Translocation of the yeast dolichol-phosphate-mannose synthase into microsomal membranes.

Dolichol-phosphate-mannose synthase catalyzes the formation of Dolichol-phosphate-mannose from Dolichol-phosphate and GDP-mannose. Analysis of the primary amino acid sequence of the yeast enzyme predicts a luminal orientation of the enzyme in the endoplasmic reticulum. We analysed the translocation of the Dolichol-phosphate-mannose synthase into dog pancreatic microsomal membranes: resistance to proteolytic attack provides evidence of its luminal orientation and asks for a reevaluation of the topology of the reaction.

Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes.

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome-targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β-oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species.

Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

A comparative study of the preventative effects exerted by two probiotics, Lactobacillus reuteri and Lactobacillus fermentum, in the trinitrobenzenesulfonic acid model of rat colitis

The intestinal anti-inflammatory effects of two probiotics isolated from breast milk, Lactobacillus reuteri and L. fermentum, were evaluated and compared in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis. Colitis was induced in rats by intracolonic administration of 10 mg TNBS dissolved in 50% ethanol (0.25 ml). Either L. reuteri or L. fermentum was daily administered orally (5 x 10(8) colony-forming units suspended in 0.5 ml skimmed milk) to each group of rats (n 10) for 3 weeks, starting 2 weeks before colitis induction. Colonic damage was evaluated histologically and biochemically, and the colonic luminal contents were used for bacterial studies and for SCFA production. Both probiotics showed intestinal anti-inflammatory effects in this model of experimental colitis, as evidenced histologically and by a significant reduction of colonic myeloperoxidase activity (P<0.05). L. fermentum significantly counteracted the colonic glutathione depletion induced by the inflammatory process. In addition, both probiotics lowered colonic TNFalpha levels (P<0.01) and inducible NO synthase expression when compared with non-treated rats; however, the decrease in colonic cyclo-oxygenase-2 expression was only achieved with L.fermentum administration. Finally, the two probiotics induced the growth of Lactobacilli species in comparison with control colitic rats, but the production of SCFA in colonic contents was only increased when L. fermentum was given. In conclusion, L. fermentum can exert beneficial immunomodulatory properties in inflammatory bowel disease, being more effective than L. reuteri, a probiotic with reputed efficacy in promoting beneficial effects on human health.

Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids

Studies in animal models and humans suggest anti-inflammatory roles on the N acylethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system in inflammatory bowel diseases. However, the presence and function of NAE-PPARα signaling system in the ulcerative colitis (UC) of humans remain unknown as well as its response to active anti-inflammatory therapies such as 5-aminosalicylic acid (5-ASA) and glucocorticoids. Expression of PPARα receptor and PPARα ligands-biosynthetic (NAPE-PLD) and -degrading (FAAH and NAAA) enzymes were analyzed in untreated active and 5-ASA/glucocorticoids/immunomodulators-treated quiescent UC patients compared to healthy human colonic tissue by RT-PCR and immunohistochemical analyses. PPARα, NAAA, NAPE-PLD and FAAH showed differential distributions in the colonic epithelium, lamina propria, smooth muscle and enteric plexus. Gene expression analysis indicated a decrease of PPARα, PPARγ and NAAA, and an increase of FAAH and iNOS in the active colitis mucosa. Immunohistochemical expression in active colitis epithelium confirmed a PPARα decrease, but showed a sharp NAAA increase and a NAPE-PLD decrease, which were partially restored to control levels after treatment. We also characterized the immune cells of the UC mucosa infiltrate. We detected a decreased number of NAAA-positive and an increased number of FAAH-positive immune cells in active UC, which were partially restored to control levels after treatment. NAE-PPARα signaling system is impaired during active UC and 5-ASA/glucocorticoids treatment restored its normal expression. Since 5-ASA actions may work through PPARα and glucocorticoids through NAE-producing/degrading enzymes, the use of PPARα agonists or FAAH/NAAA blockers that increases endogenous PPARα ligands may yield similar therapeutics advantages.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99%). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5%), followed by the wild-type A383/A553 (17.5%) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Chronic nitric oxide synthase inhibition and carotid artery distensibility in renal hypertensive rats.

The goal of the present study was to examine the viscoelastic properties of the carotid artery in genetically identical rats exposed to similar levels of blood pressure sustained by different mechanisms. Eight-week old male Wistar rats were examined 2 weeks after renal artery clipping (two-kidney, one clip [2K1C] Goldblatt rats, n = 53) or sham operation (n = 49). One half of the 2K1C and sham rats received the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 1.48 mmol/L) in their drinking water for 2 weeks after the surgical procedure. Mean blood pressure increased significantly in the 2K1C-water (182 mm Hg), 2K1C-L-NAME (197 mm Hg), and sham-L-NAME (170 mm Hg) rats compared with the sham-water rats (127 mm Hg). Plasma renin activity was not altered by L-NAME but significantly enhanced after renal artery clipping. A significant and similar increase in the cross-sectional area of the carotid artery was observed in L-NAME and vehicle-treated 2K1C rats. L-NAME per se did not modify cross-sectional area in the sham rats. There was a significant upward shift of the distensibility-pressure curve in the L-NAME- and vehicle-treated 2K1C rats compared with the sham-L-NAME rats. L-NAME treatment did not alter the distensibility-pressure curve in the 2K1C rats. These results demonstrate that the mechanisms responsible for artery wall hypertrophy in renovascular hypertension are accompanied by an increase in arterial distensibility that is not dependent on the synthesis of nitric oxide.

3-amino-1,2,4-triazole inhibits macrophage NO synthase.

Murine macrophages activated by interferon-gamma and lipopolysaccharide become leishmanicidal through a process involving L-arginine-derived nitrogen oxidation products. Both nitrite secretion and parasite killing by activated macrophages were inhibited by 3-amino-1,2,4-triazole as well as the related compound, 3-amino-1,2,4-triazine. Moreover, NO synthase activity in cytosolic extracts of activated cells was inhibited by both compounds. 4-amino-1,2,4-triazole, an isomer of 3-amino-1,2,4-triazole, was without effect. Our results suggest that besides its known inhibitory effect on catalases and peroxidases, 3-amino-1,2,4-triazole is an inhibitor of NO synthase. The resemblance between the tautomeric form of 3-amino-1,2,4-triazole and the guanidino group of L-arginine, the natural substrate for NO synthase, might be responsible for the observed inhibition.

Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae.

The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.

Etude des effets du monoxyde d'azote (NO) sur le métabolisme des acides gras libres à partir d'un modèle de souris invalidée pour le gène de la NO synthase endothéliale

RESUME GENERAL Au cours de ces dernières années, le monoxyde d'azote (NO) produit par une famille d'enzymes, les NO synthases (NOS), est apparu comme un effecteur central dans la régulation du système cardiovasculaire et du métabolisme énergétique. Chez l'homme, un défaut de production du NO est associé à des maladies cardiovasculaires et métaboliques comme la résistance à l'insuline ou le diabète de type 2. Ces pathologies se retrouvent chez les souris invalidées pour la NO synthase endothéliale (eN0S-/-) qui présentent non seulement une hypertension mais également une résistance à l'insuline et une dyslipidémie (augmentation des triglycérides et des acides gras libres). Ces anomalies sont étroitement associées et impliquées dans le développement du diabète de type 2. Dans cette étude, nous avons essayé de déterminer à partir du modèle de souris eN0S-/-, l'influence de la eNOS et de son produit, le NO, sur la régulation du métabolisme lipidique intracellulaire. Ainsi, nous avons montré que cette enzyme et le NO régulent directement l'activité β-oxydative des mitochondries isolées du muscle squelettique, du muscle cardiaque et du tissu adipeux blanc. Par ailleurs, dans le muscle de ces souris, le contenu des mitochondries et l'expression des gènes impliqués dans leur biogénèse sont diminués, ce qui suggère que la eNOS et/ou le NO contrôlent également la synthèse de ces organelles. Les mitochondries, via la β-oxydation, sont impliquées dans la production d'énergie à partir des acides gras libres. Dans notre modèle animal, la diminution de la β-oxydation dans le muscle, s'accompagne d'une accumulation des triglycérides intramyocellulaires. Cette accumulation prédispose fortement au développement de la résistance à l'insuline. Les anomalies du métabolisme β-oxydatif favorisent donc probablement l'apparition de la dyslipidémie et le développement de la résistance à l'insuline observées chez les souris eN0S-/-. Cette hypothèse est soutenue par différentes études effectuées chez l'homme et l'animal qui suggèrent qu'une dysfonction mitochondriale peut être à l'origine de la résistance à l'insuline. Ces données récentes et les résultats de ce travail apportent un regard nouveau sur le rôle du NO dans le développement des maladies métaboliques que sont la résistance à l'insuline, le diabète de type 2 et l'obésité. Elles placent aux centres de ces mécanismes une organelle, la mitochondrie, située au carrefour des métabolismes glucidiques et lipidiques. SUMMARY Over the last years, nitric oxide (NO), synthesized by a family of enzymes, the NO synthases, has become a central regulator of the cardiovascular system and energy metabolism. In humans, defective NO production is found in cardiovascular and metabolic diseases such as insulin resistance or type 2 diabetes mellitus. These alterations are also found in knockout mice for the endothelial nitric oxide synthase (eN0S-/-), which are not only hypertensive but also display insulin resistance and dyslipidemia (with increased triglyceride and free fatty acid levels). These pathologic features are tightly linked and involved in the pathogenesis of type 2 DM. In this study, using eN0S-/- mice, we determined the role played by this enzyme and its product, NO, on intracellular lipid metabolism. We show that eNOS and NO directly regulate β-oxidation in mitochondria isolated from skeletal and cardiac muscle as well as white adipose tissue. Furthermore, in the skeletal muscle of these mice, the mitochondrial content and the expression of genes involved in mitochondrial biogenesis are decreased, suggesting that eNOS and/or NO also regulate the synthesis of this intracellular organelle. Mitochondria, through β-oxidation, play a role in energy production from free fatty acids. In our animal model, decreased β-oxidation in skeletal muscle is associated with accumulation of intramyocellular lipids. This increased lipid content plays an important role in the pathogenesis of insulin resistance. Defective β-oxidation, therefore, probably favours the development of insulin resistance and dyslipidemia as seen in these animals. This hypothesis is strengthened by studies in humans and animals indicating that mitochondrial dysfunction is associated with insulin resistance. These recent data and the results of this work provide evidence for a role of NO in the development of metabolic diseases such as insulin resistance or type diabetes mellitus. They put as a central player, an organelle, the mitochondria, which lies at the crossway of carbohydrate and lipid metabolism. RESUME DIDACTIQUE Le maintien des fonctions vitales et l'accomplissement d'une activité physique nécessitent, chez l'homme, un apport quotidien d'énergie. Cette énergie est présente, dans l'alimentation, principalement sous forme de graisses (lipides) ou de sucres. La production d'énergie s'effectue en majorité dans le muscle au niveau d'une organelle particulière, la mitochondrie. La régulation du métabolisme énergétique fait intervenir de nombreux facteurs de régulation dont l'un des plus connu est l'insuline. De nombreuses maladies comme le diabète de type 2, l'obésité ou le syndrome métabolique découlent de la dérégulation du métabolisme énergétique. Un mécanisme particulier, la résistance à l'insuline, qui se caractérise par un défaut d'action de l'insuline au niveau de ses tissus cibles (foie, muscle...) est souvent impliqué dans le développement de ces pathologies. L'étude de ces anomalies métaboliques nécessite l'utilisation de modèles, notamment animaux, qui ont la particularité de reproduire partiellement un état pathologique caractéristique de certaines maladies humaines. Dans ce travail, nous avons utilisé un modèle de souris dont la particularité est de ne pas exprimer une enzyme, la monoxyde d'azote (NO) synthase endothéliale (eNOS), responsable de la synthèse d'un gaz, le NO. Ces souris présentent une hypertension artérielle, des anomalies du métabolisme des lipides et une résistance à l'insuline. Or, de récents travaux effectués chez l'homme montrent que des individus insulino-résistants ou diabétiques de type 2 ont une diminution de la production de NO. Lors de nos investigations, nous avons démontré que la quantité et la capacité des mitochondries à utiliser les lipides comme substrat énergétique est diminuée dans les muscles des souris eN0S-/-. Par ailleurs, ces deux anomalies sont associées dans ce tissu à une accumulation des lipides. De façon très intéressante, ce phénomène est décrit dans de nombreuses études effectuées chez l'homme et l'animal comme favorisant le développement de la résistance à l'insuline. Les résultats de ce travail suggèrent donc que la eNOS et/ou le NO joue un rôle important dans l'activité et la synthèse des mitochondries. Le NO pourrait donc constituer une cible thérapeutique dans le traitement des maladies métaboliques.

Analysis of the contribution of the β-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Analysis of the contribution of the beta-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae.

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase.

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.

Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation.

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.