RESUMO
BACKGROUND: Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. RESULTS: Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. CONCLUSIONS: The developed approach, based on parallel 13C tracer studies with GC-MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments.
Assuntos
Synechocystis , Aldeído Liases , Aminoácidos/metabolismo , Dióxido de Carbono/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Análise do Fluxo Metabólico , Açúcares/metabolismo , Synechocystis/metabolismoRESUMO
A convenient access to a triad of triazoles with ferrocenyl and cobaltoceniumyl substituents is reported. N-Alkylation, deprotonation and metalation with CuI /AgI /AuI synthons affords the heteroleptic triazolylidene complexes. Due to the combination of neutral, electron-donating ferrocenyl substituents and cationic, strongly electron-withdrawing cobaltocenium substituents, the mesoionic carbene (MIC) ligands of these complexes are electronically interesting "push-pull", "pull-push" and "pull-pull" metalloligands with further switchable redox states based on their fully reversible FeII /FeIII , (ferrocene/ferrocenium) and CoIII /CoII , (cobaltocenium/cobaltocene) redox couples. These are the first examples of metal complexes of (di)cationic NHC ligands based on cobaltoceniumyl substituents. DFT calculated Tolman electronic parameter (TEP) of the new MIC ligands, show these metalloligands to be extremely electron-poor NHCs with properties unmatched in other carbene chemistry. Utilization of these multimetallic electronically tunable compounds in catalytic oxazoline synthesis and in antitumor studies are presented. Remarkably, 1â mol % of the AuI complex with the dicationic MIC ligand displays full catalytic conversion, without the need for any other additives, in less than 2â hours at ambient temperatures. These results thus firmly establish these new classes of cobaltoceniumyl based (di)cationic MIC ligands as prominent players in several branches of chemistry.
RESUMO
Succinic acid is a platform chemical of recognized industrial value and accordingly faces a continuous challenge to enable manufacturing from most attractive raw materials. It is mainly produced from glucose, using microbial fermentation. Here, we explore and optimize succinate production from sucrose, a globally applied substrate in biotechnology, using the rumen bacterium Basfia succiniciproducens DD1. As basis of the strain optimization, the yet unknown sucrose metabolism of the microbe was studied, using 13C metabolic flux analyses. When grown in batch culture on sucrose, the bacterium exhibited a high succinate yield of 1molmol-1 and a by-product spectrum, which did not match the expected PTS-mediated sucrose catabolism. This led to the discovery of a fructokinase, involved in sucrose catabolism. The flux approach unraveled that the fructokinase and the fructose PTS both contribute to phosphorylation of the fructose part of sucrose. The contribution of the fructokinase reduces the undesired loss of the succinate precursor PEP into pyruvate and into pyruvate-derived by-products and enables increased succinate production, exclusively via the reductive TCA cycle branch. These findings were used to design superior producers. Mutants, which (i) overexpress the beneficial fructokinase, (II) lack the competing fructose PTS, and (iii) combine both traits, produce significantly more succinate. In a fed-batch process, B. succiniciproducens ΔfruA achieved a titer of 71gL-1 succinate and a yield of 2.5molmol-1 from sucrose.
Assuntos
Isótopos de Carbono/metabolismo , Engenharia Metabólica , Modelos Biológicos , Pasteurellaceae , Rúmen/microbiologia , Ácido Succínico/metabolismo , Sacarose/metabolismo , Animais , Pasteurellaceae/genética , Pasteurellaceae/metabolismoRESUMO
Dynamin is a mechanochemical GTPase that oligomerizes around the neck of clathrin-coated pits and catalyses vesicle scission in a GTP-hydrolysis-dependent manner. The molecular details of oligomerization and the mechanism of the mechanochemical coupling are currently unknown. Here we present the crystal structure of human dynamin 1 in the nucleotide-free state with a four-domain architecture comprising the GTPase domain, the bundle signalling element, the stalk and the pleckstrin homology domain. Dynamin 1 oligomerized in the crystals via the stalks, which assemble in a criss-cross fashion. The stalks further interact via conserved surfaces with the pleckstrin homology domain and the bundle signalling element of the neighbouring dynamin molecule. This intricate domain interaction rationalizes a number of disease-related mutations in dynamin 2 and suggests a structural model for the mechanochemical coupling that reconciles previous models of dynamin function.
Assuntos
Dinamina I/química , Nucleotídeos , Cristalografia por Raios X , Dinamina I/metabolismo , Dinamina II/genética , Dinamina II/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Transferrina/metabolismoRESUMO
The recent discovery of the Entner-Doudoroff (ED) pathway as a third glycolytic route beside Embden-Meyerhof-Parnas (EMP) and oxidative pentose phosphate (OPP) pathway in oxygenic photoautotrophs requires a revision of their central carbohydrate metabolism. In this study, unexpectedly, we observed that deletion of the ED pathway alone, and even more pronounced in combination with other glycolytic routes, diminished photoautotrophic growth in continuous light in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, we found that the ED pathway is required for optimal glycogen catabolism in parallel to an operating Calvin-Benson-Bassham (CBB) cycle. It is counter-intuitive that glycolytic routes, which are a reverse to the CBB cycle and do not provide any additional biosynthetic intermediates, are important under photoautotrophic conditions. However, observations on the ability to reactivate an arrested CBB cycle revealed that they form glycolytic shunts that tap the cellular carbohydrate reservoir to replenish the cycle. Taken together, our results suggest that the classical view of the CBB cycle as an autocatalytic, completely autonomous cycle that exclusively relies on its own enzymes and CO2 fixation to regenerate ribulose-1,5-bisphosphate for Rubisco is an oversimplification. We propose that in common with other known autocatalytic cycles, the CBB cycle likewise relies on anaplerotic reactions to compensate for the depletion of intermediates, particularly in transition states and under fluctuating light conditions that are common in nature.