ABSTRACT
The objective of this study was to investigate the mechanism underlying LW-1-induced resistance to TMV in wild-type and salicylic acid (SA)-deficient NahG transgenic tobacco plants. Our findings revealed that LW-1 failed to induce antivirus infection activity and increase SA content in NahG tobacco, indicating the crucial role of SA in these processes. Meanwhile, LW-1 triggered defense-related early-signaling nitric oxide (NO) generation, as evidenced by the emergence of NO fluorescence in both types of tobacco upon treatment with LW-1, however, NO fluorescence was stronger in NahG compared to wild-type tobacco. Notably, both of them were eliminated by the NO scavenger cPTIO, which also reversed LW-1-induced antivirus activity and the increase of SA content, suggesting that NO participates in LW-1-induced resistance to TMV, and may act upstream of the SA pathway. Defense-related enzymes and genes were detected in tobacco with or without TMV inoculation, and the results showed that LW-1 regulated both enzyme activity (ß-1,3-glucanase [GLU], catalase [CAT] and phenylalanine ammonia-lyase [PAL]) and gene expression (PR1, PAL, WYKY4) through NO signaling in both SA-dependent and SA-independent pathways.
Subject(s)
Disease Resistance , Nicotiana , Nitric Oxide , Plant Diseases , Salicylic Acid , Tobacco Mosaic Virus , Nicotiana/metabolism , Nicotiana/genetics , Salicylic Acid/metabolism , Salicylic Acid/pharmacology , Nitric Oxide/metabolism , Plants, Genetically Modified , Plant Proteins/metabolism , Plant Proteins/genetics , Signal Transduction , Gene Expression Regulation, Plant/drug effectsABSTRACT
Spent biomass from agricultural and forestry industries are substantial low-cost carbon source for reducing the input of microbial lipid production. Herein, the components of the vineyard winter prunings (VWPs) from 40 grape cultivars were analyzed. The VWPs contained (w/w) cellulose ranged from 24.8% to 32.4%, hemicellulose 9.6% to 13.8%, lignin 23.7% to 32.4%. The VWPs from Cabernet Sauvignon was processed with the alkali-methanol pretreatment, and 95.8% of the sugars was released from the regenerated VWPs after enzymatic hydrolysis. The hydrolysates from the regenerated VWPs was suitable for lipid production without further treatment as a lipid content of 59% could be achieved with Cryptococcus curvatus. The regenerated VWPs was also used for lipid production via simultaneous saccharification and fermentation (SSF), which led to a lipid yield of 0.088 g/g raw VWPs, 0.126 g/g regenerated VWPs and 0.185 g/g from the reducing sugars. This work demonstrated that the VWPs can be explored for co-production of microbial lipids.
Subject(s)
Cryptococcus , Lipids , Farms , Fermentation , Sugars , BiomassABSTRACT
The medium-chain fatty acid ethyl esters (MCFAEEs) are a group of important aroma compounds generated during wine production. Wine alcohol fermentation involves several redox processes, which are affected by the oxidation-reduction potential (ORP). However, the mechanism via which ORP regulates MCFAEE production remains unclear. To investigate the effect of ORP on MCFAEE production, wine alcohol fermentation was performed using Saccharomyces cerevisiae under different ORPs. The results demonstrated that the ORPs studied (except for 90 mV) did not significantly affect cell growth, sugar consumption, and ethanol production, while the MCFAEE concentration in the simulated wines can be manipulated by ORP operation. MCFAEE levels increased till 96 h, and then decreased. The maximum MCFAEE level of 1222.97 µg/L was obtained after 96 h at 0 mV, which was 45.32% higher than that of the control. During the increase, higher relative expression of ACC1, FAS1, FAA2 and EEB1, elevated external citric acid flux, and moderate intracellular NADP+/NADPH ratio were observed at 0 mV compared to that at other ORPs. During the decrease, lowest relative expression of POX1 was detected at 0 mV. We showed for the first time the relationship between ORP operation and MCFAEE production in winemaking, which will improve the aroma quality of wine.
