Erratum to: Characterization of a KCS-like KASII from<Emphasis Type="Italic"> Jessenia bataua</Emphasis> that Elongates Saturated and Monounsaturated Stearic Acids in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

As the world population grows, the demand for food increases. Although vegetable oils provide an affordable and rich source of energy, the supply of vegetable oils available for human consumption is limited by the “fuel vs food” debate. To increase the nutritional value of vegetable oil, metabolic engineering may be used to produce oil crops of desirable fatty acid composition. We have isolated and characterized β-ketoacyl ACP-synthase II (KASII) cDNA from a high-oleic acid palm, Jessenia bataua. Jessenia KASII (JbKASII) encodes a 488-amino acid polypeptide that possesses conserved domains that are necessary for condensing activities. When overexpressed in E. coli, recombinant His-tagged JbKASII was insoluble and non-functional. However, Arabidopsis plants expressing GFP-JbKASII fusions had elevated levels of arachidic acid (C20:0) and erucic acid (C22:1) at the expense of stearic acid (C18:0) and oleic acid (C18:1). Furthermore, JbKASII failed to complement the Arabidopsis KASII mutant, fab1-2. This suggests that the substrate specificity of JbKASII is similar to that of ketoacyl-CoA synthase (KCS), which preferentially elongates stearic and oleic acids, and not palmitic acid. Our results suggest that the KCS-like JbKASII may elongate C18:0 and C18:1 to yield C20:0 and C22:1, respectively. JbKASII may, therefore, be an interesting candidate gene for promoting the production of very long chain fatty acids in transgenic oil crops.     

Production of 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from olive oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Microbial modification of naturally occurring materials is one of the efficient ways to add new values to them. Hydroxylation of free unsaturated fatty acids by microorganism is a good example of those modifications. Among microbial strains studied for that purpose, a new bacterial isolate Pseudomonas aeruginosa PR3 has been well studied to produce several hydroxy fatty acids from different unsaturated fatty acids. Of those hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was efficiently produced from oleic acid by strain PR3. However, it was highly plausible to use vegetable oil containing oleic acid rather than free oleic acid as a substrate for DOD production by strain PR3. In this study, we firstly tried to use olive oil containing high content of oleic acid as a substrate for DOD production. DOD production from olive oil was confirmed by structural determination with GC, TLC, and GC/MS analysis. DOD production yield from olive oil was 53.5%. Several important environmental factors were also tested. Galactose and glutamine were optimal carbon and nitrogen sources, and magnesium ion was critically required for DOD production from olive oil. Results from this study demonstrated that natural vegetable oils containing oleic acid could be used as efficient substrate for the production of DOD by strain PR3.     

Metabolic engineering of fatty acid biosynthetic pathway in sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.): assembling tools to develop nutritionally desirable sesame seed oil

Vegetable oils are an essential component of human diet, in terms of their health beneficial roles. Despite their importance, the fatty acid profile of most commonly used edible oil seed crop plants are imbalanced; this skewed ratio of fatty acids in the diet has been shown to be a major reason for the occurrence of cardiovascular and autoimmune diseases. Until recently, it was not possible to exert significant control over the fatty acid composition of vegetable oils derived from different plants. However, the advent of metabolic engineering, knowledge of the genetic networks and regulatory hierarchies in plants have offered novel opportunities to tailor-made the composition of vegetable oils for their optimization in regard to food functionality and dietary requirements. Sesame (Sesamum indicum L.) is one of the ancient oilseed crop in Indian subcontinent but its seed oil is devoid of balanced proportion of ω-6:ω-3 fatty acids. A recent study by our group has shed new lights on metabolic engineering strategies for the purpose of nutritional improvement of sesame seed oil to divert the carbon flux from the production of linoleic acid (C18:2) to α-linolenic acid (C18:3). Apart from that, this review evaluates current understanding of regulation of fatty acid biosynthetic pathways in sesame and attempts to identify the major options of metabolic engineering to produce superior sesame seed oil.     

