RESUMO
This first serious attempt at an autobiographical accounting has forced me to sit still long enough to compile my thoughts about a long personal and scientific journey. I especially hope that my trajectory will be of interest and perhaps beneficial to much younger women who are just getting started in their careers. To paraphrase from Virginia Woolf's writings in A Room of One's Own at the beginning of the 20th century, "for most of history Anonymous was a Woman." However, Ms. Woolf is also quoted as saying "nothing has really happened until it has been described," a harbinger of the enormous historical changes that were about to be enacted and recorded by women in the sciences and other disciplines. The progress in my chosen field of study-the chemical basis of enzyme action-has also been remarkable, from the first description of an enzyme's 3D structure to a growing and deep understanding of the origins of enzyme catalysis.
Assuntos
Coenzimas/química , Enzimas/química , Mulheres Trabalhadoras/história , Biocatálise , Escolha da Profissão , Coenzimas/metabolismo , Ensaios Enzimáticos , Enzimas/metabolismo , Feminino , História do Século XX , História do Século XXI , Humanos , Cinética , Teoria QuânticaRESUMO
Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.
Assuntos
Genes Modificadores , Mitocôndrias/genética , Mitocôndrias/patologia , Autoantígenos/metabolismo , Morte Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Epistasia Genética/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Genoma , Glutationa Peroxidase/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Células K562 , Mitocôndrias/efeitos dos fármacos , Oligomicinas/toxicidade , Oxirredução , Fosforilação Oxidativa/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/genética , Espécies Reativas de Oxigênio/metabolismo , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
The conversion of CO2 to value-added products allows both capture and recycling of greenhouse gas emissions. While plants and other photosynthetic organisms play a key role in closing the global carbon cycle, their dependence on light to drive carbon fixation can be limiting for industrial chemical synthesis. Methanogenic archaea provide an alternative platform as an autotrophic microbial species capable of non-photosynthetic CO2 fixation, providing a potential route to engineered microbial fermentation to synthesize chemicals from CO2 without the need for light irradiation. One major challenge in this goal is to connect upstream carbon-fixation pathways with downstream biosynthetic pathways, given the distinct differences in metabolism between archaea and typical heterotrophs. We engineered the model methanogen, Methanococcus maripaludis, to divert acetyl-coenzyme A toward biosynthesis of value-added chemicals, including the bioplastic polyhydroxybutyrate (PHB). A number of studies implicated limitations in the redox pool, with NAD(P)(H) pools in M. maripaludis measured to be <15% of that of Escherichia coli, likely since methanogenic archaea utilize F420 and ferredoxins instead. Multiple engineering strategies were used to precisely target and increase the cofactor pool, including heterologous expression of a synthetic nicotinamide salvage pathway as well as an NAD+-dependent formate dehydrogenase from Candida boidinii. Engineered strains of M. maripaludis with improved NADH pools produced up to 171 ± 4 mg/L PHB and 24.0 ± 1.9% of dry cell weight. The metabolic engineering strategies presented in this study broaden the utility of M. maripaludis for sustainable chemical synthesis using CO2 and may be transferable to related archaeal species.
Assuntos
Archaea , Euryarchaeota , Archaea/metabolismo , Ciclo do Carbono , Dióxido de Carbono/metabolismo , Crescimento Quimioautotrófico , Euryarchaeota/metabolismoRESUMO
Anaerobic alcoholic fermentation, particularly in high-sugar environments, presents metabolic challenges for yeasts. Crabtree-positive yeasts, including Saccharomyces cerevisiae, prefer fermentation even in the presence of oxygen. These yeasts rely on internal NAD+ recycling and extracellular assimilation of its precursor, nicotinic acid (vitamin B3), rather than de novo NAD+ production. Surprisingly, nicotinic acid assimilation is poorly characterized, even in S. cerevisiae. This study elucidated the timing of nicotinic acid uptake during grape juice-like fermentation and its impact on NAD(H) levels, the NAD+/NADH ratio, and metabolites produced. Complete uptake of extracellular nicotinic acid occurred premid-exponential phase, thereafter small amounts of vitamin B3 were exported back into the medium. Suboptimal levels of nicotinic acid were correlated with slower fermentation and reduced biomass, disrupting redox balance and impeding NAD+ regeneration, thereby affecting metabolite production. Metabolic outcomes varied with nicotinic acid concentrations, linking NAD+ availability to fermentation efficiency. A model was proposed encompassing rapid nicotinic acid uptake, accumulation during cell proliferation, and recycling with limited vitamin B3 export. This research enhances the understanding of nicotinic acid uptake dynamics during grape juice-like fermentation. These insights contribute to advancing yeast metabolism research and have profound implications for the enhancement of biotechnological practices and the wine-making industry.
