Design Of New Synthetic Pathways Of Poly(3-Hydroxybutyrate) Based On High-quality Genome-scale Metabolic Network Models

The purpose of this study is to use high-quality metabolic network models to predict new synthetic pathways of poly?3-hydroxybutyrate?.The following researches were conducted for this purpose.A high-quality genome-scale metabolic model?GEM?of Pseudomonas putida KT2440,named as PpuQY1140,was reconstructed based on comparison and revision of four published GEMs.During this process,an effective method of quality test and control was proposed and applied to reconstruct a Pan-genome metabolic model?PGM?PgmQY3592.New Poly?3-hydroxybutyrate??PHB?synthetic pathways were predicted by using PpuQY1140 and Pgm QY3592 and experimentally verified.To reconstruct a high-quality GEM for Pseudomonas putida KT2440,we proposed a novel process.Firstly,simulated conditions,ATP generation and respiratory chain efficiency were made consistent in the four models.Then a systematic pathway comparison was carried out to identify pathway differences among four models to ensure correctness of pathways.After that,we further extended the network by integrating the latest genome annotation information.Thus,a pathway-