RESUMEN
Methane, the main component of natural gas, can be used to produce methanol which can be further converted to other valuable products. There is increasing interest in using biological systems for the production of fuels and chemicals from methanol, termed methylotrophy. In this work, we have examined methanol assimilation metabolism in a synthetic methylotrophic E. coli strain. Specifically, we applied 13C-tracers and evaluated 25 different co-substrates for methanol assimilation, including amino acids, sugars and organic acids. In particular, co-utilization of threonine significantly enhanced methylotrophy. Through our investigations, we proposed specific metabolic pathways that, when activated, correlated with increased methanol assimilation. These pathways are normally repressed by the leucine-responsive regulatory protein (lrp), a global regulator of metabolism associated with the feast-or-famine response in E. coli. By deleting lrp, we were able to further enhance the methylotrophic ability of our synthetic strain, as demonstrated through increased incorporation of 13C carbon from 13C-methanol into biomass.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Proteína Reguladora de Respuesta a la Leucina/genética , Metanol/metabolismo , Escherichia coli/genéticaRESUMEN
Previously the development of a hyper acetone-butanol-ethanol (ABE) producing Clostridium acetobutylicum BKM19 strain capable of producing 30.5% more total solvent by random mutagenesis of its parental strain PJC4BK, which is a buk mutant C. acetobutylicum ATCC 824 strain is reported. Here, BKM19 and PJC4BK strains are re-sequenced by a high-throughput sequencing technique to understand the mutations responsible for enhanced solvent production. In comparison with the C. acetobutylicum PJC4BK, 13 single nucleotide variants (SNVs), one deletion and one back mutation SNV are identified in the C. acetobutylicum BKM19 genome. Except for one SNV found in the megaplasmid, all mutations are found in the chromosome of BKM19. Among them, a mutation in the thlA gene encoding thiolase is further studied with respect to enzyme activity and butanol production. The mutant thiolase (thlAV5A ) is showed a 32% higher activity than that of the wild-type thiolase (thlAWT ). In batch fermentation, butanol production is increased by 26% and 23% when the thlAV5A gene is overexpressed in the wild-type C. acetobutylicum ATCC 824 and in its derivative, the thlA-knockdown TKW-A strain, respectively. Based on structural analysis, the mutation in thiolase does not have a direct effect on the regulatory determinant region (RDR). However, the mutation at the 5th residue seems to influence the stability of the RDR, and thus, increases the enzymatic activity and enhances solvent production in the BKM19 strain.
Asunto(s)
Acetona/metabolismo , Butanoles/metabolismo , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Etanol/metabolismo , Genoma Bacteriano/genéticaRESUMEN
BACKGROUND: Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. RESULTS: The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. CONCLUSIONS: The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.
RESUMEN
Elucidating ways to enhance megakaryopoiesis in vivo would have therapeutic applications for thrombocytopenia and transfusion medicine. Nicotinamide has been shown to enhance endomitosis in megakaryocytes cultured in vitro, suggesting that it may be beneficial for the production of platelets in culture. We hypothesized that regular injections of nicotinamide in mice would also increase platelets in vivo. However, we found that platelet counts were reduced by about 25% with daily injections of nicotinamide. Altering the schedule, duration, or nicotinamide dose did not improve platelet production. Consistent with lower platelet levels, nicotinamide also tended to decrease megakaryocyte frequency in sternum and spleen sections, as well as colony formation in vitro by bone marrow progenitor cells. However, there was no effect on the fraction or ploidy of CD41(+) cells harvested from bone marrow. Together, our results suggest that, although nicotinamide increases polyploidization of megakaryocytes in culture, it does not have translatable effects in vivo.
Asunto(s)
Plaquetas/efectos de los fármacos , Niacinamida/administración & dosificación , Recuento de Plaquetas , Animales , Células de la Médula Ósea/citología , Femenino , Masculino , Megacariocitos/citología , Ratones , Células Progenitoras Mieloides/efectos de los fármacos , Bazo/citología , Trombopoyesis/efectos de los fármacosRESUMEN
Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.
