The Metabolic Flux Optimization Analysis Of 1,3-propanediol Production Of Klebsiella Pneumoiae Based On Thermodynamic Constraints

The thermodynamically feasible flux distribution can be generated by the metabolic flux analysis based on thermodynamic constraints with the capability of improving the accuracy of prediction,which has become a hot research topic in the genome scale metabolic network in recent years.In this paper,1,3-propanediol was used as the target product,and the thermodynamic constraints were added to the the genome scale metabolic network model iYL1228 of Klebsiella pneumoniae.The metabolic flux analysis,pathway redundancy analysis and cofactor specific optimization analysis were carried out to predict the reconstruction design of strain.Firstly,the model iYL1228 is integrated with flux balance analysis based on thermodynamic constraints to generate the metabolic flux of the strain producing 1,3-propanediol?1,3-PD?with glycerol.The maximum theoretical yield of 1,3-PD to glycerol and the optimal flux distributions were obtained by using loopless flux balance analysis.In addition,the effects of side products,reducing equivalents and flexible nodes on the production of 1,3-PD were studied under anaerobic and aerobic condition.The results showed that there is no 1,3-PD production under anaerobic when formic acid and ethanol synthesis rate are smaller but also specific growth rate is bigger.The effect of ethanol on the target product is unfavorable,while acetic acid and formic acid are beneficial to the formation of the target product when its fluxes are smaller under aerobic.Moreover,the flux of the target product increases as the ratio of NADH/NADPH increases.On the flux analysis of the nodes,it is more favorable when the specific growth rate is set to less than 0.26 h^-1 under anaerobic or less than 1.05 h^-1 under aerobic.Secondly,the model iYL1228 was used to analysis the metabolic pathway redundancy by using the flux variability analysis based on thermodynamic constraint.The flux balance analysis based on thermodynamic constraint was applied to determine the optimal pathway and the regulatable NADH-dependent reactions.Then the effects of these regulatable reactions on pathway redundancy were studied with flux variability analysis based on thermodynamic constraint.Results showed that the substituable NADH-dependent acetaldehyde dehydrogenase,glutamate dehydrogenase and NAD transhydrogenase reactions in the optimal pathway had a great influence on the pathway redundancy and the pathway redundancy decreased as its flux increased.However,the essential NADH-dependent glycerol dehydrogenase and malate dehydrogenase reactions had little effect on the pathway redundancy,which could be used in the later optimization of the cofactor specificity.Finally,we implied the computational method OptSwap,which predicts bioprocessing strain designs by indentifying optimal modifications of the cofactor binding specificities of oxidoreductase enzyme and complementary reaction knockouts.OptSwap predicts some perturbation strategies: swapping cofactor specificity of three reactions and knocking out one reaction in aerobic;swapping the cofactor specificity of two reactions and knocking out two reactions in anaerobic.The growth coupling was significantly improved after optimization.Besides,it has a significant effect on the coupling between the growth and the formation of the target product to change the specificity of target reaction.The value of BPCY?Biomass-Product Coupled Yield?is 19.98 % higher than native strain under aerobic,while lower 11.66% under anaerobic.Therefore,it is effective that NADPH should be used as cofactor under aerobic while NADH as cofacor under anaerobic.