Coexpressing Escherichia coli Cyclopropane Synthase with Sterculia foetida Lysophosphatidic Acid Acyltransferase Enhances Cyclopropane Fatty Acid Accumulation

Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation.Modified fatty acids (mFAs; sometimes referred to as unusual fatty acids) obtained from plants play important roles in industrial applications as lubricants, protective coatings, plastics, inks, cosmetics, etc. The hundreds of potential industrial uses of mFAs have led to considerable interest in exploring their production in transgenic crop plants. mFAs are produced by a limited number of species, and the transfer of genes encoding mFA-producing enzymes from source plants to heterologous hosts has generally resulted in only modest accumulation, usually less than 20% of the desired mFA in transgenic seed (Napier, 2007) compared with levels found in the natural source. For example, ricinoleic acid accounts for more than 90% of the fatty acid of castor bean (Ricinus communis) seeds, and tung (Aleuites fordii) seeds accumulate more than 80% α-eleostearic acid (Thelen and Ohlrogge, 2002; Drexler et al., 2003). In order to elevate the content of mFAs in the engineered plants to that found in the native plant, it is necessary to (1) optimize the synthesis of mFA (Mekhedov et al., 2001), (2) minimize its degradation (Eccleston and Ohlrogge, 1998), and (3) optimize its incorporation into triacylglycerol (TAG; Bafor et al., 1990; Bates and Browse, 2011; van Erp et al., 2011).Cyclic fatty acids (CFAs) are desirable for numerous industrial applications. The strained bond angles of the carbocyclic ring contribute to their unique chemistry and physical properties, and hydrogenation of CFAs results in ring opening to produce methyl-branched fatty acids. Branched chain fatty acids are ideally suited for the oleochemical industry as feedstocks for the production of lubricants, plastics, paints, dyes, and coatings (Carlsson et al., 2011). Cyclopropane fatty acids (CPAs) have been found in certain gymnosperms, Malvales, Litchi spp., and other Sapindales species. They accumulate to as much as 40% in seeds of Litchi chinensis (Vickery, 1980; Gaydou et al., 1993). Sterculia foetida accumulates the desaturated CFA (i.e. cyclopropene fatty acid) to more than 60% of its seed oil (Bohannon and Kleiman, 1978; Pasha and Ahmad, 1992). The first step in its synthesis is the formation of the CPA by the cyclopropane synthase (CPS) enzyme, which transfers a methyl group to C9 of the oleoyl-phospholipid followed by cyclization to form the cyclopropane ring (Grogan and Cronan, 1997; Bao et al., 2002, 2003). None of the known natural sources of CPA are suitable for its commercial production. Therefore, it would be desirable to create an oilseed crop plant that accumulates high levels of CPA by heterologously expressing CPS in seeds. However, to date, heterologous expression of plant cyclopropane synthase genes has led to only approximately 1.0% CPA in the transgenic seeds (Yu et al., 2011).Two pathways for the biosynthesis of TAG exist in plants (Bates and Browse, 2012; Fig. 1). The de novo biosynthesis from glycerol-3-phosphate and acyl-CoA occurs via the Kennedy pathway and includes three acyltransferases: glycerol-2-phosphate acyltransferase, acyl-CoA:lysophosphatidic acid acyltransferase (LPAT), and acyl-CoA:diacylglycerol acyltransferase (DGAT; Kennedy, 1961). Alternatively, acyl-CoAs can be redirected from phosphatidylcholine (PC) via the action of a phospholipase C, choline phosphotransferase, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT; Hu et al., 2012; Lu et al., 2009), or phospholipid:diacylglycerol acyltransferase (PDAT; Dahlqvist et al., 2000). An acyl group can be released from PC to generate lysophosphatidylcholine (LPC) by the back reaction of acyl-CoA:LPC acyltransferase (Stymne and Stobart, 1984; Wang et al., 2012) or a phospholipase A/acyl-CoA synthase (Chen et al., 2011).Open in a separate windowFigure 1.Schematic representation of the plant TAG biosynthesis network. Acyl editing can provide PC-modified fatty acids for de novo diacylglycerol/TAG synthesis. ACS, acyl-CoA synthase; CPT, CDP-choline:diacylglycerol choline phosphotransferase; G3P, glycerol-3-phosphate; GPAT, acyl-CoA:glycerol-3-phosphate acyltransferase; LPC acyltransferase, acyl-CoA:LPC acyltransferase; mFAS, modified fatty acid synthase (in this work, mFAS is CPS); PAP, phosphatidic acid phosphatase; PLA, phospholipase A; PLC, phospholipase C.LPAT is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid (LPA) into different phosphatidic acids (PAs) in diverse tissues. Membrane-associated LPAT activities, identified in bacteria, yeast, plant, and animal cells, catalyze the transfer of acyl groups from acyl-CoA to LPA to synthesize PA. In plants and other organisms, LPAT activities have been identified in the endoplasmic reticulum (Kim et al., 2005), plasma membrane (Bursten et al., 1991), and mitochondria (Zborowski and Wojtczak, 1969). In higher plants, endoplasmic reticulum-localized LPAT plays an essential role transferring fatty acid from CoA esters to the sn-2 position of LPA in the synthesis of PA, a key intermediate in the biosynthesis of membrane phospholipids and storage lipids in developing seeds (Maisonneuve et al., 2010). LPAT from developing seeds of flax (Linum usitatissimum), rape (Brassica napus), and castor bean preferentially incorporate oleoyl-CoA, weakly incorporate cyclopropane acyl-CoA, and were unable to incorporate methyl-branched acyl-CoA when presented with an equimolar mix of these potential substrates (Nlandu Mputu et al., 2009). Thus, LPAT activity from agronomic plants constitutes a potential bottleneck for the incorporation of branched chain acyl-CoA into PA. In this work, we investigate the utility of an LPAT from a cyclopropanoid-syntheizing plant, S. foetida, with respect to its ability to enhance CPA accumulation. In our efforts to enhance CPA accumulation in transgenic plants, we screened CPS genes from diverse sources and identified Escherichia coli CPS (EcCPS) as an effective enzyme for the production of CPA in plants. However, EcCPS is reported to prefer the sn-2 position of E. coli phospholipid (Hildebrand and Law, 1964), and the data presented here show that its expression primarily leads to the accumulation of CPA at the stereospecific numbering (sn)-1 position. Moreover, coexpression of S. foetida lysophosphatidic acid acyltransferase (SfLPAT) results in the incorporation of CPA at the sn-2 position of LPA. Thus, coexpression of EcCPS and SfLPAT enables a cycle that enriches the accumulation of CPA at both sn-1 and sn-2 positions of PC and increases the accumulation of CPA. This work underscores the utility of coexpressing an acyltransferase from mFA-accumulating species with mFA-synthesizing enzymes to help mitigate bottlenecks in mFA TAG synthesis.     

