RESUMEN
Despite the rapid pace of biomedical innovation, research and development (R&D) productivity in the pharmaceutical industry has not improved broadly. Increasingly, firms need to leverage new approaches to product development and commercial execution, while maintaining adaptability to rapid changes in the marketplace and in biomedical science. Firms are also seeking ways to capture some of the talent, infrastructure, and innovation that depends on federal R&D investment. As a result, a major transition to external innovation is taking place across the industry. One example of these external innovation initiatives is the Sanofi-MIT Partnership, which provided seed funding to MIT investigators to develop novel solutions and approaches in areas of interest to Sanofi. These projects were highly collaborative, with information and materials flowing both ways. The relatively small amount of funding and short time frame of the awards built an adaptable and flexible process to advance translational science.
Asunto(s)
Investigación Biomédica/organización & administración , Difusión de Innovaciones , Industria Farmacéutica/organización & administración , Universidades/organización & administración , Conducta Cooperativa , Eficiencia Organizacional , Humanos , Investigación Biomédica Traslacional/organización & administraciónRESUMEN
Microparticles made from degradable polyhydroxyalkanoates of different chemical compositions a homopolymer of 3-hydroxybutyric acid, copolymers of 3-hydroxybutyric and 4-hydroxybutyric acids (P3HB/4HB), 3-hydroxybutyric and 3-hydroxyvaleric acids (P3HB/3HV), 3-hydroxybutyric and 3-hydroxyhexanoic acids (P3HB/3HHx) were prepared using the solvent evaporation technique, from double emulsions. The study addresses the influence of the chemical compositions on the size and ξ-potential of microparticles. P3HB microparticles loaded with doxorubicin have been prepared and investigated. Their average diameter and ξ-potential have been found to be dependent upon the level of loading (1, 5, and 10 % of the polymer mass). Investigation of the in vitro drug release behavior showed that the total drug released from the microparticle into the medium increased with mass concentration of the drug. In this study mouse fibroblast NIH 3T3 cells were cultivated on PHA microparticles, and results of using fluorescent DAPI DNA stain, and MTT assay showed that microparticles prepared from PHAs of different chemical compositions did not exhibit cytotoxicity to cells cultured on them and proved to be highly biocompatible. Cell attachment and proliferation on PHA microparticles were similar to those on polystyrene. The cytostatic drug encapsulated in P3HB/3HV microparticles has been proven to be effective against HeLa tumor cells.
Asunto(s)
Citostáticos/administración & dosificación , Portadores de Fármacos/síntesis química , Microesferas , Polihidroxialcanoatos/química , Implantes Absorbibles , Animales , Citostáticos/farmacocinética , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Portadores de Fármacos/química , Composición de Medicamentos/métodos , Evaluación Preclínica de Medicamentos , Ensayo de Materiales , Ratones , Microscopía Electrónica de Rastreo , Modelos Biológicos , Células 3T3 NIH , Tamaño de la Partícula , Polihidroxialcanoatos/síntesis química , Polihidroxialcanoatos/farmacocinética , Polímeros/síntesis química , Polímeros/química , Polímeros/farmacocinéticaRESUMEN
AIMS: Of the biodegradable polyhydroxyalkanoates (PHAs), poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) is often considered for fabrication of biocompatible and absorbable medical devices and other applications. Depending on the application, however, specific mechanical or processing properties must be improved. To address these required properties, we sought to alter the monomer composition of the copolymer by a combination genetic engineering in an Escherichia coli host and carbon substrate feeding. METHODS AND RESULTS: We applied a new method of 3-hydroxyvalerate (3HV) monomer synthesis to produce a co-polymer by the introduction of a propionyl-CoA transferase gene (pct), along with PHA biosynthetic genes bktB, phaB and phaC from Ralstonia eutropha into engineered E. coli to produce P(HB-co-HV). The resulting strain successfully produced the copolymer containing an ultra-high 3HV monomer composition (over 80 wt%). CONCLUSIONS: To the best of our knowledge, the P(HB-co-HV) production strain constructed here synthesized polymer with the highest 3HV content of any engineered E. coli strain. This strain could also produce P(HB-co-HV) with the use of lower concentrations of propionate in the growth medium, compared to other reported strains, which could avoid the known growth inhibition from propionate in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyhydroxyalkanoates have been emphasized as a potential alternative for petroleum-based plastics by virtue of their physical properties and environmentally friendly characteristics. The copolymer produced in this work validates our genetic engineering approach and suggests that the Pct enzyme is a more efficient method for production of propionyl-CoA, the 3-hydroxyvaleryl-CoA precursor.
