RESUMO
Cyanobacteria Synechocystis sp. PCC 6803 was exploited as green cell factory for light-powered asymmetric synthesis of aromatic chiral alcohols. The effect of temperature, light, substrate and cell concentration on substrate conversions were investigated. Under the optimal condition, a series of chiral alcohols were synthesized with conversions up to 95% and enantiomer excess (ee)â¯>â¯99%. We found that the addition of Na2S2O3 and Angeli's Salt increased the NADPH content by 20% and 25%, respectively. As a result, the time to reach 95% substrate conversion was shortened by 12â¯h, which demonstrated that the NADPH regeneration and hence the reaction rates can be regulated in cyanobacteria. This blue-green algae based biocatalysis showed its potential for chiral compounds production in future.
Assuntos
Álcoois/metabolismo , Luz , NADP/biossíntese , Synechocystis/química , Álcoois/química , Estrutura Molecular , NADP/química , Synechocystis/metabolismoRESUMO
The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reactionNAADP to NAADPHis catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.
Assuntos
Sinalização do Cálcio , Oxidases Duais , Ativação Linfocitária , NADPH Oxidases , NADP/biossíntese , Linfócitos T , Animais , Oxidases Duais/genética , Células HEK293 , Humanos , Células Jurkat , Camundongos Knockout , NADP/análogos & derivados , NADPH Oxidases/genética , Linfócitos T/enzimologiaRESUMO
Hypoxia is known to be commonly present in breast tumor microenvironments. Stem-like cells that repopulate breast tumors, termed tumor-repopulating cells (TRC), thrive under hypoxic conditions, but the underlying mechanism remains unclear. Here, we show that hypoxia promotes the growth of breast TRCs through metabolic reprogramming. Hypoxia mobilized transcription factors HIF1α and FoxO1 and induced epigenetic reprogramming to upregulate cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme that initiates gluconeogenesis. PCK1 subsequently triggered retrograde carbon flow from gluconeogenesis to glycogenesis, glycogenolysis, and the pentose phosphate pathway. The resultant NADPH facilitated reduced glutathione production, leading to a moderate increase of reactive oxygen species that stimulated hypoxic breast TRC growth. Notably, this metabolic mechanism was absent in differentiated breast tumor cells. Targeting PCK1 synergized with paclitaxel to reduce the growth of triple-negative breast cancer (TNBC). These findings uncover an altered glycogen metabolic program in breast cancer, providing potential metabolic strategies to target hypoxic breast TRCs and TNBC. SIGNIFICANCE: Hypoxic breast cancer cells trigger self-growth through PCK1-mediated glycogen metabolism reprogramming that leads to NADPH production to maintain a moderate ROS level.
Assuntos
Neoplasias da Mama/metabolismo , Gluconeogênese , Glicogênio/metabolismo , Hipóxia/metabolismo , Animais , Biomarcadores , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Redes e Vias Metabólicas , Camundongos , NADP/biossíntese , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cerebrovascular remodeling is the most common cause of hypertension and stroke. Ubiquitin E3 ligase RING finger protein 34 (RNF34) is suggested to be associated with the development of multiple neurological diseases. However, the importance of RNF34 in cerebrovascular remodeling and hypertension is poorly understood. Herein, we used mice with a global RNF34 knockout as well as RNF34 floxed mice to delete RNF34 in endothelial cells and smooth muscle cells (SMCs). Our results showed that global RNF34 knockout mice substantially promoted angiotensin II (AngII)-induced middle cerebral artery (MCA) remodeling, hypertension, and neurological dysfunction. Endothelial cell RNF34 did not regulate the development of hypertension. Rather, SMC RNF34 expression is a critical regulator of hypertension and MCA remodeling. Loss of RNF34 enhanced AngII-induced mouse brain vascular SMCs (MBVSMCs) proliferation, migration and invasion. Furthermore, MCA and MBVSMCs from SMC RNF34-deficient mice showed increased superoxide anion and reactive oxygen species (ROS) generation as well as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, but exhibited no marked effect on mitochondria-derived ROS. Knockout of RNF34 promoted p22phox expression, leading to increased binding of p22phox/p47phox and p22phox/NOX2, and eventually NADPH oxidase complex formation. Immunoprecipitation assay identified that RNF34 interacted with p22phox. RNF34 deletion increased p22phox protein stability by inhibiting ubiquitin-mediated degradation. Blockade of NADPH oxidase activity or knockdown of p22phox significantly abolished the effects of RNF34 deletion on cerebrovascular remodeling and hypertension. Collectively, our study demonstrates that SMC RNF34 deficiency promotes cerebrovascular SMC hyperplasia and remodeling by increased NADPH-derived ROS generation via reducing p22phox ubiquitin-dependent degradation.