RESUMO
Phospholipids, the most abundant membrane lipid components, are crucial in maintaining membrane structures and homeostasis for biofunctions. As a structurally diverse and tightly regulated system involved in multiple organelles, phospholipid metabolism is complicated to manipulate. Thus, repurposing phospholipids for lipid-derived chemical production remains unexplored. Herein, we develop a Saccharomyces cerevisiae platform for de novo production of oleoylethanolamide, a phospholipid derivative with promising pharmacological applications in ameliorating lipid dysfunction and neurobehavioral symptoms. Through deregulation of phospholipid metabolism, screening of biosynthetic enzymes, engineering of subcellular trafficking and process optimization, we could produce oleoylethanolamide at a titer of 8,115.7 µg l-1 and a yield on glucose of 405.8 µg g-1. Our work provides a proof-of-concept study for systemically repurposing phospholipid metabolism for conversion towards value-added biological chemicals, and this multi-faceted framework may shed light on tailoring phospholipid metabolism in other microbial hosts.
Assuntos
Endocanabinoides/biossíntese , Engenharia Metabólica/métodos , Ácidos Oleicos/biossíntese , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acil Coenzima A/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , Coenzima A Ligases/genética , Endocanabinoides/genética , Enzimas/genética , Enzimas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Lisofosfolipase/genética , Lisofosfolipase/metabolismo , Microrganismos Geneticamente Modificados , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Ácidos Oleicos/genética , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Fosfolipídeos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes into the reaction chambers and demonstrated their capability to convert acyl-ACP or acyl-CoA from canonical fatty acid biosynthesis to short/medium-chain fatty acids and methyl ketones.
Assuntos
Basidiomycota/enzimologia , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/metabolismo , Cetonas/metabolismo , Saccharomyces cerevisiae/enzimologia , Biocatálise , Ácido Graxo Sintase Tipo I/química , Ácidos Graxos/química , Cetonas/química , Modelos Moleculares , Estrutura MolecularRESUMO
A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.
Assuntos
Expressão Gênica , Engenharia Metabólica , Peptídeos/genética , Saccharomyces cerevisiae/genética , Vírus/genética , Vetores Genéticos , Maytenus/enzimologia , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Triterpenos/metabolismo , Proteínas Virais/genéticaRESUMO
Microbial synthesis of medium chain aliphatic hydrocarbons, attractive drop-in molecules to gasoline and jet fuels, is a promising way to reduce our reliance on petroleum-based fuels. In this study, we enabled the synthesis of straight chain hydrocarbons (C7-C13) by yeast Saccharomyces cerevisiae through engineering fatty acid synthases to control the chain length of fatty acids and introducing heterologous pathways for alkane or 1-alkene synthesis. We carried out enzyme engineering/screening of the fatty aldehyde deformylating oxygenase (ADO), and compartmentalization of the alkane biosynthesis pathway into peroxisomes to improve alkane production. The two-step synthesis of alkanes was found to be inefficient due to the formation of alcohols derived from aldehyde intermediates. Alternatively, the drain of aldehyde intermediates could be circumvented by introducing a one-step decarboxylation of fatty acids to 1-alkenes, which could be synthesized at a level of 3mg/L, 25-fold higher than that of alkanes produced via aldehydes.
Assuntos
Alcanos/metabolismo , Alcenos/metabolismo , Ácidos Graxos , Engenharia Metabólica , Peroxissomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Peroxissomos/genética , Peroxissomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Beta-elemene, a sesquiterpene and the major component of the medicinal herb Curcuma wenyujin, has antitumor activity against various types of cancer and could potentially serve as a potent antineoplastic drug. However, its current mode of production through extraction from plants has been inefficient and suffers from limited natural resources. Here, we engineered a yeast cell factory for the sustainable production of germacrene A, which can be transformed to beta-elemene by a one-step chemical reaction in vitro. Two heterologous germacrene A synthases (GASs) converting farnesyl pyrophosphate (FPP) to germacrene A were evaluated in yeast for their ability to produce germacrene A. Thereafter, several metabolic engineering strategies were used to improve the production level. Overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase and fusion of FPP synthase with GAS, led to a sixfold increase in germacrene A production in shake-flask culture. Finally, 190.7 mg/l of germacrene A was achieved. The results reported in this study represent the highest titer of germacrene A reported to date. These results provide a basis for creating an efficient route for further industrial application re-placing the traditional extraction of beta-elemene from plant sources.
