NADH-based kinetic model for acetone-butanol-ethanol production by Clostridium.

We present in this work a kinetic model of the acetone-butanol-ethanol (ABE) fermentation based on enzyme kinetics expressions. The model includes the effect of the co-substrate NADH as a modulating factor of cellular metabolism. The simulations obtained with the model showed an adequate fit to the experimental data reported by several authors, matching or improving the results observed with previous models. In addition, this model does not require artificial mathematical strategies such as on-off functions to achieve a satisfactory fit of the ABE fermentation dynamics. The parametric sensitivity allowed to identify the direct glucose → acetyl-CoA → butyryl-CoA pathway as being more significant for butanol production than the acid re-assimilation pathway. Likewise, model simulations showed that the increase in NADH, due to glucose concentration, favors butanol production and selectivity, finding a maximum selectivity of 3.6, at NADH concentrations above 55 mM and glucose concentration of 126 mM. The introduction of NADH in the model would allow its use for the analysis of electrofermentation processes with Clostridium, since the model establishes a basis for representing changes in the intracellular redox potential from extracellular variables.

An enhanced genome-scale metabolic reconstruction of Streptomyces clavuligerus identifies novel strain improvement strategies.

In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.

Identification of FadAB Complexes Involved in Fatty Acid ß-Oxidation in Streptomyces coelicolor and Construction of a Triacylglycerol Overproducing strain.

Oleaginous microorganisms represent possible platforms for the sustainable production of oleochemicals and biofuels due to their metabolic robustness and the possibility to be engineered. Streptomyces coelicolor is among the narrow group of prokaryotes capable of accumulating triacylglycerol (TAG) as carbon and energy reserve. Although the pathways for TAG biosynthesis in this organism have been widely addressed, the set of genes required for their breakdown have remained elusive so far. Here, we identified and characterized three gene clusters involved in the ß-oxidation of fatty acids (FA). The role of each of the three different S. coelicolor FadAB proteins in FA catabolism was confirmed by complementation of an Escherichia coliΔfadBA mutant strain deficient in ß-oxidation. In S. coelicolor, the expression profile of the three gene clusters showed variation related with the stage of growth and the presence of FA in media. Flux balance analyses using a corrected version of the current S. coelicolor metabolic model containing detailed TAG biosynthesis reactions suggested the relevance of the identified fadAB genes in the accumulation of TAG. Thus, through the construction and analysis of fadAB knockout mutant strains, we obtained an S. coelicolor mutant that showed a 4.3-fold increase in the TAG content compared to the wild type strain grown under the same culture conditions.