Acyl-lipid Desaturase Research Articles

Engineering Rhodosporidium toruloides for sustainable production of value-added punicic acid from glucose and wood residues

Rhodosporidium toruloides has emerged as a prominent candidate for producing single-cell oil from cost-effective feedstocks. In this study, the capability of R. toruloides to produce punicic acid (PuA), a representative plant unusual fatty acid, was investigated. The introduction of acyl lipid desaturase and conjugase (PgFADX) allowed R. toruloides to accumulate 3.7 % of total fatty acids as PuA. Delta-12 acyl lipid desaturase (PgFAD2) and diacylglycerol acyltransferase 2 were shown to benefit PuA production. The strain with PgFADX and PgFAD2 coexpression accumulated 12 % of its lipids as PuA from glucose, which translated into a PuA titer of 451.6 mg/L in shake flask condition. Utilizing wood hydrolysate as the feedstock, this strain produced 6.4 % PuA with a titer of 310 mg/L. Taken together, the results demonstrated that R. toruloides could serve as an ideal platform for the production of plant-derived high-value conjugated fatty acid using agricultural and forestry waste as feedstock.

Transcriptome analysis of Acinetobacter calcoaceticus HX09 strain with outstanding crude-oil-degrading ability.

Microbial remediation plays a pivotal role in the elimination of petroleum pollutants, making it imperative to investigate the capabilities of microorganisms in degrading petroleum. The present study describes the isolation of a promising strain, Acinetobacter sp. HX09, from petroleum-contaminated water. GC-MS analysis revealed a remarkable removal efficiency for short and medium-chain alkanes, with a rate of approximately 64% after a 7-days incubation at 30 °C. Transcriptome analysis of HX09 exhibited a predominant upregulation in gene expression levels by the induce of crude oil. Notably, genes such as alkane 1-monooxygenase, dehydrogenases and fatty acid metabolic enzymes exhibited fold changes range from 3.16 to 1.3. Based on the alkB gene sequences in HX09, the Phyre2 algorithm generated a three-dimensional structure that exhibited similarity to segments of acyl coenzyme desaturases and acyl lipid desaturases. Furthermore, three biodegradation-related gene clusters were predicted in HX09 based on the reference genome sequence. These findings contribute to our understanding of the hydrocarbon-degrading mechanisms employed by Acinetobacter species and facilitate the development of effective remediation strategies for crude oil- polluted environments.

Effect of Auxin on Fatty Acid Composition and Activity of Acyl-Lipid Desaturases in Seedlings of Spring Wheat Triticum aestivum L.

Effect of Auxin on Fatty Acid Composition and Activity of Acyl-Lipid Desaturases in Seedlings of Spring Wheat Triticum aestivum L.

The Composition and the Content of ∆-5 Sterols, Fatty Acids, and the Activity of Acyl-Lipid Desaturases in the Shoots of Ephedra monosperma, Introduced in the Botanical Garden of the Cryolithozone of Yakutia

Evergreen plants in permafrost ecosystems survive unfavorable autumn cooling and extremely low winter temperatures by maintaining optimal physiological activity of tissue cell membranes. To some extent, these features are due to the properties of shoot lipids performing a number of functions during adaptation. Sterols (STs) play a key role in regulating the fluidity and permeability of plant membranes (phytosterols) with a wide structural diversity. The composition of neutral lipids, STs, and fatty acids (FAs) in shoots of the evergreen shrub Ephedra monosperma growing in the Botanical Garden cryolithozone was first studied with HPTLC-UV/Vis/FLD and GC-MS. Twenty FAs were found, from C14:0 to C23:0; they included mono-, di-, tri-, and tetraene FAs. The high content of β-sitosterol among other ∆-5 sterols and an increased amount of C18:2(∆9,12) linoleic acid in lipids composition during the autumn–winter period was found to play an important role in the adaptation of ephedra shoots to the autumn–winter period, providing the cell membrane with greater plasticity, fluidity, and flexibility. The important role of diene linoleic fatty acid C18:2(∆9,12) in ephedra shoot lipids in the processes of low-temperature adaptation was shown.

Effect of Auxin on Fatty Acid Composition and Activity of Acyl-Lipid Desaturases in Sprouts of Spring Wheat <i>Triticum aestivum</i> L.

