Fermentation process for producing CFAs using Yarrowia lipolytica.

Past research has sought to improve the production of cyclopropane fatty acids by the oleaginous yeast Yarrowia lipolytica by heterologously expressing the E. coli fatty acid synthase gene and improving cultivation processes. Cyclopropane fatty acids display properties that hold promise for biofuel applications. The E. coli fatty acid synthase gene was introduced into several genetic backgrounds of the yeast Y. lipolytica to optimize lipid synthesis; the mean cyclopropane fatty acid productivity was 43 mg L-1 h-1 on glucose, and the production rate reached its maximum (3.06 g L-1) after 72 h of cultivation in a bioreactor. The best strain (JMY6851) overexpressed simultaneously the E. coli cyclopropane fatty acid synthase gene under a hybrid promoter (hp8d) and Y. lipolytica LRO1 gene. In fed-batch process using crude glycerol as carbon source, JMY6851 strain displayed high lipid accumulation (78% of dry cell weight) and high biomass production (56 g L-1). After 165 h of cultivation, cyclopropane fatty acids represented 22% of the lipids produced; cyclopropane fatty acid productivity (103.3 mg L-1 h-1) was maximal at 72.5 h of cultivation.

Optimization of cyclopropane fatty acids production in Yarrowia lipolytica.

Cyclopropane fatty acids, which can be simply converted to methylated fatty acids, are good unusual fatty acid candidates for long-term resistance to oxidization and low-temperature fluidity useful for oleochemistry and biofuels. Cyclopropane fatty acids are present in low amounts in plants or bacteria. In order to develop a process for large-scale biolipid production, we expressed 10 cyclopropane fatty acid synthases from various organisms in the oleaginous yeast Yarrowia lipolytica, a model yeast for lipid metabolism and naturally capable of producing large amounts of lipids. The Escherichia coli cyclopropane fatty acid synthase expression in Y. lipolytica allows the production of two classes of cyclopropane fatty acids, a C17:0 cyclopropanated form and a C19:0 cyclopropanated form, whereas others produce only the C17:0 form. Expression optimization and fed-batch fermentation set-up enable us to reach a specific productivity of 0.032 g·L-1 ·hr-1 with a genetically modified strain containing cyclopropane fatty acid up to 45% of the total lipid content corresponding to a titre of 2.3 ± 0.2 g/L and a yield of 56.2 ± 4.4 mg/g.

Evaluation of performance and validity limits of gas chromatography electron ionisation with Orbitrap detection for fatty acid methyl ester analyses.

RATIONALE: While the GC-Orbitrap, marketed in 2015, represents a technological breakthrough in terms of sensitivity, resolution and mass stability, many studies have reported ion ratio modification in mass spectra using the standard 70 eV electron ionisation. METHODS: We studied the influence of the acquisition and sample parameters leading to these modifications on fatty acid methyl esters (FAMEs). RESULTS: FAMEs showed that these variations in relative intensities of ions were related to the acquisition parameters such as the mass range and the offset values of the C-TRAP, but also directly related to the column concentration of the sample, and especially that it was molecule-dependent. Advantageously, it is possible to use this feature to promote the molecular ions of FAMEs sometimes not present in a spectrum under electron ionisation at 70 eV. CONCLUSIONS: The 70 eV electron ionisation mass spectra from the GC-Orbitrap were clearly molecule-dependent and could be due to metastable ions during storage states in the C-TRAP.

Glycerolipid analysis during desiccation and recovery of the resurrection plant Xerophyta humilis (Bak) Dur and Schinz.

Xerophyta humilis is a poikilochlorophyllous monocot resurrection plant used as a model to study vegetative desiccation tolerance. Dehydration imposes tension and ultimate loss of integrity of membranes in desiccation sensitive species. We investigated the predominant molecular species of glycerolipids present in root and leaf tissues, using multiple reaction monitoring mass spectrometry, and then analysed changes therein during dehydration and subsequent rehydration of whole plants. The presence of fatty acids with long carbon chains and with odd numbers of carbons were detected and confirmed by gas chromatography. Dehydration of both leaves and roots resulted in an increase in species containing polyunsaturated fatty acids and a decrease in disaturated species. Upon rehydration, lipid saturation was reversed, with this being initiated immediately upon watering in roots but only 12-24 hr later in leaves. Relative levels of species with short-chained odd-numbered saturated fatty acids decreased during dehydration and increased during rehydration, whereas the reverse trend was observed for long-chained fatty acids. X. humilis has a unique lipid composition, this report being one of the few to demonstrate the presence of odd-numbered fatty acids in plant phosphoglycerolipids.