Subject(s)
Wine , Esters/analysis , Fatty Acids/metabolism , Fermentation , Oxidation-Reduction , Saccharomyces cerevisiae/metabolism , Wine/analysisABSTRACT
ß-Glucosidase is a key enzyme that hydrolyzes nonvolatile glycosylated precursors of aroma compounds and enhances the organoleptic quality of wines. In this study, a novel ß-glucosidase from Hanseniaspora uvarum Yun268 was localized, purified, and characterized. Results indicated that ß-glucosidase activity was mainly distributed within the cells. After purification via ammonium sulfate precipitation combined with chromatography, ß-glucosidase specific activity increased 8.36 times, and the activity recovery was 56.90%. The enzyme had a molecular mass of 74.22 kDa. It has a Michaelis constant (Km ) of 0.65 mmol/L, and a maximum velocity (Vmax ) of 5.1 nmol/min under optimum conditions; and Km of 0.94 mmol/L, and Vmax of 2.8 nmol/min under typical winemaking conditions. It exhibited the highest activity at 50°C and pH 5.0 and was stable at a temperature range of 20-80°C and pH range of 3.0-8.0. The enzyme has good tolerance to Fe3+ , especially maintaining 93.68% of its activity with 10 mmol/L of Fe3+ . Ethanol (<20%) and glucose (<150 g/L) inhibited its activity only slightly. Therefore, ß-glucosidase from H. uvarum Yun268 has excellent biochemical properties and a good application potential in winemaking. PRACTICAL APPLICATION: Winemaking is a biotechnological process in which exogenous ß-glucosidase is used to overcome the deficiency of endogenous ß-glucosidase activity in grapes. By localizing, purifying, and characterizing of ß-glucosidase from Hanseniaspora uvarum Yun268, it is expected to reveal its physical and chemical characteristics to evaluate its oenological properties in winemaking. The results may provide the basis for promoting the release of varietal aroma and improving wine sensory quality in the wine industry.
Subject(s)
Hanseniaspora , Wine , Fermentation , Odorants/analysis , Wine/analysis , beta-Glucosidase/metabolismABSTRACT
BACKGROUND: Limonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc. The objective of this work was to engineer Rhodosporidium toruloides as a cell factory for the production of limonene. RESULTS: By overexpressing the limonene synthase (LS), neryl pyrophosphate synthase (NPPS)/geranyl pyrophosphate synthase and the native hydroxy-methyl-glutaryl-CoA reductase (HMGR), we established a baseline for limonene production based on the mevalonate route in Rhodosporidium toruloides. To further enhance the limonene titer, the acetoacetyl-CoA thiolase/HMGR (EfMvaE) and mevalonate synthase (EfMvaS) from Enterococcus faecalis, the mevalonate kinase from Methanosarcina mazei (MmMK) and the chimeric enzyme NPPS-LS were introduced in the carotenogenesis-deficient strain. The resulting strains produced a maximum limonene titer of 393.5 mg/L. CONCLUSION: In this study, we successfully engineered the carotenogenesis yeast R. toruloides to produce limonene. This is the first report on engineering R. toruloides toward limonene production based on NPP and the fusion protein SltNPPS-CltLS. The results demonstrated that R. toruloides is viable for limonene production, which would provide insights into microbial production of valuable monoterpenes.
ABSTRACT
Limonene and its derivatives have great market potential with diverse applications in food, pharmaceuticals, cosmetics, etc. Commercial production of limonene and its derivatives through extraction from plants suffers from the unstable market supply, while chemical synthesis of these compounds is hindered by high energy consumption and pollutant emission. Microbial biosynthesis provides a promising alternative approach for the sustainable supply of limonene and its derivatives. However, low efficiency and specificity of the biosynthetic enzymes and pathways in heterologous hosts make it still challenging for the commercialization of microbial limonene production. On the other hand, the limonene toxicity heavily reduces cellular fitness, which poses a serious challenge for improving limonene titer. Here, we critically review the recent progresses in engineering microbes for limonene biosynthesis and derivation with the emphasis on enzyme characterization and pathway optimization. In particular, we introduce the current trends in microbial limonene decoration for the biosynthesis of bio-active molecules such as α-terpineol and perillyl alcohol. We also discuss the feasible strategies for relieving limonene toxicity and enhancing the robustness of microbial cell factories.