Asunto(s)
Coenzima A Transferasas/metabolismo , Escherichia coli/metabolismo , Ácidos Pentanoicos/metabolismo , Poliésteres/metabolismo , Coenzima A Transferasas/genética , Cupriavidus necator/enzimología , Cupriavidus necator/genética , Escherichia coli/genética , Ingeniería Genética , Microbiología Industrial , Propionatos/metabolismoRESUMEN
Synthesis of 3- and 4-hydroxybutyrate copolymer (3HB-CO-4HB), the most promising member of the biodegradable polyhydroxyalcanoate (PHA) family, has been studied. Cultivation conditions of naturally occurring strains of hydrogen-oxidizing bacteria Ralstonia eutropha B5786 and Cupriavidus eutrophus B10646 have been optimized to ensure efficient synthesis of the 3HB-CO-4HB copolymer. A set of highly pure samples of the 3HB-CO-4HB copolymer with 4HB content varying from 8.7 to 24.3 mol % has been obtained. Incorporation of 4-HB into the copolymer was shown to cause a more pronounced decrease in polymer crystallinity than the incorporation of 3-hydroxyvalerate or 3-hydroxyhexanoate; samples with a degree of crystallinity below 30% have been obtained. The weight average molecular mass of the 3HB-CO-4HB copolymers was shown to be independent on the monomer ratio and to vary broadly (from 540 to 1110 kDa).
Asunto(s)
Cupriavidus necator/crecimiento & desarrollo , Cupriavidus necator/metabolismo , Hidrógeno/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Técnicas de Cultivo de Célula , Cristalografía , Medios de Cultivo/química , Cupriavidus necator/genética , Hidroxibutiratos/química , Oxidación-Reducción , Poliésteres/químicaRESUMEN
Generally, prokaryotes store carbon as polyhydroxyalkanoate, starch, or glycogen. The Gram-positive actinomycete Rhodococcus opacus strain PD630 is noteworthy in that it stores carbon in the form of triacylglycerol (TAG). Several studies have demonstrated that R. opacus PD630 can accumulate up to 76% of its cell dry weight as TAG when grown under nitrogen-limiting conditions. While this process is well studied, the underlying molecular and biochemical mechanisms leading to TAG biosynthesis and subsequent storage are poorly understood. We designed a high-throughput genetic screening to identify genes and their products required for TAG biosynthesis and storage in R. opacus PD630. We identified a gene predicted to encode a putative heparin-binding hemagglutinin homolog, which we have termed tadA (triacylglycerol accumulation deficient), as being important for TAG accumulation. Kinetic studies of TAG accumulation in both the wild-type (WT) and mutant strains demonstrated that the tadA mutant accumulates 30 to 40% less TAG than the parental strain (WT). We observed that lipid bodies formed by the mutant strain were of a different size and shape than those of the WT. Characterization of TadA demonstrated that the protein is capable of binding heparin and of agglutinating purified lipid bodies. Finally, we observed that the TadA protein localizes to lipid bodies in R. opacus PD630 both in vivo and in vitro. Based on these data, we hypothesize that the TadA protein acts to aggregate small lipid bodies, found in cells during early stages of lipid storage, into larger lipid bodies and thus plays a key role in lipid body maturation in R. opacus PD630.
Asunto(s)
Lectinas/metabolismo , Lípidos/biosíntesis , Rhodococcus/metabolismo , Secuencia de Aminoácidos , Cromatografía en Capa Delgada , Genes Bacterianos/genética , Heparina/metabolismo , Lectinas/genética , Lectinas/fisiología , Metabolismo de los Lípidos/genética , Microscopía Fluorescente , Datos de Secuencia Molecular , Rhodococcus/fisiología , Alineación de Secuencia , Triglicéridos/biosíntesis , Triglicéridos/genética , Triglicéridos/metabolismoRESUMEN
Biopharmaceuticals are often produced by recombinant E. coli or mammalian cell lines. This is usually achieved by the introduction of a gene or cDNA coding for the protein of interest into a well-characterized strain of producer cells. Naturally, each recombinant production system has its own unique advantages and disadvantages. This paper examines the current practices, developments, and future trends in the production of biopharmaceuticals. Platform technologies for rapid screening and analyses of biosystems are reviewed. Strategies to improve productivity via metabolic and integrated engineering are also highlighted.
