production at high glucose concentration. Hyperglycemia in LKO mice led to increased liver weight and accumulation of triglycerides, glycogen and cholesterol together with elevated levels of Gro3P, dihydroxyacetone phosphate, acetyl-CoA and some Krebs cycle intermediates in liver. Hyperglycemic LKO mouse liver showed elevated expression of proinflammatory cytokines and M1-macrophage markers accompanied by increased plasma triglycerides, LDL/VLDL, urea and uric acid and myocardial triglycerides. ConclusionsThe glycerol shunt orchestrated by G3PP acts as a glucose excess detoxification pathway in hepatocytes by preventing metabolic disturbances that contribute to enhanced liver fat, glycogen storage, inflammation and lipid build-up in the heart. We propose G3PP as a novel therapeutic target for hepatic disorders linked to nutrient excess.

Genome-wide transcriptional regulation in Saccharomyces cerevisiae in response to carbon dioxide

Sugar metabolism by Saccharomyces cerevisiae produces ample amounts of CO2 under both aerobic and anaerobic conditions. High solubility of CO2 in fermentation media, contributing to enjoyable sensory properties of sparkling wine and beers by S. cerevisiae, might affect yeast metabolism. To elucidate the overlooked effects of CO2 on yeast metabolism, we examined glucose fermentation by S. cerevisiae under CO2 as compared to N2 and O2 limited conditions. While both CO2 and N2 conditions are considered anaerobic, less glycerol and acetate but more ethanol were produced under CO2 condition. Transcriptomic analysis revealed that significantly decreased mRNA levels of GPP1 coding for glycerol-3-phosphate phosphatase in glycerol synthesis explained the reduced glycerol production under CO2 condition. Besides, transcriptional regulations in signal transduction, carbohydrate synthesis, heme synthesis, membrane and cell wall metabolism, and respiration were detected in response to CO2. Interestingly, signal transduction was uniquely regulated under CO2 condition, where upregulated genes (STE3, MSB2, WSC3, STE12, and TEC1) in the signal sensors and transcriptional factors suggested that MAPK signaling pathway plays a critical role in CO2 sensing and CO2-induced metabolisms in yeast. Our study identifies CO2 as an external stimulus for modulating metabolic activities in yeast and a transcriptional effector for diverse applications.

Short-term Adaptation Strategy Improved Xylitol Production by Candida guilliermondii on Sugarcane Bagasse Hemicellulosic Hydrolysate

In this work, we proposed a short-term adaptation strategy to improve xylitol production on sugarcane bagasse hemicellulosic hydrolysate through the maximization of Candida guilliermondii FTI 20037 tolerance to inhibitors. Hemicellulosic hydrolysate obtained by diluted acid hydrolysis (1.0% (wv−1) H2SO4, 1:10 solid/liquid ratio, 121 °C, 10 min) was concentrated up to fivefold to obtain hydrolysates with different concentration factors. Yeast was cultivated in each hydrolysate for 24-h and consecutively transferred to the subsequent more concentrated hydrolysate to obtain different adaptation degrees. Adapted cells were used as inoculum in fermentations with the same hydrolysate in which they were adapted. The performance of adapted and non-adapted yeast was compared to validate the adaptation strategy employed. The beneficial effects of adaptation were more pronounced in the hydrolysates with higher inhibitor concentration (twofold concentrated and non-treated, H2N; and fivefold concentrated and treated, H5). It improved xylose assimilation and xylitol production as well as xylitol yield and xylitol volumetric productivity in both hydrolysates. A 62.5% increase in productivity (0.24 to 0.39 gL−1 h−1) and a 15.7% increase in yield (0.51 to 0.59 gg−1) were observed for H5 hydrolysate, while for H2N hydrolysate these increases were 54.5 and 29.6%, respectively. Yeast adaptation also improved arabinose consumption and reduced glycerol production. The reduction in glycerol production indicates a greater tolerance of adapted cells to the inhibitors present in hydrolysates. Short-term adaptation proved to be an efficient strategy to improve yeast tolerance as well as its fermentative performance on sugarcane bagasse hemicellulosic hydrolysate.

