RESUMO
The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.
Assuntos
Proteínas de Bactérias/genética , Reatores Biológicos , Caulobacter crescentus/genética , Engenharia Metabólica/métodos , Modelos Químicos , Proteínas de Bactérias/metabolismo , Biocombustíveis , Metabolismo dos Carboidratos/genética , Caulobacter crescentus/enzimologia , Simulação por Computador , Hidroliases/genética , Hidroliases/metabolismo , Ácidos Cetoglutáricos/metabolismo , Redes e Vias Metabólicas/genética , NADP/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Xilose/metabolismoRESUMO
2-Keto-3-deoxy-sugar acids are key intermediates of central metabolism and integral constituents of bacterial (lipo)polysaccharides and cell wall components and are therefore continuously and highly demanded in related research fields. The stereospecific chemical synthesis of chiral 2-keto-deoxy-sugar acids involves a multitude of reaction steps, while in metabolic pathways only few conversions lead to the same 2-keto-3-deoxy sugar acids from easily available carbohydrate precursors. Here we present a straightforward and highly economic one-step biocatalytic synthesis procedure of 2-keto-3-deoxy-d-gluconate (KDG) from d-gluconate using recombinant gluconate dehydratase (GAD) from the hyperthermophilic crenarchaeon Thermoproteus tenax. This method is highly advantageous to KDG production schemes described so far for several reasons: (i) the d-gluconate is completely converted to stereochemically pure D-KDG without side-product formation, (ii) the final KDG yield is approximately 90%, (iii) the newly developed quantitative and qualitative LC-MS analysis method enabled the simultaneous detection of d-gluconate and KDG and (iv) the T. tenax GAD as biocatalyst can be provided by a simple and rapid procedure involving only two precipitation steps. The described utilization of dehydratases for 2-keto-3-deoxy sugar acid syntheses represents a highly resource-efficient one-step preparation and offers potential short synthetic routes toward a broad range of 2-keto-3-deoxy sugar acids and their derivatives.