Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous <Emphasis Type="Italic">Rhodococcus</Emphasis> strains

Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.     

Analysis of storage lipid accumulation in Alcanivorax borkumensis: Evidence for alternative triacylglycerol biosynthesis routes in bacteria

Marine hydrocarbonoclastic bacteria, like Alcanivorax borkumensis, play a globally important role in bioremediation of petroleum oil contamination in marine ecosystems. Accumulation of storage lipids, serving as endogenous carbon and energy sources during starvation periods, might be a potential adaptation mechanism for coping with nutrient limitation, which is a frequent stress factor challenging those bacteria in their natural marine habitats. Here we report on the analysis of storage lipid biosynthesis in A. borkumensis strain SK2. Triacylglycerols (TAGs) and wax esters (WEs), but not poly(hydroxyalkanoic acids), are the principal storage lipids present in this and other hydrocarbonoclastic bacterial species. Although so far assumed to be a characteristic restricted to gram-positive actinomycetes, substantial accumulation of TAGs corresponding to a fatty acid content of more than 23% of the cellular dry weight is the first characteristic of large-scale de novo TAG biosynthesis in a gram-negative bacterium. The acyltransferase AtfA1 (ABO_2742) exhibiting wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) activity plays a key role in both TAG and WE biosynthesis, whereas AtfA2 (ABO_1804) was dispensable for storage lipid formation. However, reduced but still substantial residual TAG levels in atfA1 and atfA2 knockout mutants compellingly indicate the existence of a yet unknown WS/DGAT-independent alternative TAG biosynthesis route. Storage lipids of A. borkumensis were enriched in saturated fatty acids and accumulated as insoluble intracytoplasmic inclusions exhibiting great structural variety. Storage lipid accumulation provided only a slight growth advantage during short-term starvation periods but was not required for maintaining viability and long-term persistence during extended starvation phases.     

Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance.

Branched-chain fatty acids of the iso and anteiso series occur in many bacteria as the major acyl constituents of membrane lipids. In addition, omega-cyclohexyl and omega-cycloheptyl fatty acids are present in several bacterial species. These two types of fatty acids are synthesized by the repeated condensation of malonyl coenzyme A with one of the branched-chain and cyclic primers by the same enzyme system. The pathway of de novo branched-chain fatty acid synthesis differs only in initial steps of synthesis from that of the common straight-chain fatty acid (palmitic acid) present in most organisms. The cell membranes composed largely of iso-, anteiso-, and omega-alicyclic acids support growth of bacteria, which inhabit normal as well as extreme environments. The occurrence of these types of fatty acids as major cellular fatty acids is an important criterion used to aid identification and classification of bacteria.     

Endoplasmic reticulum-located PDAT1-2 from castor bean enhances hydroxy fatty acid accumulation in transgenic plants

Ricinoleic acid (12-hydroxy-octadeca-9-enoic acid) is a major unusual fatty acid in castor oil. This hydroxy fatty acid is useful in industrial materials. This unusual fatty acid accumulates in triacylglycerol (TAG) in the seeds of the castor bean (Ricinus communis L.), even though it is synthesized in phospholipids, which indicates that the castor plant has an editing enzyme, which functions as a phospholipid:diacylglycerol acyltransferase (PDAT) that is specific to ricinoleic acid. Transgenic plants containing fatty acid Δ12-hydroxylase encoded by the castor bean FAH12 gene produce a limited amount of hydroxy fatty acid, a maximum of around 17% of TAGs present in Arabidopsis seeds, and this unusual fatty acid remains in phospholipids of cell membranes in seeds. Identification of ricinoleate-specific PDAT from castor bean and manipulation of the phospholipid editing system in transgenic plants will enhance accumulation of the hydroxy fatty acid in transgenic seeds. The castor plant has three PDAT genes; PDAT1-1 and PDAT2 are homologs of PDAT, which are commonly found in plants; however, PDAT1-2 is newly grouped as a castor bean-specific gene. PDAT1-2 is expressed in developing seeds and localized in the endoplasmic reticulum, similar to FAH12, indicating its involvement in conversion of ricinoleic acid into TAG. PDAT1-2 significantly enhances accumulation of total hydroxy fatty acid up to 25%, with a significant increase in castor-like oil, 2-OH TAG, in seeds of transgenic Arabidopsis, which is an identification of the key gene for oilseed engineering in production of unusual fatty acids.     