Enhancing ethanol yields in corn dry grind process by reducing glycerol production

AbstractBackground and objectivesReducing glycerol production, a major by‐product produced during fermentation by yeast Saccharomyces cerevisiae, can improve the ethanol productivity in corn dry grind process. This study investigates the potential of glycerol reduction using an advanced engineered yeast strain and determines the effect of enzyme dosage, pH, and temperature on the granular starch hydrolysis (GSH) process efficiency using both conventional yeast and advanced yeast.FindingsEthanol yields in GSH process were found maximum at enzyme dosage 4.8 kg/MT grains and remained unchanged with further increase in dosage. The glycerol production was reduced by more than 50% with use of advanced enzymes compared to the conventional yeast and resulted in 2.2–3.8% higher final ethanol concentrations. Performances of both yeast were reduced and lead to stuck fermentation at 36°C temperature.ConclusionsUse of advanced yeast strains can improve the ethanol yields by reducing glycerol production in GSH process; however, the ethanol yields are highly dependent on the process conditions, especially enzyme loadings and temperature.Significance and noveltyGSH process is expected to produce lower glycerol compared to conventional dry grind process. This research demonstrated that advanced engineered yeasts can reduce the glycerol production in GSH process also, improving the process yields and profitability.

Deletion of glycerol-3-phosphate dehydrogenase genes improved 2,3-butanediol production by reducing glycerol production in pyruvate decarboxylase-deficient Saccharomyces cerevisiae

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible.

Overexpression of THI4 and HAP4 Improves Glucose Metabolism and Ethanol Production in Saccharomyces cerevisiae.

Redox homeostasis is essential to the maintenance of cell metabolism. Changes in the redox state cause global metabolic and transcriptional changes. Our previous study indicated that the overexpression of NADH oxidase in Saccharomyces cerevisiae led to increased glucose consumption and ethanol production. Gene expression related to thiamine synthesis and osmotolerance as well as HAP4 expression was increased in response to redox change caused by the overexpression of NADH oxidase. To identify detailed relationships among cofactor levels, thiamine synthesis, expression of HAP4, and osmotolerance, and to determine whether these changes are interdependent, THI4 and HAP4 were overexpressed in S. cerevisiae BY4741. The glucose consumption rate of THI4-overexpressing strain (thi4-OE) was the highest, followed by HAP4-overexpressing strain (hap4-OE) > NADH oxidase-overexpressing strain (nox-OE) > control strain (con), while strain hap4-OE showed the highest concentration of ethanol after 26 h of fermentation. Reduced glycerol production and increased osmotolerance were observed in thi4-OE and hap4-OE, as well as in nox-OE. HAP4 globally regulated thiamine synthesis, biomass synthesis, respiration, and osmotolerance of cells, which conferred the recombinant strain hap4-OE with faster glucose metabolism and enhanced stress resistance. Moreover, overexpression of HAP4 might extend the life span of cells under caloric restriction by lowering the NADH level. Although overexpression of THI4 and HAP4 induced various similar changes at both the metabolic and the transcriptional level, the regulatory effect of THI4 was more limited than that of HAP4, and was restricted to the growth phase of cells. Our findings are expected to benefit the bio-ethanol industry.

Enhanced ethanol production and reduced glycerol formation in fps1∆ mutants of Saccharomyces cerevisiae engineered for improved redox balancing.

Ethanol is by volume the largest fermentation product. During ethanol production by Saccharomyces cerevisiae about 4-5% of the carbon source is lost to glycerol production. Different approaches have been proposed for improving the ethanol yield while reducing glycerol production. Here we studied the effect of reducing glycerol export/formation through deletion of the aquaglyceroporin gene FPS1 together with expressing gapN encoding NADP+-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans and overexpressing the ATP-NADH kinase gene UTR1 from S. cerevisiae. This strategy will allow reducing the redox balance problem observed when the glycerol pathway is blocked, and hereby improve ethanol production. We found that our strategy enabled increasing the ethanol yield by 4.6% in the case of the best producing strain, compared to the reference strain, without any major effect on the specific growth rate.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-014-0086-z) contains supplementary material, which is available to authorized users.