Subject(s)
Cyclohexenes , Metabolic Engineering , LimoneneABSTRACT
The dynamic changes of wine ester production during mixed fermentation with Hanseniaspora uvarum Yun268 and Saccharomyces cerevisiae F5 was investigated at different levels and timings of nitrogen nutrient addition. Nitrogen additions were performed by supplementing yeast assimilable nitrogen (YAN) into a synthetic grape must with defined composition. Ester precursors and extracellular metabolites involved in ester synthesis were analyzed throughout the fermentation. Results showed that nitrogen additions covering 50-200â¯mg/L YAN at the point of yeast inoculation slightly affected yeast competition and ester profiles. Interestingly, when YAN was supplemented in the mid-stage, the survival of H. uvarum Yun268 was enhanced, resulting in more than a 2-fold increase in the levels of higher alcohol acetates compared to that at the initial stage. Furthermore, carbon fluxes may be redistributed in the central pathway, which contributed to the production of medium-chain fatty acids and eventually triggered a 1.2-fold elevation in corresponding ethyl ester levels.
Subject(s)
Esters/analysis , Fermentation , Hanseniaspora/metabolism , Nitrogen/metabolism , Saccharomyces cerevisiae/metabolism , Acetic Acid/analysis , Citric Acid/analysis , Food Microbiology , Malates/analysis , Succinic Acid/analysis , Vitis/chemistry , Volatile Organic Compounds/analysis , Wine/analysisABSTRACT
The dynamic pattern of volatiles during Pinot Noir winemaking in monsoon climate with yeast extracellular extract (EE) treatment was analyzed. EE from selected Pichia fermentans and Rhodotorula mucilaginosa strains, and almond ß-glucosidase were added after 12-h alcohol fermentation, and the volatiles were determined every 24â¯h by GC-MS. After 6-month storage, wine aroma was evaluated instrumentally as well as by well-trained panelists. Results showed that enzyme treatments improved the contents of both varietal and fermentative volatiles. The levels of C6 compounds, terpenes, and higher alcohols increased constantly during alcohol fermentation, whereas acetates, short and medium chain fatty acid ethyl esters, phenylethyls, and fatty acids increased first, followed by gradual decrease. EE treatment retarded the decrease of fruity ester content in wine. Mathematical regression between wine aroma and volatiles showed that the relatively higher contents of acetates, ethyl esters, and C13-norisoprenoids in 6-month EE-treated wine were responsible for the improvement in floral aroma intensity.
Subject(s)
Fermentation , Fruit/chemistry , Volatile Organic Compounds/analysis , Wine/analysis , Wine/microbiology , Alcohols/analysis , Climate , Fatty Acids/analysis , Odorants/analysis , Pichia , Rhodotorula/metabolism , Saccharomyces cerevisiae/metabolism , Terpenes/analysis , Vitis , Yeasts/enzymology , Yeasts/metabolism , beta-GlucosidaseABSTRACT
The chemical profiles and aroma contribution of terpene compounds in Meili grapes and wine were analyzed. Bound terpene compounds were extracted using methanol, purified using Amberlite XAD-2 resin, concentrated in methanol/ethyl acetate, and enzymatically hydrolyzed to release aglycones. Free terpene compounds were identified using solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Wine aroma characteristics were quantified by a trained sensory panel. Seventeen terpene glycosides were quantified in grapes and wines as pentosyl-glucopyranoside, the content of which ranged from 804 to 836⯵g/kg, and from 155 to 192⯵g/L, respectively. Eight free terpenes were present in wines with their content ranging from 40.1 to 59.7⯵g/L. Linalool was abundant both in bound and free terpenes, and mathematical regression revealed that terpenes, especially linalool (contribution efficientâ¯>â¯0.4), contributed heavily to Meili wine aroma. Finally, a molecular rearrangement scheme based on linalool was proposed in Meili grape and wine.