Production of <Emphasis Type="Italic">cis</Emphasis>-Vaccenic Acid-oriented Unsaturated Fatty Acid in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biodiesel is produced worldwide as an alternative energy fuel and substitute for petroleum. Biodiesel is often obtained from vegetable oil, but production of biodiesel from plants requires additional land for growing crops and can affect the global food supply. Consequently, it is necessary to develop appropriate microorganisms for the development of an alternative biodiesel feedstock. Escherichia coli is suitable for the production of biodiesel feedstocks since it can synthesize fatty acids for lipid production, grows well, and is amenable to genetic engineering. Recombinant E. coli was designed and constructed for the production of biodiesel with improved unsaturated fatty acid contents via regulation of the FAS pathway consisting of initiation, elongation, and termination steps. Here, we investigated the effects of fabA, fabB, and fabF gene expression on the production of unsaturated fatty acids and observed that the concentration of cis-vaccenic acid, a major component of unsaturated fatty acids, increased 1.77-fold compared to that of the control strain. We also introduced the genes which synthesize malonyl-ACP used during initiation step of fatty acid synthesis and the genes which produce free fatty acids during termination step to study the effect of combination of genes in elongation step and other steps. The total fatty acid content of this strain increased by 35.7% compared to that of the control strain. The amounts of unsaturated fatty acids and cis-vaccenic acid increased by 3.27 and 3.37-fold, respectively.     

Production of a value-added hydroxy fatty acid, 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid,from high oleic safflower Oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFAs), originally obtained in small amounts from plant systems, are good examples of structurally modified lipids, and they render special properties such as higher viscosity and reactivity compared to normal fatty acids. Based on these properties, HFAs possess high industrial potential in a wide range of applications. Recently, various microbial strains were tested for the production of HFAs from different unsaturated fatty acids since HFA production is limited to plant systems. Among the microbial strains tested, Pseudomonas aeruginosa PR3 has been well studied for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) from oleic acid. Previously, we reported that strain PR3 could utilize triolein instead of oleic acid as a substrate for the production of DOD (Appl. Microbiol. Biotechnol. 2007, 74: 301–306). In this study, we focused on utilization of vegetable oil as a substrate for DOD production by PR3. Consequently, strain PR3 efficiently utilized high oleic safflower oil as a substrate for DOD production. Optimal initial medium pH and incubation time were pH 8.0 and 72 h, respectively. Optimal carbon and nitrogen sources were fructose and glutamine, respectively. Results from this study demonstrate that normal vegetable oils could be used as efficient substrates for the production of value-added HFAs by microbial bioconversion.     

Production of 7, 10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from triolein via lipase induction by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFA) have gained importance because of their special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. The bacterial isolate Pseudomonas aeruginosa (PR3) was reported to produce mono-, di-, and trihydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced with high yield from oleic acid by PR3. Up to now, the substrates used for microbial HFA production were free fatty acids. However, it is possible to utilize triacylglycerides, specifically triolein containing three oleic groups, as a substrate by microbial enzyme system involved in HFA production from oleic acid. In this study we used triolein as a substrate and firstly report that triolein could be efficiently utilized by PR3 to produce DOD. Triolein was first hydrolyzed into oleic acid by the triolein-induced lipase and then the released oleic acid was converted to DOD by PR3. Results from this study demonstrated that natural vegetable oils, without being intentionally hydrolyzed, could be used as efficient substrates for the microbial production of value-added hydroxy fatty acids.     

Overexpression of <Emphasis Type="Italic">SsDGAT2</Emphasis> from <Emphasis Type="Italic">Sapium sebiferum</Emphasis> (L.) Roxb Increases Seed Oleic Acid Level in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Sapium sebiferum (L.) Roxb is one of the most important oil trees in China. Diacylglycerol acyltransferases (DGATs) esterify sn-1, 2-diacylglycerol with a long-chain fatty acyl-CoA, the last step and the rate-limiting step of triacylglycerol (TAG) biosynthesis in prokaryotic and eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the SsDGAT2 gene had not been reported to date. To clarify the function of SsDGAT2, we cloned the CDS (rapid amplification of cDNA end) of SsDGAT2 by RACE technology. The full-length CDS of SsDGAT2 contains 1011 bp and encodes a protein of 336 amino acids. Recombinant SsDGAT2 restored TAG biosynthesis to the yeast strain Saccharomyces cerevisiae H1246 TAG-deficient mutant and preferentially incorporated unsaturated C18 fatty acids into lipids. To investigate the biotechnological potential of SsDGAT2, it was expressed under the control of the 35S promoter in Arabidopsis Col-4. The oleic acid content increased by 50 % in transgenic plants relative to the control. The results indicated that most of the oleic acid increase was at the expense of linolenic acid (18:3) content, which suggests that high-oleic-acid-content seeds can be created by the overexpression of SsDGAT2 in S. sebiferum (L.) Roxb.     

