A Statistical Thermodynamical Interpretation of Metabolism

The metabolic network of a cell can be decomposed into discrete elementary modes that contribute, each with a certain probability, to the overall flux through the metabolism. These modes are cell function supporting, fundamental pathways that represent permissible ‘quantum’ states of the metabolism. For the case that cellular regulatory mechanisms for pathway fluxes evolved in an unbiased way, we demonstrate theoretically that the usage probabilities of individual elementary modes are distributed according to Boltzmann’s distribution law such that the rate of entropy production is maximized. Such distribution can be observed experimentally in highly evolved metabolic networks. Therefore, cell function has a natural tendency to operate at a maximum rate of entropy generation using preferentially efficient pathways with small reaction entropies. Ultimately, evolution of metabolic networks appears to be driven by forces that can be quantified by the distance of the current metabolic state from the state of maximum entropy generation that represents the unbiased, most probable selection of fundamental pathway choices

Systematic development of biomass overproducing Scheffersomyces stipitis for high-cell-density fermentations

The development of economically feasible bio-based process requires efficient cell factories capable of producing the desired product at high titer under high-cell-density fermentation. Herein we present a combinatorial approach based on systems metabolic engineering and metabolic evolution for the development of efficient biomass-producing strain. Systems metabolic engineering guided by flux balance analysis (FBA) was first employed to rationally design mutant strains of Scheffersomyces stipitis with high biomass yield. By experimentally implementing these mutations, the biomass yield was improved by 30% in GPD1, 25% in TKL1, 30% in CIT1, and 44% in ZWF1 overexpressed mutants compared to wild-type. These designed mutants were further fine-tuned through metabolic evolution resulting in the maximal biomass yield of 0.49 g-cdw/g-glucose, which matches well with predicted yield phenotype. The constructed mutants are beneficial for biotechnology applications dealing with high cell titer cultivations. This work demonstrates a solid confirmation of systems metabolic engineering in combination with metabolic evolution approach for efficient strain development, which could assist in rapid optimization of cell factory for an economically viable and sustainable bio-based process

Minimal Escherichia coli Cell for the Most Efficient Production of Ethanol from Hexoses and Pentoses▿ †

To obtain an efficient ethanologenic Escherichia coli strain, we reduced the functional space of the central metabolic network, with eight gene knockout mutations, from over 15,000 pathway possibilities to 6 pathway options that support cell function. The remaining pathways, identified by elementary mode analysis, consist of four pathways with non-growth-associated conversion of pentoses and hexoses into ethanol at theoretical yields and two pathways with tight coupling of anaerobic cell growth with ethanol formation at high yields. Elimination of three additional genes resulted in a strain that selectively grows only on pentoses, even in the presence of glucose, with a high ethanol yield. We showed that the ethanol yields of strains with minimized metabolic functionality closely matched the theoretical predictions. Remarkably, catabolite repression was completely absent during anaerobic growth, resulting in the simultaneous utilization of pentoses and hexoses for ethanol production

MOESM1 of Model-based optimization and scale-up of multi-feed simultaneous saccharification and co-fermentation of steam pre-treated lignocellulose enables high gravity ethanol production

Additional file 1: Table S1. Contains experimental results from enzymatic hydrolysis experiment

Model-based optimization and scale-up of multi-feed simultaneous saccharification and co-fermentation of steam pre-treated lignocellulose enables high gravity ethanol production.

High content of water-insoluble solids (WIS) is required for simultaneous saccharification and co-fermentation (SSCF) operations to reach the high ethanol concentrations that meet the techno-economic requirements of industrial-scale production. The fundamental challenges of such processes are related to the high viscosity and inhibitor contents of the medium. Poor mass transfer and inhibition of the yeast lead to decreased ethanol yield, titre and productivity. In the present work, high-solid SSCF of pre-treated wheat straw was carried out by multi-feed SSCF which is a fed-batch process with additions of substrate, enzymes and cells, integrated with yeast propagation and adaptation on the pre-treatment liquor. The combined feeding strategies were systematically compared and optimized using experiments and simulations

Rational design and construction of an efficient E. coli for production of diapolycopendioic acid

We applied elementary mode analysis to a recombinant metabolic network of carotenoid-producing E. coli in order to identify multiple-gene knockouts for an enhanced synthesis of the carotenoids diapolycopendial (DPL) and diapolycopendioic acid (DPA). Based on the model, all inefficient carotenoid biosynthesis pathways were eliminated in a strain containing a combination of eight gene deletions. To validate the model prediction, the designed strain was constructed and tested for its performance. The designed mutant produces the carotenoids at significantly increased yields and rates as compared to the wild-type. The consistency between model prediction and experimental results demonstrates that elementary mode analysis is useful as a guiding tool also for the rational strain design of more complex pathways for secondary metabolite production