RESUMEN
Poly(hydroxyalkanoates) are natural polymers with thermoplastic properties. One polymer of this class with commercial applicability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) can be produced by bacterial fermentation, but the process is not economically competitive with polymer production from petrochemicals. Poly(hydroxyalkanoate) production in green plants promises much lower costs, but producing copolymer with the appropriate monomer composition is problematic. In this study, we have engineered Arabidopsis and Brassica to produce PHBV in leaves and seeds, respectively, by redirecting the metabolic flow of intermediates from fatty acid and amino acid biosynthesis. We present a pathway for the biosynthesis of PHBV in plant plastids, and also report copolymer production, metabolic intermediate analyses, and pathway dynamics.
Asunto(s)
Arabidopsis/metabolismo , Brassica/metabolismo , Poliésteres/metabolismo , Acilcoenzima A/biosíntesis , Aminación , Butiratos/metabolismo , Espectroscopía de Resonancia MagnéticaRESUMEN
The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.
Asunto(s)
Acetil-CoA C-Aciltransferasa/metabolismo , Aciltransferasas/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Arabidopsis/metabolismo , Cupriavidus necator/enzimología , Poliésteres/metabolismo , Acetil-CoA C-Aciltransferasa/genética , Aciltransferasas/genética , Oxidorreductasas de Alcohol/genética , Arabidopsis/genética , Cupriavidus necator/genética , Homocigoto , Modelos Moleculares , Estructura Molecular , Hojas de la Planta , Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes/metabolismo , SemillasRESUMEN
Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism's intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. Ralstonia eutropha produces both the homopolymer poly-beta-hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form beta-ketovaleryl-CoA. This activity has generally been attributed to the beta-ketothiolase encoded by R. eutropha phbA. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of bktB, which encodes a beta-ketothiolase from R. eutropha that is capable of forming beta-ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of beta-hydroxyvalerate derived from propionyl-CoA. We also report an additional beta-ketothiolase, designated BktC, that probably serves as a secondary route toward beta-hydroxyvalerate production.
Asunto(s)
Acetil-CoA C-Aciltransferasa/metabolismo , Alcaligenes/enzimología , Polímeros/metabolismo , Acetil-CoA C-Aciltransferasa/genética , Acetil-CoA C-Aciltransferasa/aislamiento & purificación , Alcaligenes/genética , Secuencia de Aminoácidos , Secuencia de Bases , Genes Bacterianos , Prueba de Complementación Genética , Genotipo , Cinética , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Two Mg(2+)-dependent DNA endonucleases have been isolated from mammalian cells which have a strong preference to nick within long tracts of guanine residues in vitro. One endonuclease activity is mitochondrial (mt). The other endonuclease, called Endonuclease G, is associated with isolated nuclei, and is released when the nuclear chromatin is exposed to moderate ionic strength. Our laboratory has previously purified the mt endonuclease to near homogeneity from mitochondria of bovine heart and reported the enzyme to be a homodimer of a approximately 29 kDa polypeptide [Cummings, O. W. et al. (1987) J. Biol. Chem., 262, 2005-2015]. Although the purified mt endonuclease will extensively fragment M13 viral ssDNA and plasmid dsDNAs in vitro, the enzyme displays an unusually strong preference to nick within a (dG)12:(dC)12 sequence tract which resides just upstream from the origin of DNA replication in the mitochondrial genome. The nuclear Endonuclease G first identified from its selective targeting of several (dG)n:(dC)n tracts in vitro (where N = 3-29), was subsequently purified from calf thymus nuclei and shown to be a homodimer of a approximately 26-kDa polypeptide [Côté, J. et al. (1989) J. Biol. Chem., 264, 3301-3310]. In the present study, we find that Endonuclease G partially purified from calf thymus nuclei will extensively degrade both viral ss- and dsDNAs in vitro, and that the enzyme possesses biochemical properties and specificity for nucleotide sequences in DNA that are strongly related or identical to those of the mt endonuclease. These findings and the discovery of sequence identity between the proteins strengthen the conclusion that the nuclear Endonuclease G is the same enzyme as the mt endonuclease.