All fatty acids are not equal: discrimination in plant membrane lipids

Plant membrane lipids are primarily composed of 16-carbon and 18-carbon fatty acids containing up to three double bonds. By contrast, the seed oils of many plant species contain fatty acids with significantly different structures. These unusual fatty acids sometimes accumulate to >90% of the total fatty acid content in the seed triacylglycerols, but are generally excluded from the membrane lipids of the plant, including those of the seed. The reasons for their exclusion and the mechanisms by which this is achieved are not completely understood. Here we discuss recent research that has given new insights into how plants prevent the accumulation of unusual fatty acids in membrane lipids, and how strict this censorship of membrane composition is. We also describe a transgenic experiment that resulted in an excessive buildup of unusual fatty acids in cellular membranes, and clearly illustrated that the control of membrane lipid composition is essential for normal plant growth and development.     

The phosphatidylcholine diacylglycerol cholinephosphotransferase is required for efficient hydroxy fatty acid accumulation in transgenic Arabidopsis

We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. The PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), thus enriching PC-modified fatty acids in the DAG pool prior to forming TAG. We show here that PDCT is required for the efficient metabolism of engineered hydroxy fatty acids in Arabidopsis (Arabidopsis thaliana) seeds. When a fatty acid hydroxylase (FAH12) from castor (Ricinus communis) was expressed in Arabidopsis seeds, the PDCT-deficient mutant accumulated only about half the amount of hydroxy fatty acids compared with that in the wild-type seeds. We also isolated a PDCT from castor encoded by the RcROD1 (Reduced Oleate Desaturation1) gene. Seed-specific coexpression of this enzyme significantly increased hydroxy fatty acid accumulation in wild type-FAH12 and in a previously produced transgenic Arabidopsis line coexpressing a castor diacylglycerol acyltransferase 2. Analyzing the TAG molecular species and regiochemistry, along with analysis of fatty acid composition in TAG and PC during seed development, indicate that PDCT acts in planta to enhance the fluxes of fatty acids through PC and enrich the hydroxy fatty acids in DAG, and thus in TAG. In addition, PDCT partially restores the oil content that is decreased in FAH12-expressing seeds. Our results add a new gene in the genetic toolbox for efficiently engineering unusual fatty acids in transgenic oilseeds.     

Apparent Role of Phosphatidylcholine in the Metabolism of Petroselinic Acid in Developing Umbelliferae Endosperm

