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A combined experimental and modelling approach for the Weimberg pathway optimisation

Abstract (Expand)

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.

Authors: Lu Shen, Martha Kohlhaas, Junichi Enoki, Roland Meier, Bernhard Schönenberger, Roland Wohlgemuth, Robert Kourist, Felix Niemeyer, David van Niekerk, Christopher Bräsen, Jochen Niemeyer, Jacky Snoep, Bettina Siebers

Date Published: 1st Dec 2020

Publication Type: Not specified

The Carbon Switch at the Level of Pyruvate and Phosphoenolpyruvate in Sulfolobus solfataricus P2.

Abstract (Expand)

Sulfolobus solfataricus P2 grows on different carbohydrates as well as alcohols, peptides and amino acids. Carbohydrates such as D-glucose or D-galactose are degraded via the modified, branched … Entner-Doudoroff (ED) pathway whereas growth on peptides requires the Embden-Meyerhof-Parnas (EMP) pathway for gluconeogenesis. As for most hyperthermophilic Archaea an important control point is established at the level of triosephophate conversion, however, the regulation at the level of pyruvate/phosphoenolpyruvate conversion was not tackled so far. Here we describe the cloning, expression, purification and characterization of the pyruvate kinase (PK, SSO0981) and the phosphoenolpyruvate synthetase (PEPS, SSO0883) of Sul. solfataricus. The PK showed only catabolic activity [catalytic efficiency (PEP): 627.95 mM(-1)s(-1), 70 degrees C] with phosphoenolpyruvate as substrate and ADP as phosphate acceptor and was allosterically inhibited by ATP and isocitrate (K i 0.8 mM). The PEPS was reversible, however, exhibited preferred activity in the gluconeogenic direction [catalytic efficiency (pyruvate): 1.04 mM(-1)s(-1), 70 degrees C] and showed some inhibition by AMP and alpha-ketoglutarate. The gene SSO2829 annotated as PEPS/pyruvate:phosphate dikinase (PPDK) revealed neither PEPS nor PPDK activity. Our studies suggest that the energy charge of the cell as well as the availability of building blocks in the citric acid cycle and the carbon/nitrogen balance plays a major role in the Sul. solfataricus carbon switch. The comparison of regulatory features of well-studied hyperthermophilic Archaea reveals a close link and sophisticated coordination between the respective sugar kinases and the kinetic and regulatory properties of the enzymes at the level of PEP-pyruvate conversion.

Authors: P. Haferkamp, B. Tjaden, L. Shen, C. Brasen, T. Kouril, B. Siebers

Date Published: 30th Apr 2019

Publication Type: Journal

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