Asunto(s)
Núcleo Celular/enzimología , Endodesoxirribonucleasas/aislamiento & purificación , Mitocondrias/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Centrifugación por Gradiente de Densidad , ADN/genética , ADN/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/metabolismo , Técnicas In Vitro , Mitocondrias Cardíacas/enzimología , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Timo/enzimologíaRESUMEN
Rat heart mitochondria contain a potent endonuclease activity that closely resembles the endonuclease of bovine and human heart mitochondria, and shows a striking preference for an evolutionarily conserved sequence that resides just upstream from the heavy (H)-strand origin of DNA replication (Ori H), (Low, R.L. et al. (1988) Nucleic Acids Res. 16, 6427-6425). This study reports that while the site-directed endonuclease is evident in the mt fractions of several rat organs, the levels of activity among them varies in an unexpected and marked fashion. There is nearly 200-times more of this endonuclease activity per mg of mt protein in the heart than in the liver (or spleen). Levels intermediate to those in heart and liver are found in the kidney and brain. The large variations in endonuclease activity do not correlate with reported rates of mtDNA turnover among tissues and are in contrast to the much smaller variations in levels of mtDNA and DNA polymerase-gamma activity. However, there may be some relationship between the amount of the endonuclease and the rate of oxidative phosphorylation.
Asunto(s)
Endonucleasas/metabolismo , Mitocondrias Cardíacas/enzimología , Animales , Encéfalo/enzimología , ADN/análisis , ADN Polimerasa III/análisis , Riñón/enzimología , Cinética , Hígado/enzimología , Fosforilación Oxidativa , Ratas , Bazo/enzimologíaRESUMEN
This study compared HSPG and CSPG in adhesion sites of isogenic metastatic lung tumor cells (v-Ki-ras-transformed 3T3 cells) after injection by different routes (IV and footpad) into athymic nude mice. The proportion of HSPG was significantly greater in experimental lung metastatic cell (IV injection) than in spontaneously metastatic cells. The proportion of CSPG in these two groups of metastatic lung cells did not differ significantly. In addition, the proportion of HSPG and CSPG in spontaneously metastatic lung tumor cells was similar, unlike experimental metastatic tumor cells.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/análisis , Heparitina Sulfato/análisis , Neoplasias Pulmonares/química , Animales , Adhesión Celular , Línea Celular Transformada , Genes ras , Proteoglicanos de Heparán Sulfato , Inyecciones , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
Initial studies described the significance of heparan sulfate proteoglycans of Balb/c 3T3 cells in their adhesion on fibronectin matrices, including their binding to multiple domains in FN, the importance of this binding in microfilament and close contact formation, and the cooperativity of both HS-PG and 140k glycoprotein integrin's binding to FN to achieve tight-focal contacts under cells. These analyses utilized model HS-binding proteins, such as platelet factor 4, and proteolytic fragments of FN with differing binding activities in both cell biological analyses of adhesion responses and in biochemical analyses of the HS-PG in the adhesion sites. In contrast, dermatan sulfate proteoglycans (DS-PG) inhibit 3T3 adhesion on FN but not on collagen; of special note is the discovery that certain integrin-binding fragments of FN also contain a third HS/DS-binding domain that is cryptic and that provides a more effective mechanism for inhibiting integrin: FN binding. Kirsten Ras oncogene-transformed 3T3 cells and their nude-mouse-derived primary or lung metastatic tumors are also being analyzed by similar approaches. HS-PGs in the adhesion sites of these tumor populations undergo extensive catabolism, resulting in alteration of their binding to FN affinity columns (and by implication alteration in adhesion responses of these tumor cells on FN matrices). Functions for HS-PG on the surface of neuronal cell derivatives, e.g., neuroblastoma cells derived from the neural crest of the embryo and potentially related in some ways to peripheral neurons, are also being explored. HS-binding fragments of FN or PF4 facilitate attachment and spreading of neuroblastoma cells but not neurite outgrowth, contrasting with the ability of dorsal root ganglion neurons to extend neurites on HS-binding substrata. The catabolism of HS-PG in neuroblastoma adhesion sites is minimal, indicating that this cannot be the explanation for incompetence in neurite extension. Neurite extension by neuroblastoma cells on FN results from three different and overlapping binding activities of non-PG receptors on the cell surface--RGDS-dependent binding to integrin, an RGDS-independent mechanism (perhaps a cell type-specific domain), and a ganglioside-dependent process. However, these neurite-extending reactions can be modulated either by exogenous addition of proteoglycans acting in a "trans" manner with the cell surface or by endogenous HG-PG acting in a "cis" manner with one or more of these receptors.(ABSTRACT TRUNCATED AT 400 WORDS)