Assuntos
Fermentação , NAD , Niacina , Oxirredução , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Niacina/metabolismo , NAD/metabolismo , Etanol/metabolismo , Coenzimas/metabolismoRESUMO
Clostridium thermocellum is a cellulolytic thermophile that is considered for the consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but the incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated via gene deletions in ammonium-regulated, nicotinamide adenine dinucleotide phosphate (NADPH)-supplying and NADPH-consuming pathways as well as via physiological characterization in cellobiose-limited or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt (Δppdk) or a redox-independent conversion of PEP to pyruvate (Δppdk ΔmalE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased from 46 to 60%, suggesting that the secretion of these amino acids balances the NADPH surplus from the malate shunt. The unchanged amino acid secretion in Δppdk falsified a previous hypothesis on an ammonium-regulated PEP-to-pyruvate flux redistribution. The possible involvement of another NADPH-supplier, namely, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, the deletion of glutamate synthase (gogat) in ammonium assimilation resulted in the upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, a claim which is supported by the product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of the NADH needed for ethanol formation. This suggests that metabolic engineering strategies that simplify the redox metabolism and ammonium assimilation can contribute to increased ethanol yields. IMPORTANCE Improving the ethanol yield of C. thermocellum is important for the industrial implementation of this microorganism in consolidated bioprocessing. A central role of NADPH in driving amino acid byproduct formation was demonstrated by eliminating the NADPH-supplying malate shunt and separately by changing the cofactor specificity in ammonium assimilation. With amino acid secretion diverting carbon and electrons away from ethanol, these insights are important for further metabolic engineering to reach industrial requirements on ethanol yield. This study also provides chemostat data that are relevant for training genome-scale metabolic models and for improving the validity of their predictions, especially considering the reduced degree-of-freedom in the redox metabolism of the strains generated here. In addition, this study advances the fundamental understanding on the mechanisms underlying amino acid secretion in cellulolytic Clostridia as well as on the regulation and cofactor specificity in ammonium assimilation. Together, these efforts aid in the development of C. thermocellum for the sustainable consolidated bioprocessing of lignocellulose to ethanol with minimal pretreatment.
Assuntos
Aminoácidos , Compostos de Amônio , Clostridium thermocellum , NADP , Aminoácidos/biossíntese , Aminoácidos/metabolismo , Compostos de Amônio/metabolismo , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Etanol/metabolismo , Ferredoxinas/metabolismo , Malatos/metabolismo , NAD/metabolismo , NADP/metabolismo , Piruvatos/metabolismo , OxirreduçãoRESUMO
L-Homoserine is a valuable amino acid as a platform chemical in the synthesis of various important compounds. Development of microbial strains for high-level L-homoserine production is an attractive research direction in recent years. Herein, we converted a wild-type Escherichia coli to a non-auxotrophic and plasmid-free hyperproducer of L-homoserine using systematically metabolic engineer strategies. First, an initial strain was obtained through regulating L-homoserine degradation pathway and enhancing synthetic flow. To facilitate L-homoserine production, flux-control genes were tuned by optimizing the copy numbers in chromosome, and transport system was modified to promote L-homoserine efflux. Subsequently, a strategy of cofactors synergistic utilization was proposed and successfully applied to achieve L-homoserine hyperproduction. The final engineered strain could efficiently produce 85.29 g/L L-homoserine, which was the highest production level ever reported from a plasmid-free, antibiotic-free, inducer-free and nonauxotrophic strain. These strategies used here can be considered for developing microbial cell factory of other L-aspartate derivatives.