Assuntos
Regulação Fúngica da Expressão Gênica , Engenharia Metabólica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sesquiterpenos de Germacrano/biossíntese , Sesquiterpenos/metabolismo , Técnicas de Cultura Celular por Lotes , Meios de Cultura/química , Geraniltranstransferase/genética , Geraniltranstransferase/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Fosfatos de Poli-Isoprenil , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: While oxygen limitation can be extremely damaging for many animals, some vertebrates have perfected anaerobic survival. Freshwater turtles belonging to the Trachemys and Chrysemys genera, for example, can survive many weeks without oxygen, and as such are commonly used as model animals for vertebrate anoxia tolerance. SCOPE OF REVIEW: In the present review we discuss the recent advances made in understanding the biochemical and molecular nature of natural anoxia tolerance of freshwater turtles. MAJOR CONCLUSIONS: Research in recent years has shown that activation of several important pathways occurs in response to anoxia in turtles, including those that function in the stress response, cell cycle arrest, inhibition of gene expression and metabolism. These likely contribute to anoxia tolerance in turtle tissues by minimizing cell damage in response to anoxia, as well as facilitating metabolic rate depression. GENERAL SIGNIFICANCE: The research discussed in the present review contributes to the understanding of how freshwater turtles can survive without oxygen for prolonged periods of time. This could also improve understanding of the molecular nature of hypoxic/ischemic injuries in mammalian tissues and suggest potential ways to avoid these.
Assuntos
Hipóxia/metabolismo , Oxigênio/metabolismo , Tartarugas/metabolismo , Adaptação Fisiológica , Animais , Antioxidantes/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Hipóxia/genética , Hipóxia/fisiopatologia , Estresse Oxidativo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Tartarugas/genéticaRESUMO
Engineering industrial cell factories to effectively yield a desired product while dealing with industrially relevant stresses is usually the most challenging step in the development of industrial production of chemicals using microbial fermentation processes. Using synthetic biology tools, microbial cell factories such as Saccharomyces cerevisiae can be engineered to express synthetic pathways for the production of fuels, biopharmaceuticals, fragrances, and food flavors. However, directing fluxes through these synthetic pathways towards the desired product can be demanding due to complex regulation or poor gene expression. Systems biology, which applies computational tools and mathematical modeling to understand complex biological networks, can be used to guide synthetic biology design. Here, we present our perspective on how systems biology can impact synthetic biology towards the goal of developing improved yeast cell factories. Biotechnol. Bioeng. 2016;113: 1164-1170. © 2015 Wiley Periodicals, Inc.