Subject(s)
Fruit/chemistry , Terpenes/chemistry , Vitis/chemistry , Wine/analysis , Gas Chromatography-Mass Spectrometry/methods , Odorants/analysis , Solid Phase MicroextractionABSTRACT
A novel analytical method was developed for the determination of organic acids (formic acid, acetic acid and propionic acid) in 1, 2-butylene oxide (1, 2-BO) products by valve switch-ion chromatography (IC).The samples were diluted in ethanol, and then were eluted from a concentrator column (IonPac TAC-ULP1) to an analytical column (IonPac AS11).The extracts were detected by a suppressed conductivity detector.Formic acid, acetic acid and propionic acid were separated well.Good linear relationships for the three organic acids were obtained.The spiked recoveries of the three organic acids in the samples were in the range of 92.5%-111.8%.The relative standard deviations (RSDs) were less than 5.6%(n=3).The limits of detection (LODs, S/N=3) of the formic acid, acetic acid and propionic acid were 0.60-4.80 µg/L.The method is simple, rapid, and accurate, and is suitable for the determination of the organic acids in an insoluble organic system.
ABSTRACT
Wine aroma response to a selected Hanseniaspora uvarum Yun268 strain was investigated using different inoculation strategies with commercial Saccharomyces cerevisiae yeast, namely, simultaneous fermentation (SiF), sequential fermentation (SeF), S. cerevisiae fermentation treated with extracellular extract of H. uvarum (EE), and pure S. cerevisiae fermentation (PF). Contributive volatiles in the perception of enhanced aroma traits were uncovered by partial least-squares regression. Results showed that controlled inoculation resulted into different amounts of H. uvarum Yun268, which distinctively affected the chemical and sensory profiles of wines. The concentration of aromatic compounds could be increased by H. uvarum Yun268 yeasts via high levels of ß-glucosidase activity and fatty acids. Terpenes, C13-norisoprenoids, acetate esters, ethyl esters, and fatty acids served as the impact volatiles that contributed to the enhanced aroma traits. SiF specifically increased the contents of C13-norisoprenoids, terpenes, and ethyl esters, while EE enhanced varietal volatile content rather than those of fermentative ones. However, excessive H. uvarum Yun268 in sequential inoculation elevated the concentrations of acetate esters and volatile phenols, triggering nail polish odor in Cabernet Sauvignon red wines.
Subject(s)
Fermentation , Food Microbiology/methods , Fruit/microbiology , Hanseniaspora/metabolism , Odorants/analysis , Saccharomyces cerevisiae/metabolism , Smell , Vitis/microbiology , Volatile Organic Compounds/analysis , Wine/microbiology , Ethanol/metabolism , Food Analysis , Humans , Judgment , Least-Squares Analysis , Olfactory Perception , Time FactorsABSTRACT
Medium-chain fatty acid (MCFA) ethyl esters, as yeast secondary metabolites, significantly contribute to the fruity aroma of foods and beverages. To improve the MCFA ethyl ester content of wine, mixed fermentations with Hanseniaspora uvarum Yun268 and Saccharomyces cerevisiae were performed. Final volatiles were analyzed by gas solid phase microextraction-chromatography-mass spectrometry, and aroma characteristics were quantitated by sensory analysis. Results showed that mixed fermentation increased MCFA ethyl ester content by 37% in Cabernet Gernischt wine compared to that obtained by pure fermentation. Partial least-squares regression analysis further revealed that the improved MCFA ethyl esters specifically enhanced the temperate fruity aroma of wine. The enhancement of MCFA ethyl esters was attributed to the increased contents of MCFAs that could be induced by the presence of H. uvarum Yun268 in mixed fermentation. Meanwhile, the timing of yeast inoculations significantly affected the involving biomass of each strain and the dynamics of ethanol accumulation.
Subject(s)
Hanseniaspora , Saccharomyces cerevisiae , Fatty Acids , Fermentation , Flavoring Agents , Humans , WineABSTRACT
Microbial lipids produced by oleaginous yeasts serve as promising alternatives to traditional oils and fats for the production of biodiesel and oleochemicals. To improve its techno-economics, it is pivotal to use wastes and produce high quality lipids of special fatty acid composition. In the present study, four oleaginous yeasts were tested to use free fatty acids for lipid production under non-growth conditions. Microbial lipids of exceptionally high fatty acid relative contents, e.g. those contained over 70% myristic acid or 80% oleic acid, were produced that may be otherwise inaccessible by growing cells on various carbon sources. It was found that Cryptococcus curvatus is a robust strain that can efficiently use oleic acid as well as even-numbered saturated fatty acids with carbon atoms ranging from 10 to 20. Our results provided new opportunity for the production of functional lipids and for the exploitation of organic wastes rich in free fatty acids.
Subject(s)
Fatty Acids, Nonesterified/biosynthesis , Yeasts/growth & development , Yeasts/metabolism , Chromatography, Thin LayerABSTRACT
To reduce wastes and the costs of microbial lipid production, it is imperative to recycle resources, including spent cell mass, mineral nutrients and water. In the present study, lipid production by the oleaginous yeast Rhodosporidium toruloides was used as a model system to demonstrate resources recycling. It was found that the hydrolysates of spent cell mass were good media to support cell growth of various oleaginous yeasts. When serial repitching experiments were performed using 70g/L glucose and the hydrolysates alone as nutrients, it produced 16.6, 14.6 and 12.9g/L lipids, for three successive cycles, while lipid titre remained almost constant when spent water was also recycled. The cell mass hydrolysates could be used as equivalents to the mixture of yeast extract and peptone to support lipid production from corn stalk hydrolysates. Our results showed efficient recycling of lipid production wastes and should be helpful to advance microbial lipid technology.
Subject(s)
Lipids/biosynthesis , Waste Products , Yeasts/metabolism , Basidiomycota/metabolism , Glucose/metabolism , Recycling , WastewaterABSTRACT
With ever-increasing culture of yeasts for the production of biofuels and other metabolites, spent yeast cell mass exceeds its traditional market demands. Yeast cell mass contains glucose, mannose and other sugars that may be utilized for microbial culture. Here we demonstrated that the oleaginous yeast Lipomyces starkeyi could utilize glucose and mannose simultaneously for lipid production. Overall substrate consumption rates and lipid coefficients were 0.58 g/L/h and 0.18 g lipid/g sugar, respectively, in flask cultures regardless of glucose, mannose or a mixture of both as the carbon source. L. starkeyi grew well on the hydrolysates of spent cell mass of Rhodosporidium toruloides, consumed both glucose and mannose therein, and produced lipid at a yield of 0.12 g lipid/g total reducing sugars. This co-utilization strategy expands carbon sources for lipid production. It should provide an opportunity for recycling spent cell mass and be of significant interests to biorefinery and biofuel production.
Subject(s)
Glucose/metabolism , Lipids/biosynthesis , Lipomyces/metabolism , Mannose/metabolism , Fatty Acids/metabolismABSTRACT
Fatty acid ethyl esters (FAEEs) are attractive biofuel molecules. Conventional FAEEs production process uses triglycerides and ethanol as feedstocks and is sensitive to water contents. In this work, we show that the oleaginous yeast Rhodosporidium toruloides cells are capable of converting lipids into FAEEs intracellularly in aqueous phase. Up to 73% of cellular neutral glycerides could be converted into FAEEs when lipid-rich cells were incubated for 84 h at 35°C, pH 6.0 in a broth containing 10 vol% ethanol. It was found that neutral glycerides were first hydrolyzed to free fatty acids followed by esterification and that lipid droplets played important roles in the process. This new process provides a novel opportunity for integration of microbial lipid production technology with bioethanol fermentation for more efficient production of drop-in biofuels from renewable resources.
Subject(s)
Basidiomycota/metabolism , Esters/metabolism , Fatty Acids/biosynthesis , Ethanol/metabolism , Fermentation , Magnetic Resonance Spectroscopy , Time FactorsABSTRACT
Microbial lipids are potential alternative feedstock for biofuel and oleochemical industries. The oleaginous yeast Rhodosporidium toruloides AS 2.1389 is an excellent lipid producer. To attain parameters for the understanding of the lipid production process, we performed continuous cultivation experiments under either carbon or nitrogen limitation. The maintenance coefficient and maximum cell mass yield for this yeast were determined as 5.7 mg glucose/g cell/h and 0.42 g cell/g glucose, respectively, under carbon limitation. Under nitrogen limitation, the highest lipid yield of 0.19 g/g was observed at the dilution rate of 0.02 h(-1) while the highest specific lipid formation rate of 0.058 g/g cell/h at the dilution rate of 0.08 h(-1). A kinetic model of lipid formation under steady state conditions was developed, parameters estimated, and optimal continuous cultivation conditions forecasted. These data should be very helpful to develop and design more efficient bioprocesses for microbial lipid production.
Subject(s)
Basidiomycota/cytology , Basidiomycota/metabolism , Carbon/metabolism , Lipid Metabolism , Nitrogen/metabolismABSTRACT
Lignocellulosic biomass pretreatment with ionic liquids (ILs) has been emerged as a new technology, but the effects of residual ILs on the downstream biotransformation remain largely unknown. Here, three typical ILs were tested for their effects on lipid production by the oleaginous yeast Rhodosporidium toruloides AS 2.1389. When cultures were maintained at pH 6.0 in the presence of 30mM ILs, [Emim]Cl, [Emim][DEP], or [Emim][OAc], minor inhibition effects were observed. When cultures were performed in the presence of 60mM ILs or without pH control, inhibition was largely dependent on ILs. Detailed analysis indicated that the anion of [Emim][OAc] was assimilated, leading to a rapid alkaline-pH shift and enhanced inhibition on cell growth and lipid production. Our results demonstrated that R. toruloides is a robust lipid producer tolerating ILs at low concentrations, and that care should be taken in bioprocess control and data analysis when ILs are involved.
Subject(s)
Biofuels , Ionic Liquids/toxicity , Lipid Metabolism , Yeasts/drug effects , Biomass , Yeasts/growth & development , Yeasts/metabolismABSTRACT
To evaluate the effectiveness of enzymatic assisted extraction (EAE) of lipid from the oleaginous yeast Rhodosporidium toruloides in the presence of beta-1,3-glucomannanase at a larger scale, we investigated the effects of enzymatic treatment and extraction conditions on lipid extraction yields at 10-L scale by using the broth of R. toruloides Y4 as the feed and ethyl acetate as the solvent. When it was treated for 0.5 h, the lipid extraction yield reached 71.1%, indicating that the enzymatic treatment process reached similar efficiency to that obtained at 10-mL scale. The inhibitory effect of emulsification was greatly reduced by repeated extraction. After extracted for three times, yields of lipid extraction, solvent recovery and total material recovery reached 92.9%, 87.0% and 94.2% respectively. As it can use the lipid production slurry with good extraction efficiency, EAE technology is promising for industrial production of microbial lipids.
Subject(s)
Basidiomycota/metabolism , Biofuels , Lipids/isolation & purification , beta-Mannosidase/metabolism , Bioreactors , Fermentation , Industrial Microbiology , Lipids/biosynthesisABSTRACT
Triacylglycerols are among the most attractive alternative raw materials for biofuel development. Current oil plant-based technologies are limited in terms of triacylglycerol production capacity and rate. These limitations may be circumvented by biotransformation of carbohydrates into lipids; however, our understanding of microbial oleaginicity remains limited. Here we present the results of a multi-omic analysis of Rhodosporidium toruloides, a robust triacylglycerol-producing fungus. The assembly of genome and transcriptome sequencing data reveals a genome of 20.2 Mb containing 8,171 protein-coding genes, the majority of which have multiple introns. Genes including a novel fatty acid synthase are predicted to participate in metabolic pathways absent in non-oleaginous yeasts. Transcriptomic and proteomic data suggest that lipid accumulation under nitrogen-limited conditions correlates with the induction of lipogenesis, nitrogenous compound recycling, macromolecule metabolism and autophagy. The multi-omic map of R. toruloides therefore provides a valuable resource for efforts to rationally engineer lipid-production pathways.