Cyclopropane amino acid ester dipeptide sweeteners

A series of esters of L-aspartyl-1-aminocyclopropane carboxylic acid has been prepared and their sweet tastes determined. The sweetest ester prepared was about 300 times sweeter than sucrose. An attempt to use basic conditions during preparation of the dipeptide allyl ester led to succinimide formation of the aspartyl peptide even though the beta-carboxyl group was protected by a t-butyl ester function. The X-ray structure of the propyl ester (1c) was determined and its conformation is discussed.     

Functional Analysis of Leishmania Cyclopropane Fatty Acid Synthetase

The single gene encoding cyclopropane fatty acid synthetase (CFAS) is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular) and host (intracellular) stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.     

Cyclopropane ring formation in membrane lipids of bacteria.

It has been known for several decades that cyclopropane fatty acids (CFAs) occur in the phospholipids of many species of bacteria. CFAs are formed by the addition of a methylene group, derived from the methyl group of S-adenosylmethionine, across the carbon-carbon double bond of unsaturated fatty acids (UFAs). The C1 transfer does not involve free fatty acids or intermediates of phospholipid biosynthesis but, rather, mature phospholipid molecules already incorporated into membrane bilayers. Furthermore, CFAs are typically produced at the onset of the stationary phase in bacterial cultures. CFA formation can thus be considered a conditional, postsynthetic modification of bacterial membrane lipid bilayers. This modification is noteworthy in several respects. It is catalyzed by a soluble enzyme, although one of the substrates, the UFA double bond, is normally sequestered deep within the hydrophobic interior of the phospholipid bilayer. The enzyme, CFA synthase, discriminates between phospholipid vesicles containing only saturated fatty acids and those containing UFAs; it exhibits no affinity for vesicles of the former composition. These and other properties imply that topologically novel protein-lipid interactions occur in the biosynthesis of CFAs. The timing and extent of the UFA-to-CFA conversion in batch cultures and the widespread distribution of CFA synthesis among bacteria would seem to suggest an important physiological role for this phenomenon, yet its rationale remains unclear despite experimental tests of a variety of hypotheses. Manipulation of the CFA synthase of Escherichia coli by genetic methods has nevertheless provided valuable insight into the physiology of CFA formation. It has identified the CFA synthase gene as one of several rpoS-regulated genes of E. coli and has provided for the construction of strains in which proposed cellular functions of CFAs can be properly evaluated. Cloning and manipulation of the CFA synthase structural gene have also enabled this novel but extremely unstable enzyme to be purified and analyzed in molecular terms and have led to the identification of mechanistically related enzymes in clinically important bacterial pathogens.     