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Circulação Cerebrovascular/fisiologia , Hipertensão/metabolismo , NADP/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Remodelação Vascular/fisiologia , Animais , Proteínas de Transporte/genética , Células Cultivadas , Células HEK293 , Humanos , Hipertensão/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Estresse Oxidativo/fisiologiaRESUMO
Aging of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative damage in macromolecules, which contributes to cellular malfunction, compromises cell viability, and, ultimately, leads to functional decline. Cellular detoxification relies in part on the production of NADPH, which is an important cofactor for major cellular antioxidant systems. NADPH is produced principally by the housekeeping enzyme glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the rate-limiting step in the pentose phosphate pathway. We show here that G6PD transgenic mice (G6PD-Tg), which show enhanced constitutive G6PD activity and NADPH production along life, have lower auditory thresholds than wild-type mice during aging, together with preserved inner hair cell (IHC) and outer hair cell (OHC), OHC innervation, and a conserved number of synapses per IHC. Gene expression of antioxidant enzymes was higher in 3-month-old G6PD-Tg mice than in wild-type counterparts, whereas the levels of pro-apoptotic proteins were lower. Consequently, nitration of proteins, mitochondrial damage, and TUNEL+ apoptotic cells were all lower in 9-month-old G6PD-Tg than in wild-type counterparts. Unexpectedly, G6PD overexpression triggered low-grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophage infiltration. Our results lead us to propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the production of ROS and cellular detoxification power along aging and thus prevents hearing loss progression.
Assuntos
Envelhecimento/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Presbiacusia/enzimologia , Presbiacusia/prevenção & controle , Envelhecimento/fisiologia , Animais , Apoptose , Limiar Auditivo/fisiologia , Cóclea/metabolismo , Cóclea/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NADP/biossíntese , Estresse Oxidativo , Presbiacusia/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulação para CimaRESUMO
The isocitrate dehydrogenases enzymes, IDH1 and IDH2, catalyze the conversion of isocitrate to α-ketoglutarate (αKG) in the cell cytoplasm and mitochondria, respectively, and contribute to generating the dihydronicotinamide-adenine dinucleotide phosphate (NADPH) as reductive potential in different cellular processes. Mutations in IDH1 and IDH2 genes are found collectively in about 20-25% of acute myeloid leukemia (AML) patients. Mutant IDH enzymes have neomorphic activity and convert αKG to the oncometabolite R-2-hydroxyglutarate (R-2-HG) which accumulates at high levels in the cell and hampers the function of αKG-dependent enzymes, including epigenetic regulators, thus leading to altered gene expression and block of differentiation and contributing to leukemia development. Inhibition of the neomorphic mutants induces marked decrease in R-2-HG levels and restores myeloid differentiation. Enasidenib and ivosidenib are potent and selective inhibitors of mutant IDH2 and IDH1, respectively, act as differentiating agents and showed clinical activity in relapsed/refractory (R/R) AML harboring the specific mutation. As single agents, both drugs have been approved by the Food and Drug Administration (FDA) for the treatment of R/R AML. The relevance of IDH targeting within either single agent approach or, most importantly, combinatorial treatments in AML will be discussed.