The metabolic costs of improving ethanol yield by reducing glycerol formation capacity under anaerobic conditions in Saccharomyces cerevisiae.

BackgroundFinely regulating the carbon flux through the glycerol pathway by regulating the expression of the rate controlling enzyme, glycerol-3-phosphate dehydrogenase (GPDH), has been a promising approach to redirect carbon from glycerol to ethanol and thereby increasing the ethanol yield in ethanol production. Here, strains engineered in the promoter of GPD1 and deleted in GPD2 were used to investigate the possibility of reducing glycerol production of Saccharomyces cerevisiae without jeopardising its ability to cope with process stress during ethanol production. For this purpose, the mutant strains TEFmut7 and TEFmut2 with different GPD1 residual expression were studied in Very High Ethanol Performance (VHEP) fed-batch process under anaerobic conditions.ResultsBoth strains showed a drastic reduction of the glycerol yield by 44 and 61% while the ethanol yield improved by 2 and 7% respectively. TEFmut2 strain showing the highest ethanol yield was accompanied by a 28% reduction of the biomass yield. The modulation of the glycerol formation led to profound redox and energetic changes resulting in a reduction of the ATP yield (YATP) and a modulation of the production of organic acids (acetate, pyruvate and succinate). Those metabolic rearrangements resulted in a loss of ethanol and stress tolerance of the mutants, contrarily to what was previously observed under aerobiosis.ConclusionsThis work demonstrates the potential of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Previous study showed that, contrarily to anaerobiosis, the resulting gain in ethanol yield was accompanied with no loss of ethanol tolerance under aerobiosis. Moreover those mutants were still able to produce up to 90 gl-1 ethanol in an anaerobic SSF process. Fine tuning metabolic strategy may then open encouraging possibilities for further developing robust strains with improved ethanol yield.

Effects of deletion of glycerol-3-phosphate dehydrogenase and glutamate dehydrogenase genes on glycerol and ethanol metabolism in recombinant Saccharomyces cerevisiae

Bioethanol is currently used as an alternative fuel for gasoline worldwide. For economic production of bioethanol by Saccharomyces cerevisiae, formation of a main by-product, glycerol, should be prevented or minimized in order to reduce a separation cost of ethanol from fermentation broth. In this study, S. cerevisiae was engineered to investigate the effects of the sole and double disruption of NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1) and NADPH-requiring glutamate dehydrogenase 1 (GDH1) on the production of glycerol and ethanol from glucose. Even though sole deletion of GPD1 or GDH1 reduced glycerol production, double deletion of GPD1 and GDH1 resulted in the lowest glycerol concentration of 2.31 g/L, which was 46.4% lower than the wild-type strain. Interestingly, the recombinant S. cerevisiae ∆GPD1∆GDH1 strain showed a slight improvement in ethanol yield (0.414 g/g) compared with the wild-type strain (0.406 g/g). Genetic engineering of the glycerol and glutamate metabolic pathways modified NAD(P)H-requiring metabolic pathways and exerted a positive effect on glycerol reduction without affecting ethanol production.

Reduction of glycerol production to improve ethanol yield in an engineered Saccharomyces cerevisiae using glycerol as a substrate

Ethanol plays an important role in substituting the increasingly limited oil as the high-value, renewable fuel. In our previous studies, we successfully established the conversion of glycerol to ethanol by overexpression of p GcyaDak with p Gup1Cas in Saccharomyces cerevisiae. In addition to increasing ethanol production using glycerol as substrate, we minimized the synthesis of glycerol, which is the main by-product in ethanol fermentation processing. The glycerol production pathway was impaired by deletion of the genes FPS1 and GPD2. Strains deleted for both FPS1 and GPD2 reduce glycerol production and become highly sensitive to osmotic stress. We provide osmotic protection in YPH499 fps1Δgpd2Δ by overexpression of Gup1. In this study, S. cerevisiae using glycerol as substrate was modified through one-step gene disruption for redirection of glycerol carbon flux into ethanol by the deletion of two glycerol production genes, FPS1 and GPD2. The overall ethanol production in the modified strain YPH499 fps1Δgpd2Δ (p GcyaDak, p GupCas) was about 4.4 g l −1. These results demonstrate the possibility of providing protection against osmotic stress while simultaneously increasing ethanol and reducing glycerol production in S. cerevisiae strains using glycerol as a carbon source.