Synthesis of Phosphonate Analogues of the Antiviral Cyclopropane Nucleoside A-5021

A series of phosphonate analogues of the antiviral cyclopropane nucleoside A-5021 were synthesized from (1S*, 7R*)-3,5-dioxa-4,4-diphenylbicyclo[5.1.0]octane-l-methanol by a 10-step process. In contrast to the potent antiherpetic activity of A-5021, they were all devoid of antiviral activity.     

Synthesis and Antitumor Activity of Cyclopropane Derivatives of Betulinic and Betulonic Acids

New cyclopropane derivatives of betulin were synthesized by attachment of dichlorocarbenes or dibromocarbenes to the double bond of betulin diacetate, followed by the deprotection of hydroxyl groups. The betulin cyclopropane derivative was obtained from 20,29-dihydro-20,29-dichloromethylenebetulin by treatment with lithium in tert-butanol. These compounds were converted into the corresponding derivatives of betulonic acid by oxidation with chromium trioxide. The reduction of oxo group with sodium borohydride led to the corresponding derivatives of betulinic acid. 20,29-Dihydro-20,29-dichloromethylenebetulinic acid proved to be the most cytotoxic toward human melanoma of the Colo 38 and Bro lines and human ovarian carcinoma of the CaOv line (IC₅₀ 10 μM). 20,29-Dihydro-20,29-dibromomethylenebetulinic acid inhibited the growth of the Bro melanoma cell line and the CaOv carcinoma cell line practically by 50% at a concentration of 10 μM. The other derivatives of betulinic and betulonic acids were active toward all of the three cancer cell lines at concentrations higher than 10 μM.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 320–325.Original Russian Text Copyright © 2005 by Symon, Veselova, Kaplun, Vlasenkova, Fedorova, Lyutik, Gerasimova, Shvets.     

The Effect of Cyclopropane on Ion Uptake and Protoplasmic Streaming in Barley Roots

Increasing concentrations of cyclopropane up to about 95 % C₃H₆—5 % O₂ caused an increasing inhibition of uptake by excised barley roots from KC1 solution, much more so for Cl than K. No inhibition of K uptake from K₂SO₄ occurred under similar conditions. It is suggested that the small decrease in K uptake from KC1 is related to the large decrease in Cl uptake and that C₃H₆ primarily inhibits Cl uptake with little or no effect on K uptake. Time curves of uptake in 80 % C₃H₆ 20 % O₂ revealed no indication of cumulative injury in either KC1 or K₂SO₄ for periods up to 3 hours. The amount of organic acid production associated with K uptake from K₂SO₄ was about the same in 80 % C₃H_g— 20 % O₂ as in air. Oxygen consumption in K₂SO₄ was unaffected by the gas mixture and only slightly affected in KC1. Protoplasmic streaming in epidermal cells was rapidly stopped at C₃H₆ concentrations of 20 % or higher. In 80 % C₃H₆–20 % O₂ the effect on streaming was confined mostly to the outer two or three layers of root cells. A connection between the cessation of streaming and the inhibition of Cl uptake appeared to exist but very little if any relationship between streaming and K uptake was observed.     

Configurational Relationship between Substituents on Cyclopropane Ring of Pyrethroids and Their Insecticidal Toxicity

Several methyl-substituted cyclopropanecarboxylates were prepared and their steric configurations were established. The insecticidal activity against the housefly of their 5-benzyl-3-furylmethyl esters were tested. Among the substituents on cyclopropane ring, cis-methyl group to ester linkage has been found to be the most essential part for toxicity and trans-methyl and trans-isobutenyl groups greatly enhance its toxic activity.