Assuntos
Aminopiridinas/uso terapêutico , Antineoplásicos/uso terapêutico , Glicina/análogos & derivados , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Piridinas/uso terapêutico , Triazinas/uso terapêutico , Aminopiridinas/efeitos adversos , Antineoplásicos/efeitos adversos , Diferenciação Celular/efeitos dos fármacos , Ensaios Clínicos como Assunto , Glutaratos/metabolismo , Glicina/efeitos adversos , Glicina/uso terapêutico , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Isocitratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Estudos Multicêntricos como Assunto , Mutação de Sentido Incorreto , NADP/biossíntese , Piridinas/efeitos adversos , Síndrome , Triazinas/efeitos adversosRESUMO
Reactive oxygen species (ROS) that are induced upon pathogen infection plays an important role in host defence. The rickettsial pathogen Anaplasma phagocytophilum, which is primarily transmitted by Ixodes scapularis ticks in the United States, has evolved many strategies to escape ROS and survive in mammalian cells. However, little is known on the role of ROS in A. phagocytophilum infection in ticks. Our results show that A. phagocytophilum and hemin induce activation of l-tryptophan pathway in tick cells. Xanthurenic acid (XA), a tryptophan metabolite, supports A. phagocytophilum growth in tick cells through inhibition of tryptophan dioxygenase (TDO) activity leading to reduced l-kynurenine levels that subsequently affects build-up of ROS. However, hemin supports A. phagocytophilum growth in tick cells by inducing TDO activity leading to increased l-kynurenine levels and ROS production. Our data reveal that XA and kynurenic acid (KA) chelate hemin. Furthermore, treatment of tick cells with 3-hydroxyl l-kynurenine limits A. phagocytophilum growth in tick cells. RNAi-mediated knockdown of kynurenine aminotransferase expression results in increased ROS production and reduced A. phagocytophilum burden in tick cells. Collectively, these results suggest that l-tryptophan pathway metabolites influence A. phagocytophilum survival by affecting build up of ROS levels in tick cells.
Assuntos
Anaplasma phagocytophilum/metabolismo , Ixodes/microbiologia , Triptofano/metabolismo , Animais , Hemina/metabolismo , Hemina/farmacologia , Interações Hospedeiro-Patógeno , Hidrolases/genética , Hidrolases/metabolismo , Ixodes/genética , Ixodes/metabolismo , Ácido Cinurênico/metabolismo , Ácido Cinurênico/farmacologia , Cinurenina/análogos & derivados , Cinurenina/metabolismo , Cinurenina/farmacologia , NADP/biossíntese , NADP/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transaminases/genética , Transaminases/metabolismo , Triptofano Oxigenase/antagonistas & inibidores , Triptofano Oxigenase/metabolismo , Regulação para Cima , Xanturenatos/metabolismo , Xanturenatos/farmacologiaRESUMO
Thraustochytrids of the genera Schizochytrium and Aurantiochytrium accumulate oils rich in the essential, marine n3 fatty acid docosahexaenoic acid (DHA). DHA production in Aurantiochytrium sp T66 was studied with the aim to provide more knowledge about factors that affect the DHA-productivities and the contributions of the two enzyme systems used for fatty acid synthesis in thraustochytrids, fatty acid synthetase (FAS) and PUFA-synthase. Fermentations with nitrogen starvation, which is well-known to initiate lipid accumulation in oleaginous organisms, were compared to fermentations with nitrogen in excess, obtained by oxygen limitation. The specific productivities of fatty acids originating from FAS were considerably higher under nitrogen starvation than with nitrogen in excess, while the specific productivities of DHA were the same at both conditions. Global transcriptome analysis showed significant up-regulation of FAS under N-deficient conditions, while the PUFA-synthase genes were only marginally upregulated. Neither of them was upregulated under O2-limitation where nitrogen was in excess, suggesting that N-starvation mainly affects the FAS and may be less important for the PUFA-synthase. The transcriptome analysis also revealed responses likely to be related to the generation of reducing power (NADPH) for fatty acid synthesis.