Analysis of 13C labeling amino acids by capillary electrophoresis - High resolution mass spectrometry in developing flaxseed.

In context of fluxomic studies, 13C labeling analysis of amino acids are very important for solving the carbon flow calculation, because they are synthesized in various biosynthesis pathways and cellular compartments in plant cells. Traditionally, 13C labeling analysis are performed using low resolution mass spectrometry detector by GC-MS. We compared a method using capillary electrophoresis-high resolution mass spectrometry without derivatization and with better accuracy assessment of labeling measurements comparing to classical GC-MS. Our method allowed us to show that valine, leucine, alanine are not synthesized from the same pyruvate pool during the period of reserves accumulation in flax seeds.

13C labeling analysis of sugars by high resolution-mass spectrometry for metabolic flux analysis.

Metabolic flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography-high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6].

A metabolic engineering strategy for producing conjugated linoleic acids using the oleaginous yeast Yarrowia lipolytica.

Conjugated linoleic acids (CLAs) have been found to have beneficial effects on human health when used as dietary supplements. However, their availability is limited because pure, chemistry-based production is expensive, and biology-based fermentation methods can only create small quantities. In an effort to enhance microbial production of CLAs, four genetically modified strains of the oleaginous yeast Yarrowia lipolytica were generated. These mutants presented various genetic modifications, including the elimination of ß-oxidation (pox1-6∆), the inability to store lipids as triglycerides (dga1∆ dga2∆ are1∆ lro1∆), and the overexpression of the Y. lipolytica ∆12-desaturase gene (YlFAD2) under the control of the constitutive pTEF promoter. All strains received two copies of the pTEF-oPAI or pPOX-oPAI expression cassettes; PAI encodes linoleic acid isomerase in Propionibacterium acnes. The strains were cultured in neosynthesis or bioconversion medium in flasks or a bioreactor. The strain combining the three modifications mentioned above showed the best results: when it was grown in neosynthesis medium in a flask, CLAs represented 6.5% of total fatty acids and in bioconversion medium in a bioreactor, and CLA content reached 302 mg/L. In a previous study, a CLA degradation rate of 117 mg/L/h was observed in bioconversion medium. Here, by eliminating ß-oxidation, we achieved a much lower rate of 1.8 mg/L/h.

Arabidopsis seeds altered in the circadian clock protein TOC1 are characterized by higher level of linolenic acid.

The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds.

PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics.

BACKGROUND: Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics. RESULTS: A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species. CONCLUSIONS: We have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax.

Highlighting the tricarboxylic acid cycle: liquid and gas chromatography-mass spectrometry analyses of (13)C-labeled organic acids.

The tricarboxylic acid (TCA) cycle is involved in the complete oxidation of organic acids to carbon dioxide in aerobic cells. It not only uses the acetyl-CoA derived from glycolysis but also uses breakdown products of proteins, fatty acids, and nucleic acids. Therefore, the TCA cycle involves numerous carbon fluxes through central metabolism to produce reductant power and transfer the generated electrons to the aerobic electron transport system where energy is formed by oxidative phosphorylation. Although the TCA cycle plays a crucial role in aerobic organisms and tissues, the lack of direct isotopic labeling information in its intermediates (organic acids) makes the quantification of its metabolic fluxes rather approximate. This is the major technical gap that this study intended to fill. In this work, we established and validated liquid and gas chromatography-mass spectrometry methods to determine (13)C labeling in organic acids involved in the TCA cycle using scheduled multiple reaction monitoring and single ion monitoring modes, respectively. Labeled samples were generated using maize embryos cultured with [(13)C]glucose or [(13)C]glutamine. Once steady-state labeling was reached, (13)C-labeled organic acids were extracted and purified. When applying our mass spectrometric methods to those extracts, mass isotopomer abundances of seven major organic acids were successfully determined.

Gas chromatography-mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis.