RESUMEN
Asiatic acid (AA), a triterpene, is known to be cytotoxic to several tumor cell lines. AA induces dose- and time-dependent cell death in U-87 MG human glioblastoma. This cell death occurs via both apoptosis and necrosis. The effect of AA may be cell type-specific as AA-induced cell death was mainly apoptotic in colon cancer RKO cells. AA-induced glioblastoma cell death is associated with decreased mitochondrial membrane potential, activation of caspase-9 and -3, and increased intracellular free Ca2+. Although treatment of glioblastoma cells with the caspase inhibitor zVAD-fmk completely abolished AA-induced caspase activation, it did not significantly block AA-induced cell death. AA-induced cell death was significantly prevented by an intracellular Ca2+ inhibitor, BAPTA/AM. Taken together, these results indicate that AA induces cell death by both apoptosis and necrosis, with Ca2+-mediated necrotic cell death predominating.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Necrosis/inducido químicamente , Triterpenos/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Calcio/metabolismo , Caspasa 3/biosíntesis , Caspasa 9/biosíntesis , Inhibidores de Caspasas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quelantes/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Triterpenos PentacíclicosRESUMEN
A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X2 even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X2 has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5-100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA-DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.
Asunto(s)
Dactinomicina/análogos & derivados , Microbiología del Suelo , Streptomyces/metabolismo , Técnicas de Tipificación Bacteriana , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Dactinomicina/biosíntesis , Dactinomicina/toxicidad , Células HL-60 , Humanos , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Filogenia , Pigmentos Biológicos/biosíntesis , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Streptomyces/clasificación , Streptomyces/aislamiento & purificación , Streptomyces/ultraestructuraRESUMEN
The relationship between changes in mRNA abundance and enzyme activity was determined for three genes over a span of nearly 3 h during amino acid production in Corynebacterium glutamicum. Gene expression changes during C. glutamicum fermentations were examined by complementary DNA (cDNA) microarrays and by a second method for quantitating RNA levels, competitive reverse transcriptase-PCR (RT-PCR). The results obtained independently by both methods were compared and found to be in agreement, thus validating the quantitative potential of DNA microarrays for gene expression profiling. Evidence of a disparity between mRNA abundance and enzyme activity is presented and supports our belief that it is difficult to generally predict protein activity from quantitative transcriptome data. Homoserine dehydrogenase, threonine dehydratase, and homoserine kinase are enzymes involved in the biosynthesis of l-isoleucine and other aspartate-derived amino acids in C. glutamicum. Our data suggest that different underlying regulatory mechanisms may be connected with the expression of the genes encoding each of these three enzymes. Indeed, whereas in one case the increases in enzyme activity exceeded those in the corresponding mRNA abundance, in another case large increases in the levels of gene expression were not congruent with changes in enzyme activity.
Asunto(s)
Corynebacterium/enzimología , Corynebacterium/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Ácido Aspártico/metabolismo , Corynebacterium/crecimiento & desarrollo , Fermentación , Perfilación de la Expresión Génica , Homoserina Deshidrogenasa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Treonina Deshidratasa/metabolismo , Factores de TiempoRESUMEN
Cis-aminoindanol, a key chiral precursor to the HIV protease inhibitor CRIXIVAN, can be derived from indene oxidation products of (2R) stereochemistry. A number of different microorganisms, notably strains of the genera Pseudomonas and Rhodococcus, have been isolated that catalyze the oxygenation of indene to indandiol with greater stereospecificity than is achievable through traditional chemical synthesis. The yield and ultimate optical purity of indandiol produced in such biocatalytic processes is influenced by the intrinsic stereospecificity of the oxygenase(s), enantioselective dehydrogenation, and the loss of substrate to alternate, undesirable metabolites. Metabolic engineering of any indene bioconversion system for the commercial-scale production of cis-aminoindanol must account for these influences, as well as pathway fluxes and enzyme regulation, to optimize the formation of oxygenated precursors with useful stereochemistry. As such, the process by which bacterial systems carry out the bioconversion of indene to indandiol serves as a model for biological production of industrially relevant chiral synthons.
