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.     

Environmental optimization for bioconversion of triolein into 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFAs), originally found in small amount mainly from plant systems, are well known to have special properties such as higher viscosity and reactivity compared with other normal fatty acids. Recently, various microbial strains were tested to produce HFAs from different unsaturated fatty acids. Among those microbial strains tested, Pseudomonas aeruginosa PR3 are well known to utilize various unsaturated fatty acids to produce mono-, di-, and tri-HFAs. Previously, we reported that strain PR3 could utilize triolein as a substrate for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) via the induction of lipase activity (Chang et al., Appl Microbiol Biotechnol, 74:301–306, 2007). In this study, we focused on the development of the optimal environmental conditions for DOD production from triolein by PR3. Optimal initial medium pH and incubation temperature were pH 8.0 and 25°C, respectively. Magnesium ion was essentially required for DOD production. Optimal inoculum size, time for substrate addition, and substrate concentration were 1%, 12 to 24 h, and 300 mg, respectively.     

Production and identification of a novel compound, 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-hexadecenoic acid from palmitoleic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids are considered as important value-added product for industrial application because of their special properties such as higher viscosity and reactivity. Microbial production of the hydroxy fatty acids from various fatty acid substrates have been actively studied using several microorganisms. The new bacterial isolate Pseudomonas aeruginosa (PR3) had been reported to produce mono-, di-, and tri-hydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) were produced from oleic acid and ricinoleic acid, respectively. Based on the postulated common metabolic pathway involved in DOD and TOD formation by PR3, it was assumed that palmitoleic acid containing a singular 9-cis double bond, common structural property sharing with oleic acid and ricinoleic acid, could be utilized by PR3 to produce hydroxy fatty acid. In this study, we tried to use palmitoleic acid as substrate for production of hydroxy fatty acid by PR3 and firstly confirmed that PR3 could produce 7,10-dihydroxy-8(E)-hexadecenoic acid (DHD) with 23% yield from palmitoleic acid. DHD production was peaked at 72 h after the substrate was added to the 24-h-culture.     

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.     

Diversity of Oleic Acid,Ricinoleic Acid and Linoleic Acid Conversions Among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strains*

Sixteen Pseudomonas aeruginosa strains, including patent strain NRRL B-18602, three recent isolates from composted materials amended with ricinoleic acid, and 12 randomly selected from the holdings of the ARS Culture Collection, were examined for their fatty acid converting abilities. The study examined the bioconversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and ricinoleic acid to 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD). A new DOD-like compound from linoleic acid was observed. All strains except NRRL B-247 exhibited varying levels of DOD production. NRRL B-1000, NRRL B-18602 and NRRL B-23258 with yields up to 84% were among the best DOD producers. TOD production generally paralleled DOD production at a relatively lower yield of up to 15%. Strains NRRL B-1000 and NRRL B-23260 were the best TOD producers. A DOD-like product in low yields was obtained from linoleic acid. The fatty acid bioconversion capability was related neither to growth rate nor to variation in the greenish pigmentation of the strains. Production of significant quantities of DOD and TOD from oleic and ricinoleic acids, respectively, appeared to be a characteristic trait of P. aeruginosa strains. A number of highly effective strains for DOD production were identified.     

Factors Influencing the Production of a Novel Compound, 7,10-Dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic Acid,by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3 (NRRL B-18602) in Batch Cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002     

The <Emphasis Type="Italic">Lactococcus lactis</Emphasis> FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of <Emphasis Type="Italic">Escherichia coli</Emphasis> and the FabH protein of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli, one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild-type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacyl-acyl carrier protein synthase. The strain tested was a derivative (called the ∆fabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ∆fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ∆fabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.     

Anti-cyanobacterial fatty acids released from <Emphasis Type="Italic">Myriophyllum spicatum</Emphasis>

This study was carried out to identify unknown allelochemicals released from Myriophyllum spicatum and to investigate their anti-cyanobacterial effects. A series of analyses of culture solutions and methanol extracts of M. spicatum using gas chromatograph equipped with a mass selective detector revealed that M. spicatum released fatty acids, specifically, nonanoic, tetradecanoic, hexadecanoic, octadecanoic, and octadecenoic acids. Nonanoic, cis-6-octadecenoic, and cis-9-octadecenoic acids significantly inhibited growth of Microcystis aeruginosa, whereas tetradecanoic, hexadecanoic, and octadecanoic acids did not show any effect. When the inhibitory effect of nonanoic acid was compared with those of 4 polyphenols and eugeniin, which are anti-cyanobacterial compounds previously reported to be released by M. spicatum, nonanoic acid was found to be the most inhibitory to M. aeruginosa. These results indicate that not only polyphenols and eugeniin but also fatty acids such as nonanoic acid must be studied to reveal how M. spicatum exerts its allelopathic effect on M. aeruginosa.     