Studies were conducted to characterize the metabolism of the unusual fatty acid petroselinic acid (18:1cis[delta]6) in developing endosperm of the Umbelliferae species coriander (Coriandrum sativum L.) and carrot (Daucus carota L.). Analyses of fatty acid compositions of glycerolipids of these tissues revealed a dissimilar distribution of petroselinic acid in triacylglycerols (TAG) and the major polar lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Petroselinic acid comprised 70 to 75 mol% of the fatty acids of TAG but only 9 to 20 mol% of the fatty acids of PC and PE. Although such data appeared to suggest that petroselinic acid is at least partially excluded from polar lipids, results of [1-14C]acetate radiolabeling experiments gave a much different picture of the metabolism of this fatty acid. In time-course labeling of carrot endosperm, [1-14C]acetate was rapidly incorporated into PC in high levels. Through 30 min, radiolabel was most concentrated in PC, and of this, 80 to 85% was in the form of petroselinic acid. One explanation for the large disparity in amounts of petroselinic acid in PC as determined by fatty acid mass analyses and 14C radiolabeling is that turnover of these lipids or the fatty acids of these lipids results in relatively low accumulation of petroselinic acid mass. Consistent with this, the kinetics of [1-14C]acetate time-course labeling of carrot endosperm and "pulse-chase" labeling of coriander endosperm suggested a possible flux of fatty acids from PC into TAG. In time-course experiments, radiolabel initially entered PC at the highest rates but accumulated in TAG at later time points. Similarly, in pulse-chase studies, losses in absolute amounts of radioactivity from PC were accompanied by significant increases of radiolabel in TAG. In addition, stereospecific analyses of unlabeled and [1-14C]acetate-labeled PC of coriander endosperm indicated that petroselinic acid can be readily incorporated into both the sn-1 and sn-2 positions of this lipid. Because petroselinic acid is neither synthesized nor further modified on polar lipids, the apparent metabolism of this fatty acid through PC (and possibly through other polar lipids) may define a function of PC in TAG assembly apart from its involvement in fatty acid modification reactions.     

Arabidopsis amidase 1, a member of the amidase signature family

Amidase 1 (AMI1), a specific indole-3-acetamide amidohydrolase, is an Arabidopsis thaliana amidase signature enzyme that catalyzes the synthesis of indole-3-acetic acid from indole-3-acetamide. Amidase signature family members catalyze a diverse range of enzymatic reactions and are found widespread in nature, for instance in bacteria, mammals, and plants. At the protein level, the family members share a conserved stretch of approximately 50-130 amino acids, the name-giving amidase signature. Elucidation of the crystal structures of a mammalian fatty acid amide hydrolase and the bacterial malonamidase E2 revealed an unusual Ser-cisSer-Lys catalytic triad in proteins of this family. In addition, other members, such as the amidase from Rhodococcus rhodochrous strain J1 or Sulfolobus solfataricus, seem to use an accessory Cys-cisSer-Lys center. AMI1 possesses all conserved amino-acid residues of the Ser-cisSer-Lys triad, but lacks the CX(3)C motif and therefore the Cys-cisSer-Lys catalytic site. Using a set of point-mutated variants of AMI1 and chemical modifications, we analyzed the relative importance of single amino-acid residues of AMI1 with respect to substrate conversion. These experiments revealed that a specific serine residue, Ser137, is essential for AMI1 enzymatic activity. We also report structural and functional differences of AMI1 from other amidase signature enzymes.     

Engineering industrial fatty acids in oilseeds

More than 300 types of modified fatty acids (mFA) are produced in triacylglycerols (TAG) by various plant species, with many of these unusual structures rendering unique physical and chemical properties that are desirable for a variety of bio-based industrial uses. Attempts to produce these mFA in crop species have thus far failed to reach the desired levels of production and highlighted the need to better understand how fatty acids are synthesized and accumulated in seed oils. In this review we discuss how some of the progress made in recent years, such as the improved TAG synthesis model to include acyl editing and new enzymes such as PDCT, may be utilized to achieve the goal of effectively modifying plant oils for industrial uses. Co-expressing several key enzymes may circumvent the bottlenecks for the accumulation of mFA in TAG through efficient removal of mFA from phosphatidylcholine. Other approaches include the prevention of feedback inhibition of fatty acid synthesis and improving primary enzyme activity in host transgenic plants. In addition, genomic approaches are providing unprecedented power to discover more factors that may facilitate engineering mFA in oilseeds. Based on the results of the last 20 years, creating a high mFA accumulating plant will not be done by simply inserting one or two genes; it is necessary to stack genes encoding enzymes with favorable kinetic activity or specificity along with additional complementary transgenes in optimized plant backgrounds to produce industrial fatty acids at desirable levels. Finally, we discuss the potential of Camelina as an industrial oilseed platform.     