Assuntos
Técnicas de Cultura Celular por Lotes/tendências , Reatores Biológicos/microbiologia , Microbiologia Industrial/tendências , Engenharia Metabólica/tendências , Saccharomyces cerevisiae/fisiologia , Biologia de Sistemas/tendências , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/metabolismo , Simulação por Computador , Modelos BiológicosRESUMO
BACKGROUND: ChREBP (carbohydrate response element binding protein) is a glucose-responsive transcription factor that is known to be an important regulator of glycolytic and lipogenic genes in response to glucose. We hypothesized that activation of ChREBP could be relevant to anoxia survival by the anoxia-tolerant turtle, Trachemys scripta elegans. METHODS: Expression of ChREBP in response to 5 and 20h of anoxia was examined using RT-PCR and Western immunoblotting. In addition, subcellular localization and DNA-binding activity of ChREBP protein were assessed and transcript levels of liver pyruvate kinase (LPK), a downstream gene under ChREBP control were quantified using RT-PCR. RESULTS: ChREBP was anoxia-responsive in kidney and liver, with transcript levels increasing by 1.2-1.8 fold in response to anoxia and protein levels increasing by 1.8-1.9 fold. Enhanced nuclear presence under anoxia was also observed in both tissues by 2.2-2.8 fold. A 4.2 fold increase in DNA binding activity of ChREBP was also observed in liver in response to 5h of anoxia. In addition, transcript levels of LPK increased by 2.1 fold in response to 5h of anoxia in the liver. CONCLUSIONS: The results suggest that activation of ChREBP in response to anoxia might be a crucial factor for anoxia survival in turtle liver by contributing to elevated glycolytic flux in the initial phases of oxygen limitation. GENERAL SIGNIFICANCE: This study provides the first demonstration of activation of ChREBP in response to anoxia in a natural model of anoxia tolerance, further improving our understanding of the molecular nature of anoxia tolerance.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hipóxia/metabolismo , Fígado/metabolismo , Piruvato Quinase/biossíntese , Animais , Hipóxia/patologia , Fatores de Tempo , TartarugasRESUMO
Recent concerns over the sustainability of petrochemical-based processes for production of desired chemicals have fueled research into alternative modes of production. Metabolic engineering of microbial cell factories such as Saccharomyces cerevisiae and Escherichia coli offers a sustainable and flexible alternative for the production of various molecules. Acetyl-CoA is a key molecule in microbial central carbon metabolism and is involved in a variety of cellular processes. In addition, it functions as a precursor for many molecules of biotechnological relevance. Therefore, much interest exists in engineering the metabolism around the acetyl-CoA pools in cells in order to increase product titers. Here we provide an overview of the acetyl-CoA metabolism in eukaryotic and prokaryotic microbes (with a focus on S. cerevisiae and E. coli), with an emphasis on reactions involved in the production and consumption of acetyl-CoA. In addition, we review various strategies that have been used to increase acetyl-CoA production in these microbes.
Assuntos
Acetilcoenzima A , Escherichia coli , Engenharia Metabólica , Saccharomyces cerevisiae , Acetilcoenzima A/biossíntese , Acetilcoenzima A/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Pyruvate formate lyase (PFL) is characterized as an enzyme functional at anaerobic conditions, since the radical in the enzyme's active form is sensitive to oxygen. In this study, PFL and its activating enzyme from Escherichia coli were expressed in a Saccharomyces cerevisiae strain lacking pyruvate decarboxylase and having a reduced glucose uptake rate due to a mutation in the transcriptional regulator Mth1, IMI076 (Pdc(-) MTH1-ΔT ura3-52). PFL was expressed with two different electron donors, reduced ferredoxin or reduced flavodoxin, respectively, and it was found that the coexpression of either of these electron donors had a positive effect on growth under aerobic conditions, indicating increased activity of PFL. The positive effect on growth was manifested as a higher final biomass concentration and a significant increase in transcription of formate dehydrogenases. Among the two electron donors reduced flavodoxin was found to be a better electron donor than reduced ferredoxin.
Assuntos
Acetiltransferases/biossíntese , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/genética , Aerobiose , Biomassa , Escherichia coli/enzimologia , Escherichia coli/genética , Ferredoxinas/metabolismo , Flavodoxina/metabolismo , Perfilação da Expressão Gênica , Oxirredução , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transcrição GênicaRESUMO
BACKGROUND: A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole carbon source, and requires supplementation of C2 compounds to the medium in order to meet the requirement for cytosolic acetyl-CoA for biosynthesis of fatty acids and ergosterol. RESULTS: In this study, a Pdc negative strain was adaptively evolved for improved growth in glucose medium via serial transfer, resulting in three independently evolved strains, which were able to grow in minimal medium containing glucose as the sole carbon source at the maximum specific rates of 0.138, 0.148, 0.141 h(-1), respectively. Several genetic changes were identified in the evolved Pdc negative strains by genomic DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase, and RPD3 encoding a histone deacetylase. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH1 (81D) allele restored its growth on glucose at a maximum specific rate of 0.053 h(-1) in minimal medium with 2% glucose, and the CIT1 deletion in the reverse engineered strain further increased the maximum specific growth rate to 0.069 h(-1). CONCLUSIONS: In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing expression of several hexose transporter genes. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Glucose/metabolismo , Histona Desacetilases/genética , Piruvato Descarboxilase/deficiência , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Meios de Cultura/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Histona Desacetilases/metabolismo , Mutação , Piruvato Descarboxilase/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: The forkhead class O (FoxO) transcription factors are important regulators of multiple aspects of cellular metabolism. We hypothesized that activation of these transcription factors could play crucial roles in low oxygen survival in the anoxia-tolerant turtle, Trachemys scripta elegans. METHODS: Two FoxOs, FoxO1 and FoxO3, were examined in turtle tissues in response to 5 and 20h of anoxic submergence using techniques of RT-PCR, western immunoblotting and DNA-binding assays to assess activation. Transcript levels of FoxO-responsive genes were also quantified using RT-PCR. RESULTS: FoxO1 was anoxia-responsive in the liver, with increases in transcript levels, protein levels, nuclear levels and DNA-binding of 1.7-4.8fold in response to anoxia. Levels of phosphorylated FoxO1 also decreased to 57% of control values in response to 5h of anoxia, indicating activation. FoxO3 was activated in the heart, kidney and liver in response to anoxia, with nuclear levels increasing by 1.5-3.7fold and DNA-binding activity increasing by 1.3-2.9fold. Transcript levels of two FoxO-target genes, p27kip1 and catalase, also rose by 2.4-2.5fold in the turtle liver under anoxia. CONCLUSIONS: The results suggest that the FoxO transcription factors are activated in response to anoxia in T. scripta elegans, potentially contributing to the regulation of stress resistance and metabolic depression. GENERAL SIGNIFICANCE: This study provides the first demonstration of activation of FoxOs in a natural model for vertebrate anoxia tolerance, further improving understanding of how tissues can survive without oxygen.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Hipóxia/metabolismo , Fatores de Transcrição/metabolismo , Tartarugas/metabolismo , Animais , Catalase/genética , Catalase/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas de Ligação a DNA/genética , Água Doce , Expressão Gênica , Hipóxia/genética , Rim/metabolismo , Fígado/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição/genética , Tartarugas/genéticaRESUMO
Red-eared slider turtles, Trachemys scripta elegans, can survive for several weeks without oxygen when submerged in cold water. We hypothesized that anaerobiosis is aided by adaptive up-regulation of the unfolded protein response (UPR), a stress-responsive pathway that is activated by accumulation of unfolded proteins in the endoplasmic reticulum (ER) and functions to restore ER homeostasis. RT-PCR, western immunoblotting and DNA-binding assays were used to quantify the responses and/or activation status of UPR-responsive genes and proteins in turtle tissues after animal exposure to 5 or 20 h of anoxic submergence at 4 °C. The phosphorylation state of protein kinase-like ER kinase (PERK) (a UPR-regulated kinase) and eukaryotic initiation factor 2 (eIF2α) increased by 1.43-2.50 fold in response to anoxia in turtle heart, kidney, and liver. Activation of the PERK-regulated transcription factor, activating transcription factor 4 (ATF4), during anoxia was documented by elevated atf4 transcripts and total ATF4 protein (1.60-2.43 fold), increased nuclear ATF4 content, and increased DNA-binding activity (1.44-2.32 fold). ATF3 and GADD34 (downstream targets of ATF4) also increased by 1.38-3.32 fold in heart and liver under anoxia, and atf3 transcripts were also elevated in heart. Two characteristic chaperones of the UPR, GRP78, and GRP94, also responded positively to anoxia with strong increases in both the transcript and protein levels. The data demonstrate that the UPR is activated in turtle heart, kidney, and liver in response to anoxia, suggesting that this pathway mediates an integrated stress response to protect tissues during oxygen deprivation.
Assuntos
Retículo Endoplasmático/metabolismo , Hipóxia , Tartarugas/metabolismo , Resposta a Proteínas não Dobradas , Fator 3 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Anaerobiose/fisiologia , Animais , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Coração , Proteínas de Choque Térmico/metabolismo , Rim , Fígado , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oxigênio/metabolismo , Fosforilação , Dobramento de Proteína , Análise de Sequência de DNA , Transdução de Sinais , eIF-2 Quinase/metabolismoRESUMO
Recently, butanols (1-butanol, 2-butanol and iso-butanol) have generated attention as alternative gasoline additives. Butanols have several properties favorable in comparison to ethanol, and strong interest therefore exists in the reconstruction of the 1-butanol pathway in commonly used industrial microorganisms. In the present study, the biosynthetic pathway for 1-butanol production was reconstructed in the yeast Saccharomyces cerevisiae. In addition to introducing heterologous enzymes for butanol production, we engineered yeast to have increased flux toward cytosolic acetyl-CoA, the precursor metabolite for 1-butanol biosynthesis. This was done through introduction of a plasmid-containing genes for alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA synthetase (ACS), and acetyl-CoA acetyltransferase (ERG10), as well as the use of strains containing deletions in the malate synthase (MLS1) or citrate synthase (CIT2) genes. Our results show a trend to increased butanol production in strains engineered for increased cytosolic acetyl-CoA levels, with the best-producing strains having maximal butanol titers of 16.3 mg/l. This represents a 6.5-fold improvement in butanol titers compared to previous values reported for yeast and demonstrates the importance of an improved cytosolic acetyl-CoA supply for heterologous butanol production by this organism.
Assuntos
1-Butanol/metabolismo , Acetilcoenzima A/metabolismo , Biocombustíveis/provisão & distribuição , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Vias Biossintéticas/genética , Citrato (si)-Sintase/genética , Citosol/metabolismo , Malato Sintase/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia SintéticaRESUMO
BACKGROUND: Hatchlings of the painted turtle, Chrysemys picta marginata can endure long-term freezing of their extracellular body fluids. We hypothesized that freezing survival would include adaptive up-regulation of antioxidant defenses to deal with ischemia-reperfusion injuries associated with the freeze-thaw cycle. A number of antioxidant enzymes are under the control of the NF-E2 related factor 2 (Nrf2) transcription factor including members of the glutathione S-transferase (GST) and aldo-keto reductase (AKR) families. METHODS: RT-PCR and Western immunoblotting were used to measure changes in transcript and protein levels in response to 5-h freezing exposure of hatchlings. RESULTS: Transcript levels of Nrf2 increased in turtle brain, liver, and muscle by 1.5- to 2-fold, and protein levels increased in the brain and muscle by 1.6- to 2.3-fold in response to freezing. GSTs responded strongly to freezing in turtle brain with amounts of GSTP1, M1, K1, and A3 elevated by 1.5- to 2.4-fold. GSTM3 and T1 rose by 1.8- to 2.3-fold in gut, whereas reduced levels of GSTP1, M1, M3, and K1 were found in livers of frozen animals. Levels of the AKR1B4 isozyme rose 2.1-fold during freezing in brain. CONCLUSIONS: Freezing triggered tissue-specific changes in the antioxidant defenses in C.pictamarginata organs. GENERAL SIGNIFICANCE: These data indicate that activation of an antioxidant response is an important aspect of natural freeze tolerance in turtles.