Metabolic flux analysis, using 13C labeled substrates, has become a powerful methodology for quantifying intracellular fluxes. Most often, analysis is restricted to nuclear magnetic resonance or mass spectrometry measurement of 13C label incorporation into protein amino acids. However, amino acid isotopomer distribution insufficiently covers the entire network of central metabolism, especially in plant cells with highly compartmented metabolism, and analysis of other metabolites is required. Analysis of label in saccharides provides complementary data to better define fluxes around hexose, pentose, and triose phosphate pools. Here, we propose a gas chromatography-mass spectrometry (GC-MS) method to analyze 13C labeling in glucose and fructose moieties of sucrose, free glucose, fructose, maltose, inositol, and starch. Our results show that saccharide labeling for isotopomer quantification is better analyzed by chemical ionization than by electron ionization. The structure of the generated fragments was simulated and validated using labeled standards. The method is illustrated by analysis of saccharides extracted from developing rapeseed (Brassica napus L.) embryos. It is shown that glucose 6-phosphate isomerase and plastidial glucose 6-phosphate transport reactions are not at equilibrium, and light is shed on the pathways leading to fructose, maltose, and inositol synthesis.

An in silico compartmentalized metabolic model of Brassica napus enables the systemic study of regulatory aspects of plant central metabolism.

Biochemical network reconstructions represent valuable tools for the computational metabolic modeling of organisms that present a great biotechnological interest. An in silico multi-compartmental model of the central metabolism of the plant Brassica napus (Rapeseed) was constructed, aiming to investigate the metabolic properties of the Brassicaceae family. This family comprises many plants with major importance for the energy and nutrition sector, including the model plant Arabidopsis thaliana. The model utilized as objective function to be subsequently optimized, the biomass production of rapeseed developing embryos, which are characterized by a very high, oil content, up to 60% of biomass weight. In order to study global network properties of seed metabolism, various methods were employed, like Flux Balance Analysis, Principal Component Analysis of the flux space and reaction deletion studies, which simulate the effect of gene knock-out experiments. The model successfully simulated seed growth during the stage of oil accumulation and provided insight, regarding certain aspects of network plasticity, with the emphasis given in lipid biosynthesis regulation.

A water-soluble polysaccharide from Anethum graveolens seeds: Structural characterization, antioxidant activity and potential use as meat preservative.

A novel water-soluble polysaccharide named AGP1 was successfully isolated from seeds of Anethum graveolens by hot water extraction and further purified by DEAE-Sepharose chromatography. AGP1 has a relative molecular weight of 2.1 104 Da determined by Ultra-high-performance liquid chromatography (UHPLC). The AGP1 characterization was investigated by chemical and instrumental analysis including gas chromatography mass spectrometry (GC-MS), Fourier transform infrared (FT-IR) spectroscopy and X-ray diffraction. Results showed that AGP1 was mainly composed of glucose, galactose, mannose and arabinose in a molar percent of 54.3, 23.8, 14.7 and 7.2, respectively. The thermogravimetry analysis (TGA) and the differential scanning calorimetry (DSC) were used and showed that AGP1 has good thermal stability until 275 °C. Moreover, the purified polysaccharide demonstrated an appreciable in vitro antioxidant potential. The addition of the AGP1, particularly at 0.3% (w/w), in turkey sausages instead of ascorbic acid, as preservative, reduced the lipid peroxidation, preserved the pH and color and improved the bacterial stability during cold storage at 4 °C for 12 days. Overall, the results showed that the AGP1 deserves to be developed as functional and bioactive components for the food and nutraceutical industries.

Characterization of Helianthus annuus Lipoic Acid Biosynthesis: The Mitochondrial Octanoyltransferase and Lipoyl Synthase Enzyme System.

Lipoic acid (LA, 6,8-dithiooctanoic acid) is a sulfur containing coenzyme essential for the activity of several key enzymes involved in oxidative and single carbon metabolism in most bacteria and eukaryotes. LA is synthetized by the concerted activity of the octanoyltransferase (LIP2, EC 2.3.1.181) and lipoyl synthase (LIP1, EC 2.8.1.8) enzymes. In plants, pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase or glycine decarboxylase are essential complexes that need to be lipoylated. These lipoylated enzymes and complexes are located in the mitochondria, while PDH is also present in plastids where it provides acetyl-CoA for de novo fatty acid biosynthesis. As such, lipoylation of PDH could regulate fatty acid synthesis in both these organelles. In the present work, the sunflower LIP1 and LIP2 genes (HaLIP1m and HaLIP2m) were isolated sequenced, cloned, and characterized, evaluating their putative mitochondrial location. The expression of these genes was studied in different tissues and protein docking was modeled. The genes were also expressed in Escherichia coli and Arabidopsis thaliana, where their impact on fatty acid and glycerolipid composition was assessed. Lipidomic studies in Arabidopsis revealed lipid remodeling in lines overexpressing these enzymes and the involvement of both sunflower proteins in the phenotypes observed is discussed in the light of the results obtained.

Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray.

BACKGROUND: Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. RESULTS: Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties. CONCLUSION: All results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues.

Impact of sunflower (Helianthus annuus L.) plastidial lipoyl synthases genes expression in glycerolipids composition of transgenic Arabidopsis plants.

Lipoyl synthases are key enzymes in lipoic acid biosynthesis, a co-factor of several enzyme complexes involved in central metabolism. Plant pyruvate dehydrogenase complex (PDH), located in mitochondria and plastids, catalyses the first step of fatty acid biosynthesis in these organelles. Among their different components, the E2 subunit requires the lipoic acid prosthetic group to be active. De novo lipoic acid biosynthesis is achieved by the successive action of two enzymes on octanoyl-ACP: octanoyltransferase (LIP2) and lipoyl synthase (LIP1). In this study, two plastidial lipoyl synthase genes from sunflower (Helianthus annuus L.) were identified (HaLIP1p1 and HaLIP1p2), sequenced and cloned in a heterologous production system (Escherichia coli). Gene expression studies revealed similar expression patterns for both isoforms, with a slight predominance of HaLIP1p1 in vegetative tissues and mature seeds. Tertiary structural models for these enzymes indicate they both have the same theoretical catalytic sites, using lipoyl-lys and 5-deoxyadenosine as docking substrates. The fatty acid profile of E. coli cells overexpressing HaLIP1p1 and HaLIP1p2 did not present major differences, and the in vivo activity of both proteins was confirmed by complementation of an E. coli JW0623 mutant in which lipoyl synthase is defective. Although no significant differences were detected in the total fatty acid composition of transgenic Arabidopsis thaliana seeds overexpressing any of both proteins, a lipidomic analysis revealed a redistribution of the glycerolipid species, accompanied with increased phosphatidylethanolamine (PE) content and a decrease in diacyglycerols (DAG) and phosphatidylcholine (PC). Depletion of the SAM co-factor caused by HaLIP1p1 and HaLIP1p2 overexpression in transgenic plants could explain this remodelling through its effects on PC synthesis.

Agrobacterium-mediated transformation of Ruta graveolens L.

Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.

Influence of light intensity and selection scheme on regeneration time of transgenic flax plants.

This study aimed at establishing a protocol to increase the number of regenerated shoots and to limit the recovery of "escapes" during the regeneration of transgenic flax plants (cv Barbara). Here, we describe how light, adapted media and selection scheme could stimulate the transformation process, the organogenic potentiality of calli (by a factor of 3.2) and accelerate the transgenic shoot regeneration (by a factor of about 2). On comparison of the transformation rate observed while using low light (LL) and high light (HL) a considerable enhancement from 0.12 to 5.7% was evident. The promotive effect of light might also had a direct beneficial effect on transgenic plant production time leading to a reduction of more than 4 months in the time need to obtain transgenic seeds. All data indicate that HL plays a role on growth and on protein, rubisco and pigment contents by stimulating the gene implicated in photosynthetic and Calvin cycle processes.

Cloning and molecular characterization of three lysophosphatidic acid acyltransferases expressed in flax seeds.

In the context of the growing demand for α-linolenic acid due to its high nutritional value as a polyunsaturated fatty acid, we have investigated the contribution of 2-lysophosphatidic acid acyltransferase (LPAAT) enzymes from flax (Linum usitatissimum) in the accumulation of α-linolenic acid into the oil fraction of flax seed. We have isolated the cDNAs encoding three class A microsomal LPAAT2 isoforms from developing flax seeds. The three isoforms, denominated LPAAT2A, LPAAT2A2 and LPAAT2B, are able to complement the LPAAT deficient JC201 E. coli mutant, confirming their functionality. We have performed enzymatic assays showing that the specific activity of the LPAAT2A isoform is significantly higher than that of the LPAAT2A2 and LPAAT2B toward the unsaturated oleic, linoleic and linolenic acids. Moreover, LPAAT2A presents in vitro a high specificity and selectivity for linoleic and linolenic acids as compared to saturated fatty acids. The three isoforms are expressed during all the stages of seed development and in stem and leaf tissues, as shown by an analysis of the transcription level of the corresponding genes. The heterologous expression of LPAAT2A in Arabidopsis seeds leads to an increase in the accumulation of linoleic and linolenic acids in the oil fraction of the seeds from two transgenic lines.