Assuntos
Proteínas de Escherichia coli , Engenharia Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Homosserina/genética , Homosserina/metabolismo , Plasmídeos/genéticaRESUMO
Redox cofactors mediate many enzymatic processes and are increasingly employed in biomedical and energy applications. Exploring the influence of external magnetic fields on redox cofactor chemistry can enhance our understanding of magnetic-field-sensitive biological processes and allow the application of magnetic fields to modulate redox reactions involving cofactors. Through a combination of experiments and modeling, we investigate the influence of magnetic fields on electrochemical reactions in redox cofactor solutions. By employing flavin mononucleotide (FMN) cofactor as a model system, we characterize magnetically induced changes in Faradaic currents. We find that radical pair intermediates have negligible influence on current increases in FMN solution upon application of a magnetic field. The dominant mechanism underlying the observed current increases is the magneto-hydrodynamic effect. We extend our analyses to other diffusion-limited electrochemical reactions of redox cofactor solutions and arrive at similar conclusions, highlighting the opportunity to use this framework in redox cofactor chemistry.
Assuntos
Técnicas Eletroquímicas , Mononucleotídeo de Flavina/química , Hidrodinâmica , Campos Magnéticos , Oxirredução , SoluçõesRESUMO
Environmental fluctuations in the availability of nutrients lead to intricate metabolic strategies. "Candidatus Accumulibacter phosphatis," a polyphosphate-accumulating organism (PAO) responsible for enhanced biological phosphorus removal (EBPR) from wastewater treatment systems, is prevalent in aerobic/anaerobic environments. While the overall metabolic traits of these bacteria are well described, the nonavailability of isolates has led to controversial conclusions on the metabolic pathways used. In this study, we experimentally determined the redox cofactor preferences of different oxidoreductases in the central carbon metabolism of a highly enriched "Ca Accumulibacter phosphatis" culture. Remarkably, we observed that the acetoacetyl coenzyme A reductase engaged in polyhydroxyalkanoate (PHA) synthesis is NADH preferring instead of showing the generally assumed NADPH dependency. This allows rethinking of the ecological role of PHA accumulation as a fermentation product under anaerobic conditions and not just a stress response. Based on previously published metaomics data and the results of enzymatic assays, a reduced central carbon metabolic network was constructed and used for simulating different metabolic operating modes. In particular, scenarios with different acetate-to-glycogen consumption ratios were simulated, which demonstrated optima using different combinations of glycolysis, glyoxylate shunt, or branches of the tricarboxylic acid (TCA) cycle. Thus, optimal metabolic flux strategies will depend on the environment (acetate uptake) and on intracellular storage compound availability (polyphosphate/glycogen). This NADH-related metabolic flexibility is enabled by the NADH-driven PHA synthesis. It allows for maintaining metabolic activity under various environmental substrate conditions, with high carbon conservation and lower energetic costs than for NADPH-dependent PHA synthesis. Such (flexible) metabolic redox coupling can explain the competitiveness of PAOs under oxygen-fluctuating environments.IMPORTANCE Here, we demonstrate how microbial storage metabolism can adjust to a wide range of environmental conditions. Such flexibility generates a selective advantage under fluctuating environmental conditions. It can also explain the different observations reported in PAO literature, including the capacity of "Ca Accumulibacter phosphatis" to act like glycogen-accumulating organisms (GAOs). These observations stem from slightly different experimental conditions, and controversy arises only when one assumes that metabolism can operate only in a single mode. Furthermore, we also show how the study of metabolic strategies is possible when combining omics data with functional cofactor assays and modeling. Genomic information can only provide the potential of a microorganism. The environmental context and other complementary approaches are still needed to study and predict the functional expression of such metabolic potential.