Asunto(s)
Ingeniería Genética/métodos , Indanos/metabolismo , Indenos/metabolismo , Pseudomonas putida/enzimología , Rhodococcus/enzimología , Biotecnología/métodos , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/metabolismo , Indinavir/química , Indinavir/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Pseudomonas putida/genética , Rhodococcus/genéticaRESUMEN
Class I and III polyhydroxyalkanoate (PHA) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to polyhydroxybutyrate. The Class I PHA synthase from Ralstonia eutropha has been purified by numerous labs with reported specific activities that vary between 1 and 160 U/mg. An N-terminal (His)6-PHA synthase was constructed and purified with specific activity of 40 U/mg. The variable activity is shown to be related to the protein's propensity to aggregate and not to incomplete post-translational modification by coenzyme A and a phosphopantetheinyl transferase. The substrate specificities of this enzyme and the Class III PHA synthase from Allochromatium vinosum have been determined with nine analogs of varied chain length and branching, OH group position within the chain, and thioesters. The results suggest that in vitro, both PHA synthases are very specific and provide further support for their active site structural similarities. In vitro results differ from studies in vivo.
Asunto(s)
Aciltransferasas/clasificación , Aciltransferasas/metabolismo , Betaproteobacteria/enzimología , Gammaproteobacteria/enzimología , Aciltransferasas/genética , Aciltransferasas/aislamiento & purificación , Sitios de Unión , Coenzima A/metabolismo , Cinética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por SustratoRESUMEN
Radiolabeled tracers can provide valuable information about the structure of and flux distributions in biocatalytic reaction networks. This method derives from prior studies of glucose metabolism in mammalian systems and is implemented by pulsing a culture with a radiolabeled metabolite that can be transported into the cells and subsequently measuring the radioactivity of all network metabolites following separation by liquid chromatography. Intracellular fluxes can be directly determined from the transient radioactivity count data by tracking the depletion of the radiolabeled metabolite and/or the accompanying accumulation of any products formed. This technique differs from previous methods in that it is applied within a systems approach to the problem of flux determination. It has been used for the investigation of the indene bioconversion network expressed in Rhodococcus sp. KY1. Flux estimates obtained by radioactive tracers were confirmed by macroscopic metabolite balancing and showed that indene oxidation in steady state chemostat cultures proceeds primarily through a monooxygenase activity forming (1S,2R)-indan oxide, with no dehydrogenation of trans-(1R,2R)-indandiol. These results confirmed the significance of indan oxide formation and identified the hydrolysis of indan oxide as a key step in maximizing the production of (2R)-indandiol, a chiral precursor of the HIV protease inhibitor, Crixivan.
Asunto(s)
Indenos/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Rhodococcus/metabolismo , Radioisótopos de Carbono/metabolismo , Catálisis , Cromatografía Líquida de Alta Presión , Fermentación , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/metabolismo , Hidrólisis , Indenos/química , Indinavir/química , Indinavir/metabolismo , Cinética , Oxigenasas/metabolismo , Rhodococcus/citología , Rhodococcus/enzimologíaRESUMEN
Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cupriavidus necator/metabolismo , Proteínas de Unión al ADN/metabolismo , Hidroxibutiratos/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Proteínas Bacterianas/genética , Cupriavidus necator/genética , Proteínas de Unión al ADN/genética , Activación Enzimática , Ácido Graso Sintasas/metabolismoRESUMEN
We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C. glutamicum amino acid-producing strains.
Asunto(s)
Corynebacterium/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Corynebacterium/crecimiento & desarrollo , ADN Complementario/genética , Perfilación de la Expresión Génica , Hibridación de Ácido Nucleico , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , Reproducibilidad de los ResultadosRESUMEN
Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.