Assuntos
Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/genética , Tartarugas/fisiologia , Aclimatação , Sequência de Aminoácidos , Animais , Western Blotting , Galinhas/genética , Primers do DNA , Ovos , Feminino , Congelamento , Amplificação de Genes , Humanos , Dados de Sequência Molecular , Fator 2 Relacionado a NF-E2/química , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , Homologia de Sequência de Aminoácidos , Tartarugas/genética , Xenopus/genética , Peixe-Zebra/genéticaRESUMO
The importance of epigenetics has been established in many key biological processes but the relevance of this regulatory mechanism to animal survival of low oxygen conditions has never been examined. To establish whether epigenetic mechanisms could be involved in natural anoxia tolerance, we have examined the anoxia-responsive expression of the transcriptional silencers, histone deacetylases (HDACs), in tissues of a unique model for anoxia tolerance, the freshwater turtle Trachemys scripta elegans. Transcript and protein levels of all five HDACs rose by 1.3-4.6 and 1.7-3.5-fold, respectively, in skeletal muscle in response to 20 h of anoxia exposure. In addition, HDAC activity in the muscle increased by 1.5-fold in response to 20 h of anoxia and levels of acetylated histone H3 (Lys 9 or Lys 23) decreased to 40-60% of control values. The liver displayed a milder response with HDAC1, -4, and -5 protein levels increasing by 1.6-2.1-fold after 5 h anoxia exposure; acetylated histone H3 levels also decreased to 50-75% of control values. Only HDAC5 responded to anoxia exposure in the heart; Hdac5 transcript levels increased 2.1-2.3-fold and HDAC5 protein rose by 3.3-fold. Overall, our results show a tissue-specific pattern of HDAC upregulation in response to anoxia exposure in T.s. elegans, suggesting that these enzymes play a key role in anoxia tolerance, probably by contributing to the transcriptional silencing necessary in this hypometabolic state.
Assuntos
Epigênese Genética , Histona Desacetilases/genética , Hipóxia/enzimologia , Hipóxia/genética , Acetilação , Animais , Western Blotting , Coração/fisiologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Especificidade de Órgãos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TartarugasRESUMO
This corrects the article DOI: 10.1038/ncomms15587.
RESUMO
Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae. VLCFAs can be selectively modified towards the fatty alcohol docosanol (C22H46O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l-1 in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.
Assuntos
Ácidos Graxos/biossíntese , Álcoois Graxos/metabolismo , Química Verde/métodos , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocombustíveis , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/química , Álcoois Graxos/química , Fermentação , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/genéticaRESUMO
The red-eared slider turtle, Trachemys scripta elegans, is a model organism commonly used to study the environmental stress of anoxia. It exhibits multiple biochemical adaptations to ensure its survival during the winter months where quantities of oxygen are largely depleted. We proposed that JAK-STAT signaling would display stress responsive regulation to mediate the survival of the red-eared slider turtle, Trachemys scripta elegans, during anoxic stress. Importantly, the JAK-STAT signaling pathway is involved in transmitting extracellular signals to the nucleus resulting in the expression of select genes that aid cell survival and growth. Immunoblotting was used to compare the relative phosphorylation levels of JAK proteins, STAT proteins, and two of its inhibitors, SOCS and PIAS, in response to anoxia. A clear activation of the JAK-STAT pathway was observed in the liver tissue while no significant changes were found in the skeletal muscle. To further support our findings we also found an increase in mRNA transcripts of downstream targets of STATs, namely bcl-xL and bcl-2, using PCR analysis in the liver tissues. These findings suggest an important role for the JAK-STAT pathway in exhibiting natural anoxia tolerance by the red-eared slider turtle.
Assuntos
Hipóxia/metabolismo , Janus Quinases/metabolismo , Oxigênio/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Tartarugas/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Hipóxia/genética , Janus Quinases/genética , Fosforilação , Fatores de Transcrição STAT/genética , Tartarugas/genéticaRESUMO
Many high-value metabolites are produced in nature by organisms that are not ideal for large-scale production. Therefore, interest exists in expressing the biosynthetic pathways of these compounds in organisms that are more suitable for industrial production. Recent years have seen developments in both the discovery of various biosynthetic pathways, as well as development of metabolic engineering tools that allow reconstruction of complex pathways in microorganisms. In the present review we discuss recent advances in reconstruction of the biosynthetic pathways of various high-value products in the yeast Saccharomyces cerevisiae, a commonly used industrial microorganism. Key achievements in the production of different isoprenoids, aromatics and polyketides are presented and the metabolic engineering strategies underlying these accomplishments are discussed.