Effect of Auxin on Fatty Acid Composition and Activity of Acyl-Lipid Desaturases in Sprouts of Spring Wheat <i>Triticum aestivum</i> L.

Identification of differentially expressed genes involved in amino acid and lipid accumulation of winter turnip rape (Brassica rapa L.) in response to cold stress.

Winter turnip rape (Brassica rapa L.) is an important overwintering oil crop that is widely planted in northwestern China. It considered to be a good genetic resource for cold-tolerant research because its roots can survive harsh winter conditions. Here, we performed comparative transcriptomics analysis of the roots of two winter turnip rape varieties, Longyou7 (L7, strong cold tolerance) and Tianyou2 (T2, low cold tolerance), under normal condition (CK) and cold stress (CT) condition. A total of 8,366 differentially expressed genes (DEGs) were detected between the two L7 root groups (L7CK_VS_L7CT), and 8,106 DEGs were detected for T2CK_VS_T2CT. Among the DEGs, two ω-3 fatty acid desaturase (FAD3), two delta-9 acyl-lipid desaturase 2 (ADS2), one diacylglycerol kinase (DGK), and one 3-ketoacyl-CoA synthase 2 (KCS2) were differentially expressed in the two varieties and identified to be related to fatty acid synthesis. Four glutamine synthetase cytosolic isozymes (GLN), serine acetyltransferase 1 (SAT1), and serine acetyltransferase 3 (SAT3) were down-regulated under cold stress, while S-adenosylmethionine decarboxylase proenzyme 1 (AMD1) had an up-regulation tendency in response to cold stress in the two samples. Moreover, the delta-1-pyrroline-5-carboxylate synthase (P5CS), δ-ornithine aminotransferase (δ-OAT), alanine-glyoxylate transaminase (AGXT), branched-chain-amino-acid transaminase (ilvE), alpha-aminoadipic semialdehyde synthase (AASS), Tyrosine aminotransferase (TAT) and arginine decarboxylase related to amino acid metabolism were identified in two cultivars variously expressed under cold stress. The above DEGs related to amino acid metabolism were suspected to the reason for amino acids content change. The RNA-seq data were validated by real-time quantitative RT-PCR of 19 randomly selected genes. The findings of our study provide the gene expression profile between two varieties of winter turnip rape, which lay the foundation for a deeper understanding of the highly complex regulatory mechanisms in plants during cold treatment.

Insights into triterpene synthesis and unsaturated fatty-acid accumulation provided by chromosomal-level genome analysis of Akebia trifoliata subsp. australis

Akebia trifoliata subsp. australis is a well-known medicinal and potential woody oil plant in China. The limited genetic information available for A. trifoliata subsp. australis has hindered its exploitation. Here, a high-quality chromosome-level genome sequence of A. trifoliata subsp. australis is reported. The de novo genome assembly of 682.14 Mb was generated with a scaffold N50 of 43.11 Mb. The genome includes 25,598 protein-coding genes, and 71.18% (485.55 Mb) of the assembled sequences were identified as repetitive sequences. An ongoing massive burst of long terminal repeat (LTR) insertions, which occurred ~1.0 million years ago, has contributed a large proportion of LTRs in the genome of A. trifoliata subsp. australis. Phylogenetic analysis shows that A. trifoliata subsp. australis is closely related to Aquilegia coerulea and forms a clade with Papaver somniferum and Nelumbo nucifera, which supports the well-established hypothesis of a close relationship between basal eudicot species. The expansion of UDP-glucoronosyl and UDP-glucosyl transferase gene families and β-amyrin synthase-like genes and the exclusive contraction of terpene synthase gene families may be responsible for the abundant oleanane-type triterpenoids in A. trifoliata subsp. australis. Furthermore, the acyl-ACP desaturase gene family, including 12 stearoyl-acyl-carrier protein desaturase (SAD) genes, has expanded exclusively. A combined transcriptome and fatty-acid analysis of seeds at five developmental stages revealed that homologs of SADs, acyl-lipid desaturase omega fatty acid desaturases (FADs), and oleosins were highly expressed, consistent with the rapid increase in the content of fatty acids, especially unsaturated fatty acids. The genomic sequences of A. trifoliata subsp. australis will be a valuable resource for comparative genomic analyses and molecular breeding.