Assuntos
Ácidos Docosa-Hexaenoicos/biossíntese , Ácido Graxo Sintases/metabolismo , Estramenópilas/metabolismo , Ácido Graxo Sintases/genética , Fermentação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Cinética , NADP/biossíntese , Nitrogênio/metabolismo , Oxigênio/metabolismo , Regulação para CimaRESUMO
Autosomal dominant congenital cataract (ADCC), the most common hereditary disease, is a major cause of eye disease in children. Due to its high genetic and clinical heterogeneity, the identification of ADCC-associated gene mutations is essential for the development of molecular therapies. In this study, we examined a four-generation Chinese pedigree with ADCC and identified putative mutations in ADCC candidate genes via next-generation sequencing (NGS) followed by Sanger sequencing. A novel missense mutation in GJA8 (c.T217C) in ADCC patients causes a serine-to-proline substitution at residue 73 of connexin 50 (Cx50); no mutation was found in unaffected family members and unrelated healthy individuals. Functional analysis revealed that this missense mutation disrupts protein function in human lens epithelial cells (HLEpiCs), which fails to form calcium-sensitive hemichannels. Furthermore, mutant Cx50 leads to decreased ROS scavenging by inhibiting G6PD expression and thus induces cell apoptosis via aberrant activation of the unfolded protein response (UPR). In conclusion, we report a novel GJA8 heterozygous mutation in a Chinese family with a vital role in ADCC, broadening the genetic spectrum of this disease.
Assuntos
Apoptose , Conexinas/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Cristalino/patologia , Mutação de Sentido Incorreto/genética , Sequência de Bases , Catarata/genética , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Proteínas Mutantes/metabolismo , NADP/biossíntese , Linhagem , Agregados Proteicos , Espécies Reativas de Oxigênio/metabolismo , Resposta a Proteínas não Dobradas/genéticaRESUMO
Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B cell lymphomas. How EBV remodels metabolic pathways to support rapid B cell outgrowth remains largely unknown. To gain insights, primary human B cells were profiled by tandem-mass-tag-based proteomics at rest and at nine time points after infection; >8,000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production, and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism.
Assuntos
Aminoidrolases/metabolismo , Linfócitos B/metabolismo , Linfócitos B/virologia , Transformação Celular Viral , Infecções por Vírus Epstein-Barr/metabolismo , Ácido Fólico/metabolismo , Herpesvirus Humano 4/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Enzimas Multifuncionais/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Feminino , Glicólise , Células HEK293 , Humanos , Ativação Linfocitária , Mitocôndrias/metabolismo , NADP/biossíntese , Oxirredução , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina/biossínteseRESUMO
Thraustochytrids, a group of marine protists, are continuously gaining attention due to their capability in producing lipids for various biotechnological applications towards foods, medicines, chemicals, and biofuels. Although various substrates, predominantly glucose, have been used as carbon source for this microalga, it is desirable to adopt cheaper and more diversified substrate to expand their application range. In this study, we aimed to examine the ability of acetate, which can be easily generated from various resources by acetogenic microorganisms, as a substrate of Aurantiochytrium limacinum SR21. As a result of flask-scale analysis, specific growth rates (µ) of the strain SR21 grown in 3% acetate- or glucose-based medium were 0.55 and 0.98 h-1, respectively. The maximum yield of total fatty acid in acetate medium was 4.8 g/L at 48 h while that in glucose medium was 6.8 g/L at 30 h, indicating that acetate has potential as substrate. Metabolome analysis was performed to comprehensively elucidate characteristic metabolic fluctuations caused by acetate assimilation and identify targets to improve the fatty acid productivity from acetate. It was found that the use of glyoxylate cycle, which bypasses release of energy molecules such as NADH and GTP, and the inhibition of utilization of compounds from TCA cycle for anabolic reactions, may cause the slow growth in acetate which has an effect also in lipid productivity. The activity of the pentose phosphate pathway was found to be weak in acetate cultivation, thus NADPH was mainly produced in malate-pyruvate cycle. Lastly, mevalonate pathway was found to be activated in acetate cultivation which additionally competes with acetyl-CoA as starting material of fatty acid synthesis.