Mapping the <Emphasis Type="Italic">Fas</Emphasis> locus controlling stearic acid content in soybean

Increasing the stearic acid content to improve soybean [ Glycine max (L) Merr] oil quality is a desirable breeding objective for food-processing applications. Although a saturated fatty acid, stearic acid has been shown to reduce total levels of blood cholesterol and offers the potential for the production of solid fat products (such as margarine) without hydrogenation. This would result in the reduction of the level of trans fat in food products and alleviate some current health concerns. A segregating F(2) population was developed from the cross between Dare, a normal stearic acid content cultivar, and FAM94-41, a high stearic acid content line. This population was used to assess linkage between the Fas locus and simple sequence repeat (SSR) markers. Three SSR markers, Satt070, Satt474 and Satt556, were identified to be associated with stearic acid (P < 0.0001, r(2) > 0.61). A linkage map consisting of the three SSR markers and the Fas locus was then constructed in map order, Fas, Satt070, Satt474 and Satt556, with a LOD score of 3.0. Identification of these markers may be useful in molecular marker-assisted breeding programs targeting modifications in soybean fatty acids.     

Overexpression of <Emphasis Type="Italic">FAD2</Emphasis> promotes seed germination and hypocotyl elongation in <Emphasis Type="Italic">Brassica napus</Emphasis>

The enzyme fatty acid desaturase 2 (FAD2) transforms oleic acid (C18:1) to linoleic acid (C18:2) in plants and as such is involved in fatty acid synthesis. It is also involved in plant development and self-defense, such as seed germination, leaf expansion and cold resistance. We have cloned the full coding region of the Brassica napus FAD2 gene and ectopically expressed it in B. napus expressing low levels of FAD2. Overexpression of FAD2 under the control of the CaMV 35S promoter resulted in an up-regulated FAD2 mRNA level in B. napus as expected. Further analysis revealed that the FAD2 transgenic lines varied greatly in terms of their physiological characteristics, such as enhanced seed germination and increased hypocotyl length, compared to non-transgenic plants, suggesting that up-regulated FAD2 can promote seed germination and hypocotyl elongation in B. napus. Our results demonstrate the possible roles of FAD2 in plant development and also provide a platform for further analysis of fatty acid synthesis in plants.     

Heterologous expression of a fatty acid hydroxylase gene in developing seeds of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Expression of a cDNA encoding the castor bean ( Ricinus communis L.) oleate Delta12-hydroxylase in the developing seeds of Arabidopsis thaliana (L.) Heynh. results in the synthesis of four novel hydroxy fatty acids. These have been previously identified as ricinoleic acid (12-hydroxy-octadec- cis-9-enoic acid: 18:1-OH), densipolic acid (12-hydroxy-octadec- cis-9,15-enoic acid: 18:2-OH), lesquerolic acid (14-hydroxy-eicos- cis-11-enoic acid: 20:1-OH) and auricolic acid (14-hydroxy-eicos- cis-11,17-enoic acid: 20:2-OH). Using mutant lines of Arabidopsis that lack the activity of the FAE1 condensing enzyme or FAD3 ER Delta-15-desaturase, we have shown that these enzymes are required for the synthesis of C20 hydroxy fatty acids and polyunsaturated hydroxy fatty acids, respectively. Analysis of the seed fatty acid composition of transformed plants demonstrated a dramatic increase in oleic acid (18:1) levels and a decrease in linoleic acid (18:2) content correlating to the levels of hydroxy fatty acid present in the seed. Plants in which FAD2 (ER Delta12-desaturase) activity was absent showed a decrease in 18:1 content and a slight increase in 18:2 levels corresponding to hydroxy fatty acid content. Expression of the castor hydroxylase protein in yeast indicates that this enzyme has a low level of fatty acid Delta12-desaturase activity. Lipase catalysed 1,3-specific lipolysis of triacylglycerol from transformed plants demonstrated that ricinoleic acid is not excluded from the sn-2 position of triacylglycerol, but is the only hydroxy fatty acid present at this position.     

Efficient accumulation of oleic acid in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> caused by expression of rat elongase 2 gene (<Emphasis Type="Italic">rELO2</Emphasis>) and its contribution to tolerance to alcohols

When the cells of Saccharomyces cerevisiae are exposed to high concentration of ethanol, the content of oleic acid (C18:1n-9) increased as the initial concentration of ethanol increased. Based on this observation, we attempted to confer ethanol tolerance to S. cerevisiae by manipulating fatty acid composition of the cells. Rather than altering OLE1 expression [the desaturase making both C16:1n-7 (palmitoleic acid) and C18:1n-9], we introduced elongase genes. Introduction of rat elongase 1 gene (rELO1) into S. cerevisiae gave cis-vaccenic acid (cis-C18:1n-7) by conversion from C16:1n-7, and the increase in this C18:1 fatty acid did not confer ethanol tolerance to the cells. On the other hand, the introduction of rat elongase 2 gene (rELO2), which elongates C16:0 to C18:0, drastically increased C18:1n-9 content, and the cells acquired ethanol tolerance, emphasizing the specific role of C18:1n-9. Furthermore, the transformant of rELO2 also conferred tolerance to n-butanol, n-propanol, and 2-propanol.     

Physicochemical properties of cold pressed sunflower,peanut, rapeseed,mustard and olive oils grown in the Eastern Mediterranean region

Fatty acid composition and stability of vegetable oils have taken more attention as an essential source of biologically active compounds in a good balanced diet. The purpose of the study was to determine peroxide value, free fatty acids, unsaponifiable matter, total carotenoid content, iodine value and fatty acid composition of sunflower, rapeseed, mustard, peanut and olive oils. Rapeseed and peanut oils had the highest peroxide values, while sunflower oil had the lowest peroxide values. The free fatty acid value of the tested oils varied between 0.43 and 1.36% oleic. The peanut oil had the highest free acid value and the mustard oil had the lowest one. Total carotenoid contents of mustard and rape seed oil were higher than those of the other oils tested. Palmitic acid (C16:0), oleic acid (C18:1) and stearic acid (C18:0) were the common main fatty acid components of the vegetable oils tested. Followed by linoleic acid, the amount of oleic acid was the highest among other fatty acid components. Mustard oil had the highest erucic acid (C22:1) with the amount of 11.38%, indicating that it cannot be used for human consumption. Among the oils investigated, sunflower and mustard oils were more stable than rapeseed, peanut and olive oils.     

Sophorolipid production by <Emphasis Type="Italic">Candida bombicola</Emphasis> on oils with a special fatty acid composition and their consequences on cell viability

Sophorolipids production by the yeast Candia bombicola is most favourable when glucose is used as a carbon source in combination with a hydrophobic carbon source such as a common vegetable oil. Most vegetable oils are comprised of C16–C18 fatty acids, an ideal range for sophorolipid production. The use of oils with either shorter or longer fatty acids, such has coconut oil or meadowfoam oil, respectively, was evaluated. Such oils did not contribute to enhanced sophorolipid production when compared to cultures run on glucose as the sole carbon source. Moreover, a toxic effect of medium-chain fatty acids towards stationary C. bombicola cells was demonstrated.     

<Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> associated bioactive bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NJ6-3-1

Among marine bacteria isolated from the cytotoxic sponge Hymeniacidon perleve, one strain NJ6-3-1 classified as Pseudomonas sp. showed both cytotoxic and antimicrobial activities. Fatty acid analysis indicated that the bacterial strain consists mainly of C16:1, C16:0, C18:1, C18:0, C15:0, C14:0. One unusual 9,10-cyclopropane-C17:0 fatty acid and C26:0 also constitute major components, as well as the existence of squalene, the precursor of triterpenoids. The major metabolites in the culture broth were identified as alkaloids, including diketopiperazines and indole compounds, namely 3,6-diisopropylpiperazine-2,5-dione, 3-benzyl-3-isopropylpiperazine-2,5-dione, 3,6-bis-(2-methylpropyl)-piperazine-2,5-dione, indole-3-carboxaldehyde, indole-3-carboxylic acid methyl ester, indole-3-ethanol, and quinazoline-2,4-dione.From Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 35–39.Original English Text Copyright © 2005 by Li Zheng, Xiaojun Yan, Jilin Xu, Haimin Chen, Wei Lin.This article was submitted by the authors in English.     

Temperature-related non-homogeneous fatty acid desaturation in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) seeds

The fatty acid compositions of half-seeds and whole seeds of the temperature-dependent high-stearic-acid sunflower (Helianthus annuus L.) mutant CAS-14 were unexpectedly different. We found that there is a longitudinal gradient starting from the embryo up to the end of the cotyledon. The stearic acid content varied from 9.7 to 34.6% in seeds produced in a growth chamber (39/24 degrees C; day/night), and from 14.0 to 34.4% in seeds produced in the field during the summer season (35-40 degrees C in daylight and 20-25 degrees C at night). The gradient occurs throughout seed formation, and is due to a spatial and non-temporal regulation of stearic acid desaturation. A similar temperature-regulated behaviour, but for oleic and linoleic acid contents, was found in normal sunflower seeds. Since the deposition of oil bodies was homogeneous during seed formation, seeds showed the gradient throughout their development. This non-homogeneous distribution must be due to differences in the enzymatic pathway of de-novo fatty acid desaturation along the seed, resembling a morphogen gradient. Other high-stearic-acid mutant lines, such as CAS-3, did not show any gradient. This is the first time that a gradient and an inheritable maternal control of the fatty acid composition have been found in oilseeds.