Role of Fatty Acid ω3 Acyl-Lipid Desaturases in Low-Temperature Hardening of Solanum tuberosum L.

The role of fatty acid ω3 acyl-lipid desaturases in low-temperature hardening (7 days at 3°C) of potato plants (Solanum tuberosum L., cv. Yubilei Zhukova) was studied. It was found that transcriptions of the three genes of ω3 acyl-lipid desaturases present in the potato genome significantly differed in their response to low temperature. The content of the FAD3 gene transcripts dramatically fell in the first day of cooling and was not restored until the end of the hardening period. The FAD7 gene was constitutively expressed through almost the entire adaptation period. The level of the FAD8 gene transcripts sharply increased in the first day of the hardening. The total fraction of trienoic fatty acids, which are synthesized by ω3 acyl-lipid desaturases, rose by 3% during the hardening, which is a relatively large value for potato. The hardened plants, in comparison with the unhardened ones, manifested a higher net photosynthesis/dark respiration ratio, higher (2.5-fold) concentration of soluble sugars, and stronger resistance to negative (–2 or –3°C) temperatures. It is suggested that the augmentation of the trienoic FA fraction, which are mainly localized in the thylakoid membranes of the potato chloroplasts, sustains the photosynthesis under low temperatures. Therefore, the maintenance of photosynthetic activity during the low-temperature acclimation supplies the plant with photoassimilates that are the principal sources of energy and metabolites necessary for the establishment of potato plant resistance to hypothermia.

Acyl-lipid desaturases and Vipp1 cooperate in cyanobacteria to produce novel omega-3 PUFA-containing glycolipids

BackgroundDietary omega-3 (n-3), long chain (LC-, ≥ 20 carbons), polyunsaturated fatty acids (PUFAs) derived largely from marine animal sources protect against inflammatory processes and enhance brain development and function. With the depletion of natural stocks of marine animal sources and an increasing demand for n-3 LC-PUFAs, alternative, sustainable supplies are urgently needed. As a result, n-3 18-carbon and LC-PUFAs are being generated from plant or algal sources, either by engineering new biosynthetic pathways or by augmenting existing systems.ResultsWe utilized an engineered plasmid encoding two cyanobacterial acyl-lipid desaturases (DesB and DesD, encoding Δ15 and Δ6 desaturases, respectively) and “vesicle-inducing protein in plastids” (Vipp1) to induce production of stearidonic acid (SDA, 18:4 n-3) at high levels in three strains of cyanobacteria (10, 17 and 27% of total lipids in Anabaena sp. PCC7120, Synechococcus sp. PCC7002, and Leptolyngbya sp. strain BL0902, respectively). Lipidomic analysis revealed that in addition to SDA, the rare anti-inflammatory n-3 LC-PUFA eicosatetraenoic acid (ETA, 20:4 n-3) was synthesized in these engineered strains, and ~ 99% of SDA and ETA was complexed to bioavailable monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) species. Importantly, novel molecular species containing alpha-linolenic acid (ALA), SDA and/or ETA in both acyl positions of MGDG and DGDG were observed in the engineered Leptolyngbya and Synechococcus strains, suggesting that these could provide a rich source of anti-inflammatory molecules.ConclusionsOverall, this technology utilizes solar energy, consumes carbon dioxide, and produces large amounts of nutritionally important n-3 PUFAs and LC-PUFAs. Importantly, it can generate previously undescribed, highly bioavailable, anti-inflammatory galactosyl lipids. This technology could therefore be transformative in protecting ocean fisheries and augmenting the nutritional quality of human and animal food products.