Assuntos
Acetatos/metabolismo , Meios de Cultura , Ácidos Graxos/biossíntese , Fermentação/fisiologia , Metabolismo dos Lipídeos/fisiologia , Estramenópilas/metabolismo , Acetilcoenzima A/metabolismo , Meios de Cultura/química , Ácido Glucárico/metabolismo , Ácido Mevalônico/metabolismo , NADP/biossíntese , Via de Pentose Fosfato , Estramenópilas/crescimento & desenvolvimentoRESUMO
NADPH donates high energy electrons for antioxidant defense and reductive biosynthesis. Cytosolic NADP is recycled to NADPH by the oxidative pentose phosphate pathway (oxPPP), malic enzyme 1 (ME1) and isocitrate dehydrogenase 1 (IDH1). Here we show that any one of these routes can support cell growth, but the oxPPP is uniquely required to maintain a normal NADPH/NADP ratio, mammalian dihydrofolate reductase (DHFR) activity and folate metabolism. These findings are based on CRISPR deletions of glucose-6-phosphate dehydrogenase (G6PD, the committed oxPPP enzyme), ME1, IDH1, and combinations thereof in HCT116 colon cancer cells. Loss of G6PD results in high NADP, which induces compensatory increases in ME1 and IDH1 flux. But the high NADP inhibits dihydrofolate reductase (DHFR), resulting in impaired folate-mediated biosynthesis, which is reversed by recombinant expression of E. coli DHFR. Across different cancer cell lines, G6PD deletion produced consistent changes in folate-related metabolites, suggesting a general requirement for the oxPPP to support folate metabolism.
Assuntos
Ácido Fólico/metabolismo , NADP/biossíntese , Via de Pentose Fosfato , Citosol/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Células HCT116 , Homeostase , Humanos , NADP/metabolismo , Oxirredução , Estresse OxidativoRESUMO
Cofactor supply is a rate-limiting step in the bioconversion of xylose to xylitol. Strain WZ04 was first constructed by a novel simultaneous deletion-insertion strategy, replacing ptsG, xylAB and ptsF in wild-type Escherichia coli W3110 with three mutated xylose reductase genes (xr) from Neurospora crassa. Then, the pfkA, pfkB, pgi and/or sthA genes were deleted and replaced by xr to investigate the influence of carbon flux toward the pentose phosphate pathway and/or transhydrogenase activity on NADPH generation. The deletion of pfkA/pfkB significantly improved NADPH supply, but minimally influenced cell growth. The effects of insertion position and copy number of xr were examined by a quantitative real-time PCR and a shake-flask fermentation experiment. In a fed-batch fermentation experiment with a 15-L bioreactor, strain WZ51 produced 131.6 g L-1 xylitol from hemicellulosic hydrolysate (xylitol productivity: 2.09 g L-1 h-1). This study provided a potential approach for industrial-scale production of xylitol from hemicellulosic hydrolysate.
Assuntos
Aldeído Redutase/metabolismo , Escherichia coli/metabolismo , NADP/biossíntese , Via de Pentose Fosfato , Xilitol/metabolismo , Aldeído Redutase/genética , Escherichia coli/genética , Fermentação , Glicólise , Neurospora crassa/metabolismo , Xilose/metabolismoRESUMO
Nicotinamide adenine dinucleotide phosphate (NADP+) is essential for producing NADPH, the primary cofactor for reductive metabolism. We find that growth factor signaling through the phosphoinositide 3-kinase (PI3K)-Akt pathway induces acute synthesis of NADP+ and NADPH. Akt phosphorylates NAD kinase (NADK), the sole cytosolic enzyme that catalyzes the synthesis of NADP+ from NAD+ (the oxidized form of NADH), on three serine residues (Ser44, Ser46, and Ser48) within an amino-terminal domain. This phosphorylation stimulates NADK activity both in cells and directly in vitro, thereby increasing NADP+ production. A rare isoform of NADK (isoform 3) lacking this regulatory region exhibits constitutively increased activity. These data indicate that Akt-mediated phosphorylation of NADK stimulates its activity to increase NADP+ production through relief of an autoinhibitory function inherent to its amino terminus.
Assuntos
NADP/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Animais , Cromatografia Líquida , Citosol/enzimologia , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Domínios Proteicos , Serina/genética , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
Multi-enzyme cascade reactions capture the essence of nature's efficiency by increasing the productivity of a process. Here we describe one such three-enzyme cascade for the synthesis of 6-hydroxyhexanoic acid. Whole cells of Escherichia coli co-expressing an alcohol dehydrogenase and a Baeyer-Villiger monooxygenase (CHMO) for internal cofactor regeneration were used without the supply of external NADPH or NADP+. The product inhibition caused by the ε-caprolactone formed by the CHMO was overcome by the use of lipase CAL-B for in situ conversion into 6-hydroxyhexanoic acid. A stirred tank reactor under fed-batch mode was chosen for efficient catalysis. By using this setup, a product titre of >20 g L-1 was achieved in a 500 mL scale with an isolated yield of 81% 6-hydroxyhexanoic acid.
Assuntos
Álcool Desidrogenase/genética , Caproatos/síntese química , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Proteínas Fúngicas/química , Hidroxiácidos/síntese química , Lipase/química , Oxigenases de Função Mista/genética , Álcool Desidrogenase/metabolismo , Técnicas de Cultura Celular por Lotes , Biocatálise , Reatores Biológicos , Caproatos/química , Caproatos/metabolismo , Coenzimas/biossíntese , Coenzimas/química , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Expressão Gênica , Hidroxiácidos/metabolismo , Cinética , Lactonas/química , Lactonas/metabolismo , Lipase/metabolismo , Oxigenases de Função Mista/metabolismo , NADP/biossíntese , NADP/químicaRESUMO
Lycopene is a red carotenoid pigment with strong antioxidant activity. Saccharomyces cerevisiae is considered a promising host to produce lycopene, but lycopene toxicity is one of the limiting factors for high-level production. In this study, we used heterologous lycopene biosynthesis genes crtE and crtI from Xanthophyllomyces dendrorhous and crtB from Pantoea agglomerans for lycopene production in S. cerevisiae. The crtE, crtB, and crtI genes were integrated into the genome of S. cerevisiae CEN.PK2-1C strain, while deleting DPP1 and LPP1 genes to inhibit a competing pathway producing farnesol. Lycopene production was further improved by inhibiting ergosterol production via downregulation of ERG9 expression and by deleting ROX1 or MOT3 genes encoding transcriptional repressors for mevalonate and sterol biosynthetic pathways. To further increase lycopene production, CrtE and CrtB mutants with improved activities were isolated by directed evolution, and subsequently, the mutated genes were randomly integrated into the engineered lycopene-producing strains via delta-integration. To relieve lycopene toxicity by increasing unsaturated fatty acid content in cell membranes, the OLE1 gene encoding stearoyl-CoA 9-desaturase was overexpressed. In combination with the overexpression of STB5 gene encoding a transcription factor involved in NADPH production, the final strain produced up to 41.8 mg/gDCW of lycopene, which is approximately 74.6-fold higher than that produced in the initial strain.
Assuntos
Licopeno/metabolismo , Microrganismos Geneticamente Modificados , NADP/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Basidiomycota/genética , Membrana Celular/metabolismo , Evolução Molecular Direcionada , Farneseno Álcool/metabolismo , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Ácidos Graxos Insaturados/metabolismo , Regulação Fúngica da Expressão Gênica , Pantoea/genética , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Bacitracin, a kind of cyclic peptide antibiotic mainly produced by Bacillus, has wide ranges of applications. NADPH generation plays an important role in amino acid synthesis, which might influence precursor amino acid supply for bacitracin production. In this study, we want to improve bacitracin yield by enhancing intracellular precursor amino acids via strengthening NAPDH generation pathways in the bacitracin industrial production strain Bacillus licheniformis DW2. Based on our results, strengthening of NADPH pathway genes (zwf, gnd, ppnk, pntAB, and udhA) could all improve bacitracin yields in DW2, and the glucose-6-phosphate dehydrogenase Zwf overexpression strain DW2::Zwf displayed the best performance, the yield of which (886.43 U/mL) was increased by 12.43% compared to DW2 (788.40 U/mL). Then, the zwf transcriptional level and Zwf activity of DW2::Zwf were increased by 12.24-fold and 1.57-fold; NADPH and NADPH/NADH were enhanced by 61.24% and 90.63%, compared with those of DW2, respectively. Moreover, the concentrations of intracellular precursor amino acids (isoleucine, leucine, cysteine, ornithine, lysine, glutamic acid) were all enhanced obviously for bacitracin production in DW2::Zwf. Collectively, this research constructed a promising B. licheniformis strain for industrial production of bacitracin, more importantly, which revealed that strengthening of NADPH generation is an efficient strategy to improve precursor amino acid supplies for bacitracin production.
Assuntos
Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Bacitracina/biossíntese , Biotecnologia , Glucosefosfato Desidrogenase/genética , NADP/biossíntese , Bacillus licheniformis/citologia , Espaço Extracelular/metabolismo , Fermentação , Expressão Gênica , Espaço Intracelular/metabolismo , NAD/metabolismo , Transcrição GênicaRESUMO
Ginseng (Panax ginseng) and its bioactive components, ginsenosides, are popular medicinal herbal products, exhibiting various pharmacological effects. Despite their advocated use for medication, the long cultivation periods of ginseng roots and their low ginsenoside content prevent mass production of this compound. Yeast Saccharomyces cerevisiae was engineered for production of protopanaxadiol (PPD), a type of aglycone characterizing ginsenoside. PPD-producing yeast cell factory was further engineered by obtaining a balance between enzyme expressions and altering cofactor availability. Different combinations of promoters (PGPD, PCCW12, and PADH2) were utilized to construct the PPD biosynthetic pathway. Rerouting the redox metabolism to improve NADPH availability in the engineered S. cerevisiae also increased PPD production. Combining these approaches resulted in more than an 11-fold increase in PPD titer over the initially constructed strain. The series of metabolic engineering strategies of this study provides a feasible approach for the microbial production of PPD and development of microbial platforms producing other industrially-relevant terpenoids.
Assuntos
Vias Biossintéticas , NADP/biossíntese , Saccharomyces cerevisiae/metabolismo , Sapogeninas/metabolismo , Carbono/farmacologia , Engenharia Metabólica , Oxirredução , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sapogeninas/químicaRESUMO
Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), as two kinds of well-known cofactor, are widely used in the most of enzymatic redox reactions, playing an important role in industrial catalysis. In general, supply of NAD(P)H is a major challenged factor in redox fermentation systems due to its high cost and low stability, which have stimulated the development of NADH regeneration systems in recent years. Until now, a series of NAD(P)H regeneration systems have been developed. This review focuses primarily on new approaches of NAD(P)H cofactor regeneration in the biosynthesis systems, such as single cell in vivo NADH regeneration system, double cell coupling NADH regeneration system, in vitro enzyme-coupled NADH regeneration system, microbial cell surface display NADH regeneration system. Finally, the prospect and tendency of NADH regeneration are discussed.
Assuntos
NADP/biossíntese , NAD/biossíntese , Bactérias/metabolismo , Catálise , Fermentação , Redes e Vias Metabólicas , OxirreduçãoRESUMO
Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme.