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Plants naturally produce the lipid-derived polyester cutin, which is found in the plant cuticle that is deposited at the outermost extracellular matrix of the epidermis covering nearly all aboveground tissues. Being at the interface between the cell and the external environment, cutin and the cuticle play important roles in the protection of plants from several stresses. A number of enzymes involved in the synthesis of cutin monomers have recently been identified, including several P450s and one acyl-CoA synthetase, thus representing the first steps toward the understanding of polyester formation and, potentially, polyester engineering to improve the tolerance of plants to stresses, such as drought, and for industrial applications. However, numerous processes underlying cutin synthesis, such as a controlled polymerization, still remain elusive. Suberin is a second polyester found in the extracellular matrix, most often synthesized in root tissues and during secondary growth. Similar to cutin, the function of suberin is to seal off the respective tissue to inhibit water loss and contribute to resistance to pathogen attack. Being the main constituent of cork, suberin is a plant polyester that has already been industrially exploited. Genetic engineering may be worth exploring in order to change the polyester properties for either different applications or to increase cork production in other species. Polyhydroxyalkanoates (PHAs) are attractive polyesters of 3-hydroxyacids because of their properties as bioplastics and elastomers. Although PHAs are naturally found in a wide variety of bacteria, biotechnology has aimed at producing these polymers in plants as a source of cheap and renewable biodegradable plastics. Synthesis of PHA containing various monomers has been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modified in order to achieve this, including the isoprenoid pathway, the fatty acid biosynthetic pathway, and the fatty acid β-oxidation pathway. PHA synthesis has been demonstrated in a number of plants, including monocots and dicots, and up to 40% PHA per gram dry weight has been demonstrated in Arabidopsis thaliana. Despite some successes, production of PHA in crop plants remains a challenging project. PHA synthesis at high level in vegetative tissues, such as leaves, is associated with chlorosis and reduced growth. The challenge for the future is to succeed in synthesis of PHA copolymers with a narrow range of monomer compositions, at levels that do not compromise plant productivity. This goal will undoubtedly require a deeper understanding of plant biochemical pathways and how carbon fluxes through these pathways can be manipulated, areas where plant "omics" can bring very valuable contributions.

Production of polyesters in transgenic plants.

Polyhydroxyalkanoates (PHAs) are bacterial polyesters having the properties of biodegradable thermoplastics and elastomers. Synthesis of PHAs has been demonstrated in transgenic plants. Both polyhydroxybutyrate and the co-polymer poly(hydroxybutyrate-co-hydroxyvalerate) have been synthesized in the plastids of Arabidopsis thaliana and Brassica napus. Furthermore, a range of medium-chain-length PHAs has also been produced in plant peroxisomes. Development of agricultural crops to produce PHA on a large scale and at low cost will be a challenging task requiring a coordinated and stable expression of several genes. Novel extraction methods designed to maximize the use of harvested plants for PHA, oil, carbohydrate, and feed production will be needed. In addition to their use as plastics, PHAs can also be used to modify fiber properties in plants such as cotton. Furthermore, PHA can be exploited as a novel tool to study the carbon flux through various metabolic pathways, such as the fatty acid beta-oxidation cycle.

Metabolic engineering of plants for the synthesis of polyhydroxyalkanoates

Synthesis of polyhydroxyalkanoates (PHAs) in crop is viewed as an attractive approach for the production of this family of biodegradable plastics in large quantities and at low costs. Synthesisof PHAs containing various monomers has so far been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modifies to achieve this, including the isoprenois pathway, the fatty acid biosynthetic pathway, and the fatty acid

Roles of multiple acyl-CoA oxidases in the routing of carbon flow towards β-oxidation and polyhydroxyalkanoate biosynthesis in Yarrowia lipolytica.

The oleaginous yeast Yarrowia lipolytica possesses six acyl-CoA oxidase (Aox) isoenzymes encoded by genes POX1-POX6. The respective roles of these multiple Aox isoenzymes were studied in recombinant Y. lipolytica strains that express heterologous polyhydroxyalkanoate (PHA) synthase (phaC) of Pseudomonas aeruginosa in varying POX genetic backgrounds, thus allowing assessment of the impact of specific Aox enzymes on the routing of carbon flow to β-oxidation or to PHA biosynthesis. Analysis of PHA production yields during growth on fatty acids with different chain lengths has revealed that the POX genotype significantly affects the PHA levels, but not the monomer composition of PHA. Aox3p function was found to be responsible for 90% and 75% of the total PHA produced from either C9:0 or C13:0 fatty acid, respectively, whereas Aox5p encodes the main Aox involved in the biosynthesis of 70% of PHA from C9:0 fatty acid. Other Aoxs, such as Aox1p, Aox2p, Aox4p and Aox6p, were not found to play a significant role in PHA biosynthesis, independent of the chain length of the fatty acid used. Finally, three known models of β-oxidation are discussed and it is shown that a 'leaky-hose pipe model' of the cycle can be applied to Y. lipolytica.

Analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes.

Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. Two alternative pathways have been described to degrade these fatty acids. One pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme A (CoA) reductase and Delta(3)-Delta(2)-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases. Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also called R-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway. We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the beta-oxidation cycle. Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids.

Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserve. PHAs have properties of biodegradable thermoplastics and elastomers and their synthesis in crop plants is seen as an attractive system for the sustained production of large amounts of polymers at low cost. A variety of PHAs having different physical properties have now been synthesized in a number of transgenic plants, including Arabidopsis thaliana, rape and corn. This has been accomplished through the creation of novel metabolic pathways either in the cytoplasm, plastid or peroxisome of plant cells. Beyond its impact in biotechnology, PHA production in plants can also be used to study some fundamental aspects of plant metabolism. Synthesis of PHA can be used both as an indicator and a modulator of the carbon flux to pathways competing for common substrates, such as acetyl-coenzyme A in fatty acid biosynthesis or 3-hydroxyacyl-coenzyme A in fatty acid degradation. Synthesis of PHAs in plant peroxisome has been used to demonstrate changes in the flux of fatty acids to the beta-oxidation cycle in transgenic plants and mutants affected in lipid biosynthesis, as well as to study the pathway of degradation of unusual fatty acids.

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.

Produção e caracterização de polihidroxialcanoatos obtidos por fermentação da glicerina bruta residual do biodiesel

The aim of this study was to evaluate the production of polyhydroxyalkanoates (PHAs) by fermentation of Crude Glycerin, a byproduct of the biodiesel industry, by Cupriavidus necator IPT 026, 027 and 028. The influence of fermentation time and temperature in shake flasks were evaluated. The highest PHA production (2.82 g L-1) occurred at 35 ºC for 72 h of fermentation. The melting and initial thermal degradation temperatures of this PHA were 177.9 ºC and 306.33 ºC, respectively, with 55% crystallinity. FTIR spectrum was similar to those reported in literature. The polymer obtained presented three different methyl esters of hydroxyalkanoates in its composition, with molecular weight of 630 kDa. Bacteria can use Crude Glycerin as an inexpensive substrate to produce value-added biodegradable products, such as PHA.

MÉTODOS DE EXTRAÇÃO DE POLI-HIDROXIALCANOATOS A PARTIR DE BIOMASSA BACTERIANA

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters intracellularly accumulated by many bacteria as an energy reserve material and carbon source. These biopolymers may be extracted from cells after their production phase, and the extraction process involves various individual operations to ensure adequate removal of the biopolymer from the cells. During this process, the following aspects should be considered: reduction of product losses during different stages of the process to obtain a highly pure product, preservation of physical and thermal characteristics, and use of low toxicity chemicals to achieve sustainable production and avoid harming the environment. The impact of the costs of PHA extraction on the total cost of the production process may account for over 50% of the end-value of the product. Within this context, several methods of PHA extraction have been reported in the literature. These methods include the use of solvents, chemical digestion, enzymatic digestion, mechanical extraction with high-pressure homogenization and ultrasound, extraction using supercritical fluids, or a combination of these methods. The present review of the literature shows strategies for extraction processes of PHAs produced by bacteria involving cell destabilization and/or breakage, recovery, and purification of the biopolymer.

Mise au point de nanoparticules polymères pour l'administration parentérale d'agents anticancéreux hydrophobes

Plusieurs agents anticancéreux très puissants sont caractérisés par une solubilité aqueuse limitée et une toxicité systémique importante. Cette dernière serait liée d’une part à la solubilisation des agents anticancéreux à l’aide de surfactifs de bas poids moléculaire, connus pour leur toxicité intrinsèque, et d’autre part, par le manque de spécificité tissulaire des anticancéreux. Les vecteurs colloïdaux à base de polymères permettraient de résoudre certains défis liés à la formulation d’agents anticancéreux hydrophobes. D’abord, les polymères peuvent être sélectionnés afin de répondre à des critères précis de compatibilité, de dégradation et d’affinité pour le médicament à formuler. Ensuite, le fait d’encapsuler l’agent anticancéreux dans un vecteur peut améliorer son efficacité thérapeutique en favorisant son accumulation au niveau du tissu cible, i.e. la tumeur, et ainsi limiter sa distribution au niveau des tissus sains. Des travaux antérieurs menés au sein de notre laboratoire ont mené à la mise au point de micelles à base de poly(N-vinyl-pyrrolidone)-bloc-poly(D,L-lactide) (PVP-b-PDLLA) capables de solubiliser des agents anticancéreux faiblement hydrosolubles dont le PTX. Ce dernier est commercialisé sous le nom de Taxol® et formulé à l’aide du Crémophor EL (CrEL), un surfactif de bas poids moléculaire pouvant provoquer, entre autres, des réactions d’hypersensibilité sévères. Bien que les micelles de PVP-b-PDLLA chargées de PTX aient démontré une meilleure tolérance comparée au Taxol®, leur potentiel de ciblage tumoral et leur efficacité thérapeutique étaient similaires à la forme commerciale à doses égales. Ceci était possiblement dû au fait que les micelles étaient rapidement déstabilisées et ne pouvaient retenir leur cargo suite à leur administration intraveineuse. Nous avons donc décidé de poursuivre les travaux avec un autre type de vecteur, soit des nanoparticules, qui possèdent une stabilité intrinsèque supérieure aux micelles. L’objectif principal de cette thèse de doctorat était donc de mettre au point des nanoparticules polymères pour l’administration parentérale d’agents anticancéreux faiblement solubles dans l’eau. Les nanoparticules devaient permettre d’encapsuler des agents anticancéreux hydrophobes et de les libérer de manière contrôlée sur plusieurs jours. De plus, elles devaient démontrer un temps de circulation plasmatique prolongée afin de favoriser l’accumulation passive du médicament encapsulé au niveau de la tumeur. La première partie du travail visait à employer pour la première fois le copolymère amphiphile PVP-b-PDLLA comme émulsifiant dans la préparation de nanoparticules polymères. Ainsi, une méthode de fabrication des nanoparticules par émulsion huile-dans-eau a été appliquée afin de produire des nanoparticules à base de PDLLA de taille inférieure à 250 nm. Grâce aux propriétés lyoprotectrices de la couronne de PVP présente à la surface des nanoparticules, celles-ci pouvaient retrouver leur distribution de taille initiale après lyophilisation et redispersion en milieu aqueux. Deux anticancéreux hydrophobes, soit le PTX et l’étoposide (ETO), ont été encapsulés dans les nanoparticules et libérés de ces dernières de façon contrôlée sur plusieurs jours in vitro. Une procédure de « salting-out » a été appliquée afin d’améliorer le taux d’incorporation de l’ETO initialement faible étant donnée sa solubilité aqueuse légèrement supérieure à celle du PTX. Le second volet des travaux visait à comparer le PVP comme polymère de surface des nanoparticules au PEG, le polymère le plus fréquemment employé à cette fin en vectorisation. Par le biais d’études d’adsorption de protéines, de capture par les macrophages et de biodistribution chez le rat, nous avons établi une corrélation in vitro/in vivo démontrant que le PVP n’était pas un agent de surface aussi efficace que le PEG. Ainsi, malgré la présence du PVP à la surface des nanoparticules de PDLLA, ces dernières étaient rapidement éliminées de la circulation sanguine suite à leur capture par le système des phagocytes mononucléés. Par conséquent, dans le troisième volet de cette thèse, le PEG a été retenu comme agent de surface, tandis que différents polymères biodégradables de la famille des polyesters, certains synthétiques (PDLLA et copolymères d’acide lactique/acide glycolique), d’autres de source naturelle (poly(hydroxyalkanoates)(PHAs)), ont été investiguées comme matériaux formant le cœur des nanoparticules. Il en est ressorti que les propriétés physicochimiques des polyesters avaient un impact majeur sur l’efficacité d’encapsulation du PTX et son profil de libération des nanoparticules in vitro. Contrairement aux PHAs, les polymères synthétiques ont démontré des taux d’incorporation élevés ainsi qu’une libération contrôlée de leur cargo. Des études de pharmacocinétique et de biodistribution ont démontré que les nanoparticules de PDLLA dotées d’une couronne de PEG conféraient un temps de circulation plasmatique prolongé au PTX et favorisaient son accumulation tumorale. Les nanoparticules polymères représentent donc une alternative intéressante au Taxol®.

Biocompatible polyhydroxybutyrate (PHB) production by marine Vibrio azureus BTKB33 under submerged fermentation

Polyhydroxybutyrate (PHB) is known to have applications as medical implants and drug delivery carriers and is consequently in high demand. In the present study the possibilities of harnessing potential PHB-producing vibrios from marine sediments as a new source of PHB was investigated since marine environments are underexplored. Screening of polyhydroxyalkanoate (PHA)-producing vibrios from marine sediments was performed using a fluorescent plate assay followed by spectrophotometric analysis of liquid cultures. Out of 828 isolates, Vibrio sp. BTKB33 showed maximum PHA production of 0.21 g/L and PHA content of 193.33 mg/g of CDW. The strain was identified as Vibrio azureus based on phenotypic characterization and partial 16S rDNA sequence analysis. The strain also produced several industrial enzymes: amylase, caseinase, lipase, gelatinase, and DNase. The FTIR analysis of extracted PHA and its comparison with standard PHB indicated that the accumulated PHA is PHB. Bioprocess development studies for enhancing PHA production were carried out under submerged fermentation conditions. Optimal submerged fermentation conditions for enhanced intracellular accumulation of PHA production were found to be 35 °C, pH −7, 1.5 % NaCl concentration, agitation at 120 rpm, 12 h of inoculum age, 2.5 % initial inoculum concentration, and 36 h incubation along with supplementation of magnesium sulphate, glucose, and ammonium chloride. The PHA production after optimization was found to be increased to 0.48 g/L and PHA content to426.88 mg/g of CDW, indicating a 2.28-fold increase in production. Results indicated that V. azureus BTKB33 has potential for industrial production of PHB.

Screening of bacteria to produce polyhydroxyalkanoates from xylose

Although xylose is a major constituent of lignocellulosic feedstock and the second most abundant sugar in nature, only 22% of 3,152 screened bacterial isolates showed significant growth in xylose in 24 h. Of those 684, only 24% accumulated polyhydroxyalkanoates after 72 h. A mangrove isolate, identified as Bacillus sp. MA3.3, yielded the best results in literature thus far for Gram-positive strains in experiments with glucose and xylose as the sole carbon source. When glucose or xylose were supplied, poly-3-hydroxybutyrate (PHB) contents of cell dry weight were, respectively, 62 and 64%, PHB yield 0.25 and 0.24 g g(-1) and PHB productivity (P(PHB)) 0.10 and 0.06 g l(-1) h(-1). This 40% P(PHB) difference may be related to the theoretical ATP production per 3-hydroxybutyrate (3HB) monomer calculated as 3 mol mol(-1) for xylose, less than half of the ATP/3HB produced from glucose (7 mol mol(-1)). In PHB production using sugar mixtures, all parameters were strongly reduced due to carbon catabolite repression. PHB production using Gram-positive strains is particularly interesting for medical applications because these bacteria do not produce lipopolysaccharide endotoxins which can induce immunogenic reactions. Moreover, the combination of inexpensive substrates and products of more value may lead to the economical sustainability of industrial PHB production.

PHA(MCL) biosynthesis systems in Pseudomonas aeruginosa and Pseudomonas putida strains show differences on monomer specificities

The production of PHA from plant oils by Pseudomonas species soil isolated from a sugarcane crop was evaluated. Out of 22 bacterial strains three were able to use efficiently plant oils to grow and to accumulate PHA. Pseudomonas putida and Pseudomonas aeruginosa strains produced PHA presenting differences on monomer composition compatible with variability on monomer specificity of their PHA biosynthesis system. The molar fraction of 3-hydroxydodecanoate detected in the PHA was linearly correlated to the oleic acid supplied. A non-linear relationship between the molar fractions of 3-hydroxy-6-dodecenoate (3HDd Delta(6)) detected in PHA and the linoleic acid supplied was observed, compatible with saturation in the biosynthesis system capability to channel intermediate of P-oxidation to PHA synthesis. Although P. putida showed a higher 3HDd Delta(6) yield from linoleic acid when compared to P. aeruginosa, in both species it was less than 10% of the maximum theoretical value. These results contribute to the knowledge about the biosynthesis of PHA with a controlled composition from plant oils allowing in the future establishing the production of these polyesters as tailor-made polymers. (C) 2009 Elsevier B.V. All rights reserved.

Análise do polímero PHA em Saccharomyces cerevisiae recombinante : superexpressão de phaG, e a contribuição in vivo das enzimas auxiliares envolvidas na beta-oxidação de ácidos graxos insaturados em peroxissomos

Os polímeros do tipo poli-hidroxialcanoatos (PHAs) são poliésteres bacterianos que apresentam as propriedades de termoplásticos e elastômeros biodegradáveis. A síntese deste polímero em plantas de interesse agroindustrial tem sido vista como uma área promissora dentro da biotecnologia de polímeros para a produção em grande escala com baixos custos. Contudo, esta tarefa requer o aprimoramento de diferentes metodologias bioquímicas e moleculares, além de maximizar os processos de extração destes polímeros biológicos. A produção de PHAs em peroxissomos ou no citoplasma de Saccharomyces cerevisiae, por meio da expressão de uma PHA-sintase bacteriana, pode servir como indicador e modulador do fluxo de carbonos que percorre vias biossintéticas como a síntese de novo de ácidos graxos e a β-oxidação. Esta levedura tem sido usada como eucarioto modelo para manipular as rotas envolvidas na síntese de PHAs do tipo MCL (PHA com um número médio de carbonos), um polímero menos cristalino e com menor ponto de fusão quando comparado ao PHA-SCL (PHA com um número pequeno de carbonos). A enzima PhaG (3-hidroxidecanoil-ACP-CoA transacilase) é responsável pela conexão entre a síntese de ácidos graxos e a produção de PHA-MCL em bactérias do gênero Pseudomonas, em meios de cultura contendo uma fonte de carbono nãorelacionada como gliconato, etanol ou acetato. Para tentar estabelecer esta rota metabólica em S. cerevisiae, o presente trabalho avaliou a coexpressão de PhaGPa e PhaC1Pa (PHA-sintase) de P. aeruginosa para a síntese de PHA-MCL a partir de uma fonte de carbono não-relacionada em leveduras. Contudo, a presença de PhaGPa não alterou a composição ou a quantidade de PHA-MCL em relação à cepa controle contendo apenas PhaC1Pa citoplasmática ou direcionada ao peroxissomo, independentemente da fonte de carbono utilizada (rafinose ou ácido oléico). Este resultado permite sugerir que a ligação entre a síntese de ácidos graxos e a produção de PHA-MCL em S. cerevisiae não foi estabelecida, provavelmente devido à ausência de algum passo enzimático que limita o desvio de substratos da síntese de ácidos graxos para a produção de PHA-MCL em organismos que não são capazes de acumular naturalmente este polímero quando cultivados em fontes de carbono não-relacionadas.A levedura S. cerevisiae tem sido usada como um sistema modelo para estudar a β-oxidação de ácidos graxos insaturados em peroxissomos. A produção de PHA-MCL pela expressão de PhaC1Pa em peroxissomos de cepas selvagens e mutantes nulos de S. cerevisiae para as enzimas auxiliares da β-oxidação (Eci1p, Sps19p e Dci1p), multiplicadas em meio de cultivo contendo um ácido graxo insaturado como fonte de carbono, permitiu monitorar o fluxo de carbonos que percorre as vias dependente de isomerase, redutase e di-isomerase. Desta forma, o presente estudo permitiu avaliar a β- oxidação in vivo dos ácidos graxos linoléico conjugado, 9-cis,11-trans-CLA (ácido rumênico) ou 10-cis,13-cis-nonadecadienóico, para determinar a contribuição das vias alternativas na degradação destes substratos pela utilização de cepas selvagens, mutantes nulos e linhagens contendo um plasmídio multicópia para os genes ECI1 (Δ3- Δ2-enoil-CoA isomerase), SPS19 (2,4-dienoil-CoA redutase) e DCI1 (Δ3,5-Δ2,4-dienoil- CoA isomerase). As linhagens selvagens foram capazes de sintetizar PHA-MCL quando cultivadas em ácido rumênico, mas a atividade da enzima Eci1p foi essencial para a degradação deste CLA, indicando que a via dependente de isomerase é a única rota in vivo necessária para a β-oxidação do ácido rumênico em peroxissomos de S. cerevisiae. A contribuição da enzima di-isomerase (Dci1p) para a degradação do ácido 10- cis,13-cis-nonadecadienóico foi avaliada em cepas selvagens, mutantes nulos dci1Δ e linhagens de S. cerevisiae contendo os plasmídeos multicópia. De acordo com o conteúdo e a quantidade de PHA formado, a β-oxidação de ácidos graxos cisinsaturados em um carbono ímpar é, in vivo, independente da di-isomerase. Embora este resultado possa indicar o mesmo padrão de envolvimento de Dci1p na degradação de ácidos graxos cis-insaturados em um carbono ímpar em mitocôndrias de mamíferos, esta via alternativa deve ser mais bem investigada em eucariotos superiores.