Asunto(s)
Proteínas Bacterianas/fisiología , Cupriavidus necator/metabolismo , Proteínas de Unión al ADN/fisiología , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Secuencia de Bases , Elementos Transponibles de ADN , Immunoblotting , Datos de Secuencia MolecularRESUMEN
The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J., and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence alignments between the Class I and III synthases revealed similar residues in the R. eutropha synthase. Site-directed mutants of these residues were prepared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase possesses an essential catalytic dyad (C319-H508) in which the C319 is involved in covalent catalysis. A conserved Asp, D480, was shown not to be required for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studies of the [(3)H]sT-CoA with wild-type and mutant synthases reveal that 0.5 equiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest that the Class I and III synthases are mechanistically similar and structurally homologous, despite their physical and kinetic differences.
Asunto(s)
Aciltransferasas/química , Aciltransferasas/clasificación , Cupriavidus necator/enzimología , Acilcoenzima A/síntesis química , Acilcoenzima A/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Catálisis , Chromatium/enzimología , Cupriavidus necator/genética , Dimerización , Activación Enzimática/genética , Cinética , Extensión de la Cadena Peptídica de Translación/genética , Especificidad por Sustrato , TritioRESUMEN
Carbon destined for lysine synthesis in Corynebacterium glutamicum ATCC 21799 can be diverted toward threonine by overexpression of genes encoding a feedback-insensitive homoserine dehydrogenase (hom(dr)) and homoserine kinase (thrB). We studied the effects of introducing two different threonine dehydratase genes into this threonine-producing system to gauge their effects on isoleucine production. Co-expression of hom(dr), thrB, and ilvA, which encodes a native threonine dehydratase, caused isoleucine to accumulate to a final concentration of 2.2+/-0.2 g l(-1), five-fold more than accumulates in the wild-type strain, and approximately twice as much as accumulates in the strain expressing only hom(dr) and thrB. Comparing these data with previous results, we found that overexpression of the three genes, hom(dr), thrB, and ilvA, in C. glutamicum ATCC 21799 is no better in terms of isoleucine production than the expression of a single gene, tdcB, encoding a catabolic threonine dehydratase from Escherichia coli. Co-expression of hom(dr), thrB, and tdcB, however, caused the concentration of isoleucine to increase 20-fold compared to the wild-type strain, about four times more than the corresponding ilvA-expressing strain. In this system, the apparent yield of isoleucine production was multiplied by a factor of two [2.1 mmol (g dry cell weight)(-1)]. While the balance of excreted metabolites showed that the carbon flow in this strain was completely redirected from the lysine pathway into the isoleucine pathway, it also showed that more pyruvate was diverted into amino acid synthesis.
Asunto(s)
Corynebacterium/metabolismo , Isoleucina/metabolismo , Treonina Deshidratasa/metabolismo , Treonina/biosíntesis , Secuencia de Bases , Biotecnología , Corynebacterium/enzimología , Corynebacterium/genética , ADN Bacteriano/genética , Fermentación , Expresión Génica , Genes Bacterianos , Plásmidos/genética , Treonina Deshidratasa/genéticaRESUMEN
Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.
Asunto(s)
Aciltransferasas/química , Lipasa/química , Modelos Moleculares , Aciltransferasas/genética , Secuencia de Aminoácidos , Chromatium , Lipasa/genética , Mecánica , Datos de Secuencia Molecular , Conformación Proteica , Alineación de SecuenciaRESUMEN
The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.
Asunto(s)
Corynebacterium/enzimología , Escherichia coli/enzimología , Isoleucina/biosíntesis , Treonina Deshidratasa/genética , Treonina Deshidratasa/metabolismo , Biomasa , Clonación Molecular , Corynebacterium/genética , Corynebacterium/crecimiento & desarrollo , Medios de Cultivo , Escherichia coli/genética , Fermentación , Plásmidos/genética , Proteínas Recombinantes/metabolismoRESUMEN
Rhodococcus strain 124 is able to convert indene into indandiol via the actions of at least two dioxygenase systems and a putative monooxygenase system. We have identified a cosmid clone from 124 genomic DNA that is able to confer the ability to convert indene to indandiol upon Rhodococcus erythropolis SQ1, a strain that normally can not convert or metabolize indene. HPLC analysis reveals that the transformed SQ1 strain produces cis-(1R,2S)-indandiol, suggesting that the cosmid clone encodes a naphthalenetype dioxygenase. DNA sequence analysis of a portion of this clone confirmed the presence of genes for the dioxygenase as well as genes encoding a dehydrogenase and putative aldolase. These genes will be useful for manipulating indene bioconversion in Rhodococcus strain 124.