Cloning and expression of a lipoxygenase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> 42A2

In order to produce (S) 10-monohydroxy-8E-octadecenoic acid (MHOD) from oleic acid, a full-length probable lipoxygenase cDNA from Pseudomonas aeruginosa 42A2 was cloned and expressed in Escherichia coli BL21(DE3). The recombinant protein was purified by affinity chromatography to electrophoretic homogeneity and specifically stained. Its molecular mass was 70 kDa. The activity of the rec-LOX with oleic acid was about 30% of that of the prefered substrate, linoleic acid (100%). Bacterial LOX forms a new subfamily in the lipoxygenase phylogenetic tree.     

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.     

Influence of nitrogen deficiency on biochemical composition of the green alga <Emphasis Type="Italic">Botryococcus</Emphasis>

A study has been carried out to investigate the influence of nitrogen deficiency on intracellular lipid composition, including total fatty acid composition of lipids, polar lipids, and triacylglycerols, of the alga Botryococcus braunii Kütz IPPAS H-252 in batch culture. Under nitrogen limitation, the alga accumulates lipids as triacylglycerols and the total fatty acid (FA) composition changes: trienoic acids decrease (from 52.8–57.2 to 19.5–24.7% of the total FAs) and the oleic acid increases (from 1.1–1.2 to 17.1–24.4%) as does the saturated acids (from 23.7–26 to 32.9–46.1%). A similar rearrangement in the FA spectrum occurs at later times in the control culture, but it is less pronounced. Under nitrogen limitation, considerable changes in the polar lipid FAs are registered at day 13: saturated acids increase (from 28.6–35.5 to 76.8%) and all polyenoic acids markedly decrease (from 56.9–64.1 to 6.8%). Changes in the triacylglycerol fatty acid spectrum are seen on day 7: the oleic acid increases (from 14.7 to 34.2%) and remains at a high level till the end of the culture. In the control, triacylglycerols with large contents of oleic acid are detected at day 13, the total lipids and triacylglycerols still remaining unchanged.     

Overexpression of Acetyl-CoA Carboxylase Gene of <Emphasis Type="Italic">Mucor rouxii</Emphasis> Enhanced Fatty Acid Content in <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Malonyl-CoA is an essential precursor for fatty acid biosynthesis that is generated from the carboxylation of acetyl-CoA. In this work, a gene coding for acetyl-CoA carboxylase (ACC) was isolated from an oleaginous fungus, Mucor rouxii. According to the amino acid sequence homology and the conserved structural organization of the biotin carboxylase, biotin carboxyl carrier protein, and carboxyl transferase domains, the cloned gene was characterized as a multi-domain ACC1 protein. Interestingly, a 40% increase in the total fatty acid content of the non-oleaginous yeast Hansenula polymorpha was achieved by overexpressing the M. rouxii ACC1. This result demonstrated a significant improvement in the production of fatty acids through genetic modification in this yeast strain.     

Expression of a lipid-inducible,self-regulating form of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> lipase <Emphasis Type="Italic">LIP2</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Saccharomyces cerevisiae is frequently used as a bioreactor for conversion of exogenously acquired metabolites into value-added products, but has not been utilized for bioconversion of low-cost lipids such as triacylglycerols (TAGs) because the cells are typically unable to acquire these lipid substrates from the growth media. To help circumvent this limitation, the Yarrowia lipolytica lipase 2 (LIP2) gene was cloned into S. cerevisiae expression vectors and used to generate S. cerevisiae strains that secrete active Lip2 lipase (Lip2p) enzyme into the growth media. Specifically, LIP2 expression was driven by the S. cerevisiae PEX11 promoter, which maintains basal transgene expression levels in the presence of sugars in the culture medium but is rapidly upregulated by fatty acids. Northern blotting, lipase enzyme activity assays, and gas chromatographic measurements of cellular fatty acid composition after lipid feeding all confirmed that cells transformed with the PEX11 promoter–LIP2 construct were responsive to lipids in the media, i.e., cells expressing LIP2 responded rapidly to either free fatty acids or TAGs and accumulated high levels of the corresponding fatty acids in intracellular lipids. These data provided evidence of the creation of a self-regulating positive control feedback loop that allows the cells to upregulate Lip2p production only when lipids are present in the media. Regulated, autonomous production of extracellular lipase activity is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion of low-value lipids to value-added TAGs and other novel lipid products.     

