Metabolic Flux Analysis And Dynamic Simulation Of Glycerol Bioconversion Into 1, 3-Propanediol

Bio-refinery or white-biotechnology has been paid much attention due to the high price of petroleum.As an alternative fuel,biodiesel is increasedly produced and 10 percent of glycerol is accompanied as a by-product.Much attention has been paid on the bioconversion of glycerol into a high valuable chemical such as 1,3-propanediol(1,3-PD).In this paper,the process of glycerol bioconversion into 1,3-PD in Klebsiella pneumoniae was analyzed by metabolic flux analysis,and the dynamic process was simulated by a hybrid model based on metabolic elementary modes.The main work of this paper was summarized as followed:Firstly,simplifed metabolic network was constructed according to the glycerol metabolism in K.pneumoniae.A model of metabolic flux balance was developed for 1,3-PD production by K.pneumoniae under anaerobic and microaerobic conditions based on the metabolic network.The maximum theoretical yield of 1,3-PD to glycerol and the optimal flux distributions were obtained by flux balance analysis(FBA) with the maximum flux of 1,3-PD as the objective function.Furthermore,the influences of the specific growth rates,the molar fraction of NADH₂ oxidized by molecular oxygen in TCA(tricarboxylic acid ) cycle and the respiratory quotient(RQ) on the maximum theoretical yield of 1,3-PD to glycerol were analyzed.The limitation potential of various fluxes,such as ethanol,acetic,lactic,H₂, 2,3-butanediol,for the maximum theoretical yield of 1,3-PD were calculated.Additionally, the objective function driving the cellular metabolism was identified by a nonlinear bilevel programming(NBP) model.The results showed that the almost same objective functions in term of different experimental data were obtained and the important coefficient of biomass growth flux which was the main power of driving the cellular metabolism was the largest in the fitness function and then was 1,3-PD and 2,3-butaniol in turn.Secondly,the metabolic network of glycerol metabolism in K.pneumoniae under anaerobic conditions was improved and extended according to the KEGG database and related references.The measured fluxes obtained under steady-state conditions were used to estimate intracellular fluxes and identify the robustness of branch points of the anaerobic glycerol metabolism in K.pneumoniae for the production of 1,3-PD by metabolic flux analysis(MFA). The biomass concentration increased as NADH₂/NAD⁺ decreased at low initial glycerol concentrations but inversed at high initial glycerol concentrations.The absolute flux distribution revealed that the branch points of glycerol and dihydroxyacetonephosphate (DHAP) were rigid to the environmental conditions.However,the pyruvate and acetyl coenzyme A(ACCOA) metabolisms gave cells the flexibility to regulate the energy and intermediate fluxes under various environmental conditions.Additionally,it was found that the fluxes of ethanol and pyruvate formate lyase(PFL) appeared visible fluctuations at high glycerol uptake rates.Thirdly,a model that utilized existing knowledge of oxygen and redox sensing/regulatory system to assist elementary flux modes(EFMs),was developed and was carried out to predict the metabolic potential of K.pneumoniae for the production of 1,3-propanediol(1,3-PD) under anaerobic and aerobic conditions.It was found that the theoretical optimal 1,3-PD yield could reach to 0.844 mol mol^-1 if the pentose phosphate pathway(PPP) and transhydrogenase had a high flux under anaerobic conditions.However,PPP had little influence on the theoretical 1,3-PD yield and the flux through tricarboxylic acid(TCA) cycle was high under aerobic conditions.Both conditions exhibited a different distribution of NADH₂ supplied during maximum 1,3-PD production.Under anaerobic conditions,66.67%of NADH₂ was generated via the PPP.Whereas,NADH2 supply relied on the TCA-cycle in addition to the PPP under aerobic conditions.Additionally,the effect of oxygen level on the 1,3-PD and biomass was further analyzed.Finally,on the basis of glycerol metabolic network in K.penumoniae,the elementary flux modes were computed and translated into a set of macro-reactions connecting the extracellular substrates and products.A model combining macroscopical with microcosmic metabolic network was developed.As the elementary macroscopical dynamics were described by a classical dynamic model under balanced growth conditions,the model was suitable for describing the dynamic process during the exponential growth phase in batch fermentations. As the elementary macroscopical dynamics were described by a bioreactor dynamical hybrid model which combined first principles modeling with artificial neural networks(ANNs),the results showed that the model could describe the dynamic processes of batch,fed-batch and oscillation simultaneously.This approach allowed the quantification of fluxes carried by individual elementary modes which was of great help to understand the dynamics properties.