Quantifying the Metabolic Capabilities of Engineered Zymomonas Mobilis for Ethanol Production from Hexoses and Pentoses Using Linear Programming Analysis

Ivi C. Tsantili, Faculty of Mathematics, School of Applied Mathematics & Physical Sciences (S.A.M.P.S),, National Technical University of Athens (N.T.U.A), Zografou Campus, Athens, Greece, M. Nazmul Karim, Chemical Engineering Department, Texas Tech University, Lubbock, TX 79409-3121, and Maria I. Klapa, Institute of Chemical Engineering and High-Temperature Chemical Processes (ICEHT), Foundation for Research and Technology-Hellas (FORTH), PATRAS, Greece.

In the highly energy-consuming and earth-polluting era of the early 21st century, the need for discovery of alternative, renewable, environmentally friendly energy sources and the development of cost-efficient, environmentally clean methods for their conversion into higher fuels is more than imperative. Plant biomass constitutes one of the main renewable energy sources on earth. More importantly, plant biomass, i.e. the mixture of hexose and pentose sugars from the depolymerization of cellulose and hemicellulose, has the potential to be transformed into more effective fuels, ethanol being one of them, through microbial fermentation. Recently, the engineered (to catabolize pentoses) anaerobic bacterium Zymomonas mobilis has been widely discussed among if not the most promising microorganisms for the microbial conversion of plant biomass into ethanol fuel due to its numerous advantages over the currently utilized industrial microorganisms. The recent interest in Z. mobilis could explain the small number of studies regarding its in vivo physiology and the limited use of the experimental and computational tools of metabolic engineering to understand its metabolic pathway interconnectivity and regulation towards the optimization of plant biomass conversion into ethanol. One very significant step towards this direction was the recent publication of Z. mobilis full genome.

The main objective of the presented work was (a) to reconstruct the metabolic network of the engineered Z. mobilis based on the available genome annotation, the published experimental data regarding its metabolism under various conditions, other literature and metabolic databases to a level that it could be modeled based on the available metabolic engineering methodologies, and (b) identify the metabolic boundaries of the microorganism with respect to various biological objectives based only on the stoichiometric connectivity of the network using linear programming (LP) analysis. The LP model has been widely used in various organisms being the first level of metabolic modeling that enables the identification of the main factors influencing the accomplishment of certain biological objectives due to the metabolic network connectivity only. This model forms the basis for the incorporation of more complex regulatory mechanisms and the formation of more realistic models towards the simulation of the in vivo physiology.

The results of the present study indicated that ethanol and biomass production are directly related to anaerobic respiration. This suggests that better knowledge and, more importantly, improved means of analyzing anaerobic respiration in vivo are needed to yield further conclusions about possible genetic targets, which could also lead to improved strains of Z. mobilis. Moreover, this study allowed for the identification of the reactions that are essential for bacterial growth and elucidated the connectivity between the various network reactions, especially regarding main product and byproduct formation.