Assuntos
Acil Coenzima A/metabolismo , Betaproteobacteria/metabolismo , Redes e Vias Metabólicas , Betaproteobacteria/enzimologia , Análise do Fluxo Metabólico , Modelos Biológicos , NAD/metabolismo , NADP/metabolismo , OxirreduçãoRESUMO
Although there have been approximately 60 chemical compounds identified as potent fermentation inhibitors in lignocellulose hydrolysate, our research group recently discovered glycolaldehyde as a key fermentation inhibitor during second generation biofuel production. Accordingly, we have developed a yeast S. cerevisiae strain exhibiting tolerance to glycolaldehyde. During this glycolaldehyde study, we established novel approaches for rational engineering of inhibitor-tolerant S. cerevisiae strains, including engineering redox cofactors and engineering the SUMOylation pathway. These new technical dimensions provide a novel platform for engineering S. cerevisiae strains to overcome one of the key barriers for industrialization of lignocellulosic ethanol production. As such, this review discusses novel biochemical insight of glycolaldehyde in the context of the biofuel industry.
Assuntos
Acetaldeído/análogos & derivados , Saccharomyces cerevisiae/efeitos dos fármacos , Acetaldeído/farmacologia , Acetaldeído/toxicidade , Biocombustíveis , Etanol/metabolismo , Etilenoglicol/metabolismo , Fermentação/efeitos dos fármacos , Glutationa/metabolismo , Lignina/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , SumoilaçãoRESUMO
BACKGROUND: L-Leucine is a high-value amino acid with promising applications in the medicine and feed industries. However, the complex metabolic network and intracellular redox imbalance in fermentative microbes limit their efficient biosynthesis of L-leucine. RESULTS: In this study, we applied rational metabolic engineering and a dynamic regulation strategy to construct a plasmid-free, non-auxotrophic Escherichia coli strain that overproduces L-leucine. First, the L-leucine biosynthesis pathway was strengthened through multi-step rational metabolic engineering. Then, a cooperative cofactor utilization strategy was designed to ensure redox balance for L-leucine production. Finally, to further improve the L-leucine yield, a toggle switch for dynamically controlling sucAB expression was applied to accurately regulate the tricarboxylic acid cycle and the carbon flux toward L-leucine biosynthesis. Strain LEU27 produced up to 55 g/L of L-leucine, with a yield of 0.23 g/g glucose. CONCLUSIONS: The combination of strategies can be applied to the development of microbial platforms that produce L-leucine and its derivatives.
RESUMO
The alcoholic fermentation of organic carbon sources by Saccharomyces cerevisiae produces many by-products, with the most abundant originating from central carbon metabolism. The production of these metabolites involves redox reactions and largely depends on the maintenance of redox homeostasis. Despite the metabolic pathways being mostly conserved across strains of S. cerevisiae, their production of various amounts of metabolic products suggests that their intracellular concentration of redox cofactors and/or redox balance differ. This study explored the redox status dynamics and NAD(H) and NADP(H) cofactor ratios throughout alcoholic fermentation in four S. cerevisiae strains that exhibit different carbon metabolic fluxes. This study focussed on the molecular end-products of fermentation, redox cofactor ratios and the impact thereof on redox homeostasis. Strain-dependent differences were identified in the redox cofactor levels, with NADP(H) ratios and levels remaining stable while NAD(H) levels decreased drastically as the fermentation progressed. Changes in the NAD+/NADH ratio were also observed. Total levels of NAD(H) decreased drastically as the fermentation progressed despite the cells remaining viable until the end of fermentation. NAD+ was found to be favoured initially while NADH was favoured towards the end of the fermentation. The change in the NAD+/NADH redox cofactor ratio during fermentation was linked with the production of end-products. The findings in this study could steer further research in the selection of S. cerevisiae wine strains for desirable aroma contributions based on their intracellular redox balance management.
Assuntos
NAD , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , NAD/metabolismo , Fermentação , NADP/metabolismo , OxirreduçãoRESUMO
Glucose-6-phosphate dehydrogenase (Zwf) is an important enzyme in glucose metabolism via the Entner-Doudoroff pathway and the first enzyme in the oxidative pentose-phosphate pathway. It generates NAD(P)H during the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, thus aiding in anabolic processes, energy yield, and oxidative stress responses. Pseudomonas bharatica CSV86T preferentially utilized aromatic compounds over glucose and exhibited a significantly lower growth rate on glucose (0.24 h-1) with a prolonged lag phase (~10 h). In strain CSV86T, glucose was metabolized via the intracellular phosphorylative route only because it lacked an oxidative (gluconate and 2-ketogluconate) route. The genome harbored three genes zwfA, zwfB, and zwfC encoding three Zwf isozymes. The present study aimed to understand gene arrangement, gene expression profiling, and molecular and kinetic properties of the purified enzymes to unveil their physiological significance in the strain CSV86T. The zwfA was found to be a part of the zwfA-pgl-eda operon, which was proximal to other glucose transport and metabolic clusters. The zwfB was found to be arranged as a gnd-zwfB operon, while zwfC was present independently. Among the three, zwfA was transcribed maximally, and the purified ZwfA displayed the highest catalytic efficiency, cooperativity with respect to G6P, and dual cofactor specificity. Isozymes ZwfB and ZwfC were NADP+-preferring and NADP+-specific, respectively. Among other functionally characterized Zwfs, ZwfA from strain CSV86T displayed poor catalytic efficiency and the further absence of oxidative routes of glucose metabolism reflected its lower growth rate on glucose compared to P. putida KT2440 and could be probable reasons for the unique carbon source utilization hierarchy. IMPORTANCE Pseudomonas bharatica CSV86T metabolizes glucose exclusively via the intracellular phosphorylative Entner-Doudoroff pathway leading the entire glucose flux through Zwf as the strain lacks oxidative routes. This may lead to limiting the concentration of downstream metabolic intermediates. The strain CSV86T possesses three isoforms of glucose-6-phosphate dehydrogenase, ZwfA, ZwfB, and ZwfC. The expression profile and kinetic properties of purified enzymes will help to understand glucose metabolism. Isozyme ZwfA dominated in terms of expression and displayed cooperativity with dual cofactor specificity. ZwfB preferred NADP+, and ZwfC was NADP+ specific, which may aid in redox cofactor balance. Such beneficial metabolic flexibility facilitated the regulation of metabolic pathways giving survival/fitness advantages in dynamic environments. Additionally, multiple genes allowed the distribution of function among these isoforms where the primary function was allocated to one of the isoforms.
Assuntos
Pseudomonas putida , Pseudomonas , Pseudomonas/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , NADP/metabolismo , Glucose/metabolismoRESUMO
Artificial metalloenzymes (ArMs) consist of an unnatural metal or cofactor embedded in a protein scaffold, and are an excellent platform for applying the concepts of protein engineering to catalysis. In this Focused Review, we describe the application of ArMs as simple, tunable artificial models of the active sites of complex natural metalloenzymes for small-molecule activation. In this sense, ArMs expand the strategies of synthetic model chemistry to protein-based supporting ligands with potential for participation from the second coordination sphere. We focus specifically on ArMs that are structural, spectroscopic, and functional models of enzymes for activation of small molecules like CO, CO2, O2, N2, and NO, as well as production/consumption of H2. These ArMs give insight into the identities and roles of metalloenzyme structural features within and near the cofactor. We give examples of ArM work relevant to hydrogenases, acetyl-coenzyme A synthase, superoxide dismutase, heme oxygenases, nitric oxide reductase, methyl-coenzyme M reductase, copper-O2 enzymes, and nitrogenases.