Altered fatty acid composition of Nicotiana benthamiana and Nicotiana excelsior leaves under transient overexpression of the cyanobacterial desC gene

Transient heterologous gene expression in two model plant species, Nicotiana benthamiana and N. excelsior, has been used to study the localization of the heterologous Δ9 acyl-lipid desaturase (Δ9 desaturase) of Synechococcus vulcanus in different cell compartments and its functional activity in the cases of the cytosol, chloroplast, and endoplasmic reticulum (ER) localization. The functional activity and substrate specificity of the heterologous desaturase under the conditions of transient expression have been confirmed by comparison of fatty acid (FA) profiles. The Δ9 desaturase, responsible for the synthesis of oleic and palmitoleic acids, has also been shown to strongly promote the accumulation of polyunsaturated FAs. The results convincingly demonstrate that the Δ9 desaturase of the thermophilic cyanobacterium transiently expressed in two Nicotiana species considerably alters lipid metabolism in their leaves towards a higher FA unsaturation. The functional activity of Δ9 desaturase depends on both the model plant species, N. benthamiana or N. excelsior, and the cellular localization of the enzyme. The method of transient expression of heterologous genes in plants is highly effective, inexpensive, and not time-consuming, which makes it attractive for estimating the functional activity and/or substrate specificity of heterologous desaturases.

Adaptive Alterations in the Ultrastructure of Chloroplasts and the Contents of Pigments and Sugars under Low Temperature Hardening of Potato Plants: Role of Δ12 Acyl-Lipid Desaturase

Alterations in the ultrastructure of chloroplasts and the content of sugars during low-temperature hardening (5°C, 6 days) of potato plants (Solanum tuberosum L., cv. Desnitsa) and a transformed line with the desA gene of Δ12 acyl-lipid desaturase Synechocystis sp. RCC 6803 were studied. It was noted that during hardening, sugars were accumulated in the leaves of both lines, but with greater speed in the leaves of the transformants. In control plants after hardening, an increase in the total number of thylakoid membranes and in the number of plastoglobules per chloroplast were obtained accompanied by a decrease in the chloroplast cross-sectional area and starch grain area. It was found that chloroplasts of transformed plants, which were different from the control plants in the higher absolute content of membrane lipids and the amount of polyunsaturated fatty acids, corresponded to the hardened plants before cold exposure by some ultrastructural parameters. In the early period of low-temperature hardening, the transformed plants were more adaptable, according to the data on the sugar content.

Structural determinant of functionality in acyl lipid desaturases

Little is known about the structure-function relationship of membrane-bound lipid desaturases. Using a domain-swapping strategy, we found that the N terminus (comprising the two first transmembrane segments) region of Bacillus cereus DesA desaturase improves Bacillus subtilis Des activity. In addition, the replacement of the first two transmembrane domains from Bacillus licheniformis inactive open reading frame (ORF) BL02692 with the corresponding domain from DesA was sufficient to resurrect this enzyme. Unexpectedly, we were able to restore the activity of ORF BL02692 with a single substitution (Cys40Tyr) of a cysteine localized in the first transmembrane domain close to the lipid-water interface. Substitution of eight residues (Gly90, Trp104, Lys172, His228, Pro257, Leu275, Tyr282, and Leu284) by site-directed mutagenesis produced inactive variants of DesA. Homology modeling of DesA revealed that His228 is part of the metal binding center, together with the canonical His boxes. Trp104 shapes the hydrophobic tunnel, whereas Gly90 and Lys172 are probably involved in substrate binding/recognition. Pro257, Leu275, Tyr282, and Leu284 might be relevant for the structural arrangement of the active site or interaction with electron donors. This study reveals the role of the N-terminal region of Δ5 phospholipid desaturases and the individual residues necessary for the activity of this class of enzymes.

Isolation and Characterization of Gene for Expression of Delta 9 acyl-lipid Desaturase from Nostoc sp. Involved in Lipid Production

Isolation and Characterization of Gene for Expression of Delta 9 acyl-lipid Desaturase from Nostoc sp. Involved in Lipid Production

Changes in the content of fatty acid desaturases gene transcripts for Arabidopsis plants under adaptation to hypothermia

Changes in the content of acyl-lipid desaturases gene transcripts in Arabidopsis (Arabidopsis thaliana Heynh. (L.), ecotype Columbia) plants subjected to low temperature hardening for 5 days at 2°C were investigated. In the course of the experiments with hourly dynamics of plant chilling, it was established that the genes under study by their responses to cold action can be separated into two groups. The genes of the first group represented by ADS2, FAD2, and FAD7 are characterized by the amount of their transcripts that rapidly increase even for the first 2–4 h of cold exposition, while the same value for the genes FAD3, FAD6, and FAD8 belonging to the second group remained unchanged under the same conditions. The experiments with the above hardening dynamics provided evidence for consecutive character of genes ADS2, FAD2, and FAD7 transcript accumulation, where the process for ADS2 was sharply intensified even for the first days of hardening, while that for other two genes achieved maximal intensity only on the fourth day of hardening. These results, taken together, lead to the conclusion that the agreeing in time increase in the transcript content of genes ADS2, FAD2, and FAD7 encoding Δ9-, Δ12-, and ω3-desaturases resulted in the change of FAs composition of chloroplast lipids. Increasing portion in them of triene FAs during hardening time provided the maintenance of chloroplast membranes in a native state allowing the Arabidopsis plant to successfully adapt to hypothermia.

Polyphasic characterization of the thermotolerant cyanobacterium Desertifilum sp. strain IPPAS B-1220.

A cyanobacterial strain from Lake Shar-Nuur, a freshwater lake in Mongolia, was isolated and characterized by a polyphasic approach. According to the 16S ribosomal RNA gene sequence, this strain (IPPAS B-1220) belongs to a newly described genus Desertifilum. In general, strains of Desertifilum maintain their genetic stability, as seen from the analysis of the 16S rRNA gene and 16S-23S rRNA internal transcribed spacer sequences from strains collected at distant locations. The newly discovered strain is characterized by an unusual fatty acid composition (16:1Δ7 and 16:2Δ7,10). Analysis of its draft genomic sequence reveals the presence of six genes for the acyl-lipid desaturases: two Δ9-desaturases, desC1 and desC2; two Δ12-desaturases, desA1 and desA2; one desaturase of unknown specificity, desX; and one gene for the bacillary-type desaturase, desG, which supposedly encodes an ω9-desaturase. A scheme for a fatty acid desaturation pathway that describes the biosynthesis of 16:1Δ7 and 16:2Δ7,10 fatty acids in Desertifilum is proposed.

Physiological and biochemical research characteristics of plant Nicotiana tabacum, expressing genes desA or desC under different temperatures stress

Aim. For modifying of the plant organisms with genetic engineering techniques to produce genus stress resistant low temperatures or frosts it is necessary to check their physiological characteristics at high temperatures stress. Methods. In this study we used Nicotiana tabacum plants, expressing of cyanobacterial acyl-lipid desaturases genes (desA or desC), plants were tested for the level of malondialdehyde accumulation and gene expression by the reporter protein thermostable lichenase after exposure to thermal stress. Results. We discovered the reduced malondialdehyde accumulation in plants and increased expression desaturases genes after cold stress and high temperature stress. Conclusions. Cyanobacterial desaturases gene expression in Nicotiana tabacum plants did not increase their sensitivity to the high temperatures stress.Keywords: acyl-lipid desaturases, malondialdehyde, thermostable lichenase

Construction of a cyanobacterium synthesizing cyclopropane fatty acids

Microalgae have received much attention as a next-generation source of biomass energy. However, most of the fatty acids (FAs) from microalgae are multiply unsaturated; thus, the biofuels derived from them are fluid, but vulnerable to oxidation. In this study, we attempted to synthesize cyclopropane FAs in the cyanobacterium Synechocystis sp. PCC 6803 by expressing the cfa gene for cyclopropane FA synthase from Escherichia coli with the aim of producing FAs that are fluid and stable in response to oxidization. We successfully synthesized cyclopropane FAs in Synechocystis with a yield of ~30% of total FAs. Growth of the transformants was altered, particularly at low temperatures, but photosynthesis and respiration were not significantly affected. C16:1∆9 synthesis in the desA−/desD− strain by expression of the desC2 gene for sn-2 specific ∆9 desaturase positively affected growth at low temperatures via promotion of various cellular processes, with the exceptions of photosynthesis and respiration. Estimation of the apparent activities of desaturases suggested that some acyl-lipid desaturases might recognize the lipid side chain.

ДИНАМИКА ЖИРНОКИСЛОТНОГО СОСТАВА И АКТИВНОСТИ АЦИЛ-ЛИПИДНЫХ ДЕСАТУРАЗ В ХВОЕ PINUS SYLVESTRIS L., ПРОИЗРАСТАЮЩЕЙ В ИРКУТСКОЙ ОБЛАСТИ

Temperature – a significant ecological factor of the environment, which is characterized by very high variation. First of all, the cell membranes are subjected to the temperature factor, whose resistance associated with the presence of polyunsaturated fatty acids in the membrane structure and the activity of acyl-lipid desaturases responsible for membrane fluidity.The change in the fatty acid composition on needles of during vegetation period was investigated. The dynamics of saturated and unsaturated fatty acids and the role of acyl-lipid desaturases in adaptive changes in fatty acid composition are shows.27 fatty acids are found in the needles of Pinus sylvestris L. growing in the vicinity of the Melnichnaya Pad, which content are varies depending on the season.The fatty acid composition of Pinus sylvestris L. needles were studied during the year. The dynamics of saturated and unsaturated fatty acids and the role of acyl-lipid desaturases to adaptive changes in fatty acid composition were shows.The unsaturated fatty acids are predominate in general content of fatty acids, the maximum content of which is observed during the period of low freezing temperatures, saturated fatty acids-in the spring period.Whereas the highest values of the of double bonds index (1,84–1,88) are observed in winter due to the low content of saturated fatty acids, the lowest value of the index observed in the summer.Also there was relationship between content of oleic, linoleic and α-linolenic acids with modification activity of acyl-lipid ω-3, ω-6 and ω-9 desaturases.

Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth

Bacillus cereus sensu lato is composed of a set of ubiquitous strains including human pathogens that can survive a range of food processing conditions, grow in refrigerated food, and sometimes cause food poisoning. We previously identified the two-component system CasK/R that plays a key role in cold adaptation. To better understand the CasK/R-controlled mechanisms that support low-temperature adaptation, we performed a transcriptomic analysis on the ATCC 14579 strain and its isogenic ∆casK/R mutant grown at 12°C. Several genes involved in fatty acid (FA) metabolism were downregulated in the mutant, including desA and desB encoding FA acyl-lipid desaturases that catalyze the formation of a double-bond on the FA chain in positions ∆5 and ∆10, respectively. A lower proportion of FAs presumably unsaturated by DesA was observed in the ΔcasK/R strain compared to the parental strain while no difference was found for FAs presumably unsaturated by DesB. Addition of phospholipids from egg yolk lecithin rich in unsaturated FAs, to growth medium, abolished the cold-growth impairment of ΔcasK/R suggesting that exogenous unsaturated FAs can support membrane-level modifications and thus compensate for the decreased production of these FAs in the B. cereus ∆casK/R mutant during growth at low temperature. Our findings indicate that CasK/R is involved in the regulation of FA metabolism, and is necessary for cold adaptation of B. cereus unless an exogenous source of unsaturated FAs is available.

Characterization of Nicotiana tabacum plants expressing hybrid genes of cyanobacterial Δ9 or Δ12 acyl-lipid desaturases and thermostable lichenase

We established transgenic lines of Nicotiana tabacum expressing hybrid genes of Synechocystis sp. PCC 6803 Δ12 (desA) acyllipid desaturase and Synechococcus vulcanus Δ9 (desC) acyllipid desaturase with or without sequence coding for transit peptide of Rubisco small subunit of Arabidopsis thaliana under control of a constitutive promoter. Reliable increase of linoleic acid portion (C18:2; Δ9,12) accompanied with decrease of α-linolenic acid (C18:3; Δ9,12,15) relative amount was detected for plants expressing hybrid desA::licBM3 gene. No reliable changes were detected in fatty acid profiles and unsaturation index of plants transformed with Δ9 desaturase gene desC::licBM3 lacking signals of intracellular targeting while expression of this gene with Arabidopsis thaliana Rubisco small subunit transit peptide sequence caused growth of C18:3 α-linolenic acid part simultaneously with reduction of C18:2 linoleic acid part, as well as increase of unsat-uration index. No changes in relative amount of Δ9-monounsaturated fatty acids were observed in any of studied lines. All plants expressing desaturase genes exhibited enhanced levels of superoxide dismutase (SOD) activity after cold treatment in contrast to control lines with suppressed SOD activity after cold treatment.