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.     

<Emphasis Type="Italic">Trans</Emphasis> Fatty Acids in Membranes: The Free Radical Path

The double bond geometry of most of the naturally occurring unsaturated fatty acid residues is cis. Due to the relevance of fatty acids as structural components of cell membranes and as biologically active molecules, the change of the cis geometry means a change of the associated functions and activities. The finding that the cis to trans isomerization is effective in phospholipids by the intervention of radical species led to the discovery that there can indeed occur an endogenous formation of trans fatty acids, whose significance in biological systems started to be addressed with in vitro and in vivo studies. Studies of liposome models simulating the formation of isomerizing species and evaluating their ability to interact with the hydrophobic part of the membrane bilayer has contributed to the gain in knowledge of the fundamental features of the lipid isomerization in membranes. Further work is in progress for the identification of the real culprits of the in vivo lipid isomerization, and recent results are shown on oleic acid micelles, where ^•NO₂ radicals are not able to induce double bond isomerization in comparison with amphiphilic thiol, such as 2-mercaptoethanol. H₂S and sulfur-containing amino acid residues are two of the possible species involved in this process at a biological level. An update of the scenario of the geometrical isomerization in membranes by free radicals is provided, together with applications and perspectives in life sciences.     

Thermostable lipoxygenase is a key enzyme in the conversion of linoleic acid to trihydroxy-octadecenoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Lipoxygenases (LOXs) constitute a family of lipid-peroxidizing enzymes that catalyze the oxidation of unsaturated fatty acid containing a (1Z,4Z)-pentadiene structural unit, leading to formation of conjugated (Z,E)-hydroperoxydienoic acid. LOXs are known to be widely distributed in plants and animals. Recently, several microbial LOXs were reported to be involved in the production of hydroperoxy fatty acids. Among the microorganisms that produce hydroxy fatty acids, Pseudomonas aeruginosa PR3 is known to convert linoleic acid to trihydroxy fatty acid, which suggests the involvement of a LOX enzyme. Based on these reports, we identified a novel thermostable LOX from P. aeruginosa PR3 strain. The protein was purified 34.3-fold with a recovery rate of 5.14%. The K_m and V_max values of the purified enzyme were 3.57 mM and 0.73 μmol/min//mg, respectively. Heat stability of the purified enzyme was unexpectedly high with an LD₅₀ of 90 min at 80°C, although P. aeruginosa PR3 is known as a mesophilic bacterium. Substrate specificity of the purified enzyme was restricted only to unsaturated fatty acids carrying a (1Z,4Z)-pentadiene unit.     

Substrate specificity of <Emphasis Type="Italic">Stenotrophomonas nitritireducens</Emphasis> in the hydroxylation of unsaturated fatty acid

An isolated bacterium that converted unsaturated fatty acids to hydroxy fatty acids was identified as Stenotrophomonas nitritireducens by API analysis, cellular fatty acids compositions, sequencing the full 16S ribosomal ribonucleic acid, and evaluating its nitrite reduction ability. S. nitritireducens has unique regio-specificity for C16 and C18 cis-9 unsaturated fatty acids. These fatty acids are converted to their 10-hydroxy fatty acids without detectable byproducts. Among the cis-9-unsaturated fatty acids, S. nitritireducens showed the highest specificity for linoleic acid. The cells converted 20 mM linoleic acid to 13.5 mM 10-hydroxy-12(Z)-octadecenoic acid at 30°C and pH 7.5 with a yield of 67.5% (mol/mol).     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

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.     

Fatty acid synthesis in isolated leucoplasts of developing seeds of <Emphasis Type="Italic">Brassica campestris</Emphasis> L

Under in vitro conditions, the fatty acid synthesis from labelled substrates was studied in the leucoplasts isolated from developing seeds of Brassica campestris L. The rate of fatty acid synthesis with Na-(1-¹⁴C) acetate was higher at lower concentrations (up to 1 mM). However, with ¹⁴C(U)-D-glucose, the rate was higher at higher concentrations (3–4 mM) at all the three stages of seed development. ATP and NAD(P)H were absolutely required in acetate utilization. Even for glucose utilization, the exogenous supply of ATP and NAD(P)H was required. At the early stage of seed development, the maximum reduction in labelled glucose and acetate utilization for fatty acid synthesis was observed with pyruvate and glucose, respectively. However, at mid-early and mid-late stages, maximum reduction in their utilization for fatty acid synthesis was observed with glc-6-P. This suggests a shift in the utilization of substrates for fatty acid synthesis during the development of seeds probably via different translocators activated at different stages.