Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis

Abstract

Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments xylose along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting with the native glycolytic Entner Doudoroff (ED) pathway has been hypothesized. We have investigated this putative reaction network by developing a kinetic model incorporating all of the enzymatic reactions of the PP and ED pathways, including those catalyzed by the heterologous enzymes. Starting with the experimental literature on in vitro characterization of each enzymatic reaction, we have developed a kinetic model to enable dynamic simulation of intracellular metabolite concentrations along the network of interacting PP and ED metabolic pathways. This kinetic model is useful for performing in silico simulations to predict how varying the different enzyme concentrations will affect intracellular metabolite concentrations and ethanol production rate during continuous fermentation of glucose and xylose mixtures. Among the five enzymes whose concentrations were varied as inputs to the model, ethanol production in the continuous fermentor was optimized when xylose isomerase (XI) was present at the highest level, followed by transaldolase (TAL). Predictions of the model that the interconnecting enzyme phosphoglucose isomerase (PGI) does not need to be overexpressed were recently confirmed through experimental investigations. Through such systematic analysis, we can develop efficient strategies for maximizing the fermentation of both glucose and xylose, while minimizing the expression of heterologous enzymes.

Kinetic modelling of the Zymomonas mobilis Entner–Doudoroff pathway: insights into control and functionality

“…In spite of these diverse studies, this accumulated knowledge has scarcely yet been exploited as the basis for building a comprehensive kinetic model of this key component of Z. mobilis central metabolism. The only recent attempt focused on the aspects of interaction between the engineered nonoxidative part of the pentose phosphate pathway and the native Z. mobilis E-D glycolysis for xylose fermentation, assuming constant intracellular concentrations of the essential metabolic cofactors ADP, ATP, NAD(P) + and NAD(P)H (Altintas et al, 2006). Whilst such a simplification certainly reduces model complexity, since the E-D pathway itself is a major component of ATP and NAD(P)(H) turnover, this assumption of their constant concentrations significantly limits applicability of the model.…”

Comparison of glucose/xylose co-fermentation by recombinant Zymomonas mobilis under different genetic and environmental conditions

“…The two enzymes have been implicated as rate-limiting enzymes in xylose metabolism. Their limited expression levels lead to a significant accumulation of key metabolites in the pentose metabolism pathway (Altintas et al 2006). The effects of enhanced talB and tktA gene expression on ethanol production from xylose by recombinant Z. mobilis have not yet been experimentally investigated directly.…”

Effect H2SO4and Zymomonas mobilis concentration to bioethanol production bybintarofruit (cerbera manghas) substrate

“…The addition of Zymomonas mobilis bacterium into 90.1mL filtrate was done in 0.901mL, 2.703mL, and 4.505mL respectively. The holding time for the fermentation using Zymomonas mobilis bacterium was three days at room temperature [12]. After the 3-day fermentation, the filtrate was heated at 50°C to kill the Zymomonas mobilis bacterium.…”