Using regulatory information to manipulate glycerol metabolism in Saccharomyces cerevisiae

Metabolic engineering has emerged as an attractive alternative to random mutagenesis and screening to design cell factories for industrial fermentation processes. The design of metabolic networks has been realized by gene deletions or strong overexpression of heterologous genes. There is an increasing body of evidence that indicates complete inactivation of native genes and high-level activity of heterologous enzymes may be deleterious to the cell. To moderately implement their expression, genes of interest are expressed under the control of promoters with different strengths. Constructing a promoter library is labor-intensive and requires precise quantification of the promoter strength. However, when the mechanisms of pathway regulation are known, it is possible to exploit this information to effect genetic changes efficiently. We report the implementation of this concept to reducing glycerol production during aerobic growth of Saccharomyces cerevisiae. Glycerol is produced to dispose excess cytosolic reduced nicotinamide adenine dinucleotide (NADH), and the regulating step in the pathway is mediated by glycerol 3-phosphate dehydrogenase (encoded by GPD1 and GPD2 genes). We expressed NADH oxidase in S. cerevisiae under the control of the GPD2 promoter to modulate the decrease in cytosolic NADH to the right level where the heterologous enzyme does not compete with oxidative phosphorylation while at the same time, decreasing glycerol production. This metabolic design resulted in substantially decreasing glycerol production and indeed, the excess carbon was redirected to biomass, resulting in a 14% increase in the specific growth rate. We believe that such strategies are more efficient than conventional methods and will find applications in bioprocesses.

Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway.

Glycerol 3-phosphate dehydrogenase, a key enzyme in the production of glycerol, is encoded by GPD1 and GPD2. The isoforms encoded by these genes have different functions, in osmoregulation and redox balance, respectively. We investigated the roles of GPD1, GPD2 and HOG1-the kinase involved in the response to osmotic stress-in glycerol production during wine fermentation. We found that the deletion of GPD2 in a wine yeast-derived strain did not affect growth or fermentation performance and reduced glycerol production by only 20%. In contrast, a gpd1delta mutant displayed a prolonged lag phase, and produced 40% less glycerol than the wild-type strain. The deletion of HOG1 resulted in a slight decrease in growth rate and a 20% decrease in glycerol production, indicating that the HOG pathway operates under wine fermentation conditions. However, the hog1delta mutant was not as severely affected as the gpd1delta mutant during the first few hours of fermentation, and continued to express GPD1 strongly. The hog1delta mutant was able to increase glycerol production in response to high sugar concentration (15-28% glucose), to almost the same extent as the wild-type, whereas this response was totally abolished in the gpd1delta mutant. These data show that Gpd1p plays a major role in glycerol formation, particularly during the first few hours of exposure to high sugar concentration, and that GPD2 is only of little significance in anaerobic fermentation by wine yeast. The results also demonstrate that the HOG pathway exerts only limited control over GPD1 expression and glycerol production during wine fermentation.

Metabolism of adipose tissue from normal and hypothyroid rats.

The metabolism of adipose tissue from normal and hypothyroid rats was studied in vitro. Oxygen consumption was reduced in fat cells obtained from thyroidectomized rats, but the oxidation of 14C-labeled glucose, pyruvate and acetate proceeded at normal rates. The oxidation of palmitate-1-14C to 14CO2, however, was significantly reduced following thyroidectomy. Synthesis of long-chain fatty acids from a variety of precursors was also reduced in the thyroprivic tissues. In addition to the wellknown reduction in FFA release, thyroidectomy also reduced glycerol production by adipose tissue. Thus, thyroidectomy interferes with the conversion of 2 carbon units to long-chain fatty acids and with the conversion of long-chain fatty acids to 2 carbon fragments. It is suggested that abnormalities in the function of mitochondrial membranes may account for the metabolic defects of thyroidectomy in adipose tissue. (Endocrinology 82: 860, 1968)