Asunto(s)
2-Propanol/metabolismo , Butanoles/metabolismo , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Etanol/metabolismo , Ingeniería Metabólica , Acetona/metabolismo , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/metabolismo , Biocombustibles , Clostridium acetobutylicum/enzimología , Clostridium beijerinckii/enzimología , Clostridium beijerinckii/genética , Fermentación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
HoxA10 is a homeodomain transcription factor that is frequently overexpressed in human acute myeloid leukemia. In murine bone marrow transplantation studies, HoxA10 overexpression induces a myeloproliferative disorder with accumulation of mature phagocytes in the peripheral blood and tissues. Over time, differentiation block develops in these animals, resulting in acute myeloid leukemia. In immature myeloid cells, HoxA10 represses transcription of some genes that confer the mature phagocyte phenotype. Therefore, overexpressed HoxA10 blocks differentiation by repressing myeloid-specific gene transcription in differentiating myeloid cells. In contrast, target genes involved in myeloproliferation due to HoxA10 overexpression have not been identified. To identify such genes, we screened a CpG island microarray with HoxA10 co-immunoprecipitating chromatin. We identified the DUSP4 gene, which encodes mitogen-activated protein kinase phosphatase 2 (Mkp2), as a HoxA10 target gene. We analyzed the DUSP4 5'-flank and identified two proximal-promoter cis elements that are activated by HoxA10. We find that DUSP4 transcription and Mkp2 expression decrease during normal myelopoiesis. However, this down-regulation is impaired in myeloid cells overexpressing HoxA10. In hematopoietic cells, c-Jun N-terminal kinases (Jnk) are the preferred substrates for Mkp2. Therefore, Mkp2 inhibits apoptosis by dephosphorylating (inactivating) Jnk. Consistent with this, HoxA10 overexpression decreases apoptosis in differentiating myeloid cells. Therefore, our studies identify a mechanism by which overexpressed HoxA10 contributes to inappropriate cell survival during myelopoiesis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células Mieloides/enzimología , Mielopoyesis , Proteínas Tirosina Fosfatasas/biosíntesis , Animales , Apoptosis/genética , Diferenciación Celular/genética , Supervivencia Celular/genética , Islas de CpG/genética , Fosfatasas de Especificidad Dual , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica/genética , Regulación Leucémica de la Expresión Génica/genética , Proteínas Homeobox A10 , Proteínas de Homeodominio/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Células Mieloides/patología , Mielopoyesis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos/genética , Fagocitos/enzimología , Fagocitos/patología , Proteína Fosfatasa 2 , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Elementos de Respuesta/genética , Transcripción Genética/genética , Células U937RESUMEN
OBJECTIVE: Interleukin 15 (IL)-15 controls proliferation and survival of T cells, but its effects and the underlying cellular regulation are not well understood. Previous studies have focused on its effects on short-term T-cell cultures. In view of the potential problems associated with using IL-2 alone in adoptive immunotherapy protocols, we investigated the impact of IL-15 on T-cell cultures and the global transcriptional effects it elicits in such cultures. MATERIALS AND METHODS: DNA microarrays and flow cytometry were used to examine the differential effect of 20 ng/mL IL-15 on primary serum-free T-cell cultures activated and cultured in the presence of IL-2. Quantitative reverse transcriptase polymerase chain reaction confirmed select microarray data. RESULTS: IL-15 significantly increased ex vivo expansion of primary human T cells over the entire 11-day expansions without affecting viability. The 1133 genes were consistently differentially expressed among three donor samples. Ontological analysis demonstrated that IL-15 increases expression of genes involved in inflammatory response (e.g., tumor necrosis factor [TNF]-alpha, Oncostatin M, CD40L, and CD33) and apoptosis (e.g., TNF-related apoptosis-inducing ligand). IL-15 also induced expression of four suppressors of cytokine signaling (SOCS) family genes (SOCS1-3, cytokine-inducible SH2-containing protein), which are classical negative regulators of cytokine signaling. IL-15 strongly suppressed the expression of inhibitory natural killer cell receptor genes, including three C-type lectins (KLRB1, KLRC1, and KLRD1), as well as IL-7Ra and Granzyme H. Finally, IL-15 induced differential expression of TNF receptor superfamily members (CD27 and CD30). CONCLUSION: These findings suggest that exogenous IL-15 may have a potential role in adoptive immunotherapy by both enhancing proliferation and modulating functionality during ex vivo T-cell expansion.
Asunto(s)
Perfilación de la Expresión Génica , Inflamación/inmunología , Interleucina-15/inmunología , Proteínas Supresoras de la Señalización de Citocinas/genética , Linfocitos T/inmunología , Antígenos CD/efectos de los fármacos , Antígenos CD/genética , Apoptosis/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Análisis por Conglomerados , Citometría de Flujo/métodos , Granzimas/efectos de los fármacos , Granzimas/genética , Humanos , Interleucina-15/farmacología , Interleucina-2/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Inmunológicos/efectos de los fármacos , Receptores Inmunológicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Proteínas Supresoras de la Señalización de Citocinas/efectos de los fármacos , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Transcripción GenéticaRESUMEN
Prokaryotic genes are frequently organized in multicistronic operons (or transcriptional units, TUs), and usually the regulatory motifs for the whole TU are located upstream of the first TU gene. Although the number of sequenced genomes has increased dramatically, experimental information on TU organization is extremely limited. Even for organisms as extensively studied as Escherichia coli and Bacillus subtilis, TU annotation is far from complete. It therefore becomes imperative to rely on computational approaches to complement experimental information. Here we present a TU map for the obligate anaerobe Clostridium acetobutylicum ATCC 824. This map is largely based on the distance between pairs of consecutive genes but enhanced and refined by predictions of several types of promoters (sigmaA, sigmaE and sigmaF/G) and rho-independent terminator structures. Based on the set of known C.acetobutylicum TUs, the presented TU map offers an 88% prediction accuracy.
Asunto(s)
Clostridium/genética , Genoma Bacteriano , Transcripción Genética , Mapeo Cromosómico , Biología Computacional/métodos , Regiones Promotoras Genéticas , Reproducibilidad de los ResultadosRESUMEN
An intuitive normalization and gene identification method is proposed. After segmentation of the entire expression range into intensity intervals, the mean and standard deviation of the logarithm of expression ratios are calculated for each interval using the nearest neighbor genes. Genes with high differential expression are excluded from these calculations. For glass arrays, normalization is performed for each interval by using the mean of the logarithm of expression ratios in the interval. For nylonplastic membranes, the average of the means of the logarithm of ratios across the intervals of higher intensities is used for normalization. Compared with other normalization methods, this method delivered the smallest normalization errors for 42 nylonplastic arrays used to analyze cultured T cells and 22 Clostridium acetobutylicum glass arrays. For identifying differentially expressed genes, upper and lower boundaries are constructed for each interval by using the standard deviation of the expression ratio logarithms. When a C. acetobutylicum pSOL1 megaplasmid-deficient strain M5 was used, this method identified more "down-regulated" pSOL1 genes with fewer misidentifications in a comparative array analysis of M5 versus the parent strain. A comparison of quantitative RT-PCR results with different gene identification methods indicates that the proposed method is superior to other methods.