Triacylglycerol accumulation of Phaeodactylum tricornutum with different supply of inorganic carbon

The diatom Phaeodactylum tricornutum produces large quantities of lipids, especially triacylglycerols (TAGs) under nitrogen or phosphorus limitation. In this study, production of lipids and TAGs during this process was compared under conditions with different inputs of inorganic carbon. With an abundant supply of inorganic carbon, considerable accumulation of biomass, lipids, and TAGs was identified after a nitrogen/phosphorus-limiting “induction incubation.” TAGs were still synthesized and accumulated even under inorganic carbon limitation with a cessation in the production of biomass and cellular lipids. This part of accumulated TAGs could be synthesized through recycling and transformation of other lipids such as glycolipids and phospholipids. Additionally, some alterations in the fatty acid profile following TAG accumulation were found. The content of the C16:0 fatty acid increased with decreases in C16:3 and C20:5, which could have been caused by enzymatic selectivity for these fatty acids during the process of TAG synthesis. It was concluded that nitrogen and phosphorus metabolism regulates the synthesis of TAG, while carbon metabolism promotes it by providing sufficient substrates.     

新型可再生工业用油脂的代谢工程

植物种子油是一种可再生资源,亦用作生物燃油和化学工业原料. 一些野生植物能高水平合成积累羟化、环氧化和共轭脂肪酸等具有重要工业应用价值的特异脂肪酸.催化这些特异脂肪酸合成的酶主要是类脂肪酸去胞和酶2(类FAD2). 由特异脂肪酸合成到三酰基甘油脂 (TAG) 形成还需要酰基转移酶 (如DGAT) 的参与. 在油料作物种子中表达类FAD2酶及其相关基因(如DGAT),已培育出了能合成积累一定含量特异脂肪酸的工程油料品系,为基于农作物生产高附加值工业用油脂开辟了新途径. 本文论述了参与特异脂肪酸生物合成途径的关键酶基因、油料作物代谢工程策略,以及应用工程油料作物大规模生产重要工业用脂肪酸的研究进展、存在问题和应用前景等.     

Inhibition of Δ-6 desaturase reduces fatty acid re-esterification in 3T3-L1 adipocytes independent of changes in n3-PUFA cellular content

Δ-6 desaturase (D6D) is a key enzyme in the synthesis of long-chain polyunsaturated fatty acids (LC-PUFA). Evidence suggests that reduced D6D activity not only disrupts LC-PUFA production, but also impacts whole body lipid handling and body weight; however, the mechanisms remain largely unexplored. Therefore, we investigated the effect of D6D inhibition on the regulation of lipid accumulation in 3T3-L1 adipocytes with and without changes in n-3 PUFA content. 3T3-L1 cells were treated with a D6D inhibitor (SC-26196) in the presence or absence of α-linolenic acid (ALA) throughout differentiation. We found that D6D inhibition blocked the conversion of ALA to eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPAn-3) when ALA was supplemented, while no changes in n-3 PUFA content were observed in cells treated with the D6D inhibitor alone. D6D inhibited cells had reduced triacylglycerol (TAG) accumulation despite an EPA/DPA deficiency. In addition, analyses of cellular protein markers, as well as non-esterified fatty acids and glycerol release in medium, suggested an increase in lipolysis and a decrease in fatty acid re-esterification in D6D-inhibited cells, independent of n-3 PUFA changes. To provide further evidence, we treated cells with the D6D inhibitor in the presence or absence of EPA and compared them with ALA-treated cells. Although EPA further reduced TAG content, the reduced markers of fatty acid re-esterification were not affected by ALA or EPA. Collectively, this study provides new insight showing that D6D inhibition reduces TAG accumulation and fatty acid re-esterification in adipocytes independent of changes in n-3 PUFA cellular content.     

Good Fat, Essential Cellular Requirements for Triacylglycerol Synthesis to Maintain Membrane Homeostasis in Yeast

Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.     

Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.     

Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds

Expression of Delta(12)-oleic acid desaturase-related fatty acid conjugases from Calendula officinalis, Momordica charantia, and Vernicia fordii in seeds of soybean (Glycine max) or an Arabidopsis thaliana fad3/fae1 mutant was accompanied by the accumulation of the conjugated fatty acids calendic acid or alpha-eleostearic acid to amounts as high as 20% of the total fatty acids. Conjugated fatty acids, which are synthesized from phosphatidylcholine (PC)-linked substrates, accumulated in PC and phosphatidylethanolamine, and relative amounts of these fatty acids were higher in PC than in triacylglycerol (TAG) in the transgenic seeds. The highest relative amounts of conjugated fatty acids were detected in PC from seeds of soybean and A. thaliana that expressed the C. officinalis and M. charantia conjugases, where they accounted for nearly 25% of the fatty acids of this lipid class. In these seeds, >85% of the conjugated fatty acids in PC were detected in the sn-2 position, and these fatty acids were also enriched in the sn-2 position of TAG. In marked contrast to the transgenic seeds, conjugated fatty acids composed <1.5% of the fatty acids in PC from seeds of five unrelated species that naturally synthesize a variety of conjugated fatty acid isomers, including seeds that accumulate conjugated fatty acids to >80% of the total fatty acids. These results suggest that soybean and A. thaliana seeds are deficient in their metabolic capacity to selectively catalyze the flux of conjugated fatty acids from their site of synthesis on PC to storage in TAG.     

Detailed Identification of Fatty Acid Isomers Sheds Light on the Probable Precursors of Triacylglycerol Accumulation in Photoautotrophically Grown Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a model alga for studying triacylglycerol (TAG) accumulation in the photosynthetic production of biofuel. Previous studies were conducted under photoheterotrophic growth conditions in medium supplemented with acetate and/or ammonium. We wanted to demonstrate TAG accumulation under truly photoautotrophic conditions without reduced elements. We first reidentified all lipid components and fatty acids by mass spectrometry, because the currently used identification knowledge relies on data obtained in the 1980s. Accordingly, various isomers of fatty acids, which are potentially useful in tracing the flow of fatty acids leading to the accumulation of TAG, were detected. In strain CC1010 grown under photoautotrophic conditions, TAG accumulated to about 57.5 mol% of total lipids on a mole fatty acid basis after the transfer to nitrogen-deficient conditions. The content of monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol decreased drastically. The accumulated TAG contained 16:0 as the major acid and 16:4(4,7,10,13), 18:2(9,12), and 18:3(9,12,15), which are typically found in chloroplast lipids. Additionally, 18:1(11) and 18:3(5,9,12), which are specific to extrachloroplast lipids, were also abundant in the accumulated TAG. Photosynthesis and respiration slowed markedly after the shift to nitrogen-deficient conditions. These results suggest that fatty acids for the production of TAG were supplied not only from chloroplast lipids but also from other membranes within the cells, although the possibility of de novo synthesis cannot be excluded. Under nitrogen-replete conditions, supplementation with a high concentration of CO₂ promoted TAG production in the cells grown photoautotrophically, opening up the possibility to the continuous production of TAG using CO₂ produced by industry.     

Fatty Acids and Polar Lipids of Extremely Thermophilic Filamentous Bacterial Masses from Two Yellowstone Hot Springs

The fatty acid composition of filamentous bacterial masses from two very hot Yellowstone Park springs is not unusual despite the extreme environment. Both populations have a series of C(14) to C(20) straight-chain acids with a maximum at C(18), and a series of saturated iso acids with a maximum at C(17) in one case and C(19) in the other. The fatty acid pattern of this anomalous group of organisms is like that of bacteria but not of blue-green algae. Both populations have similar polar lipids and identical carotenoids. It is speculated that these organisms may be adapted to their high-temperature environment by means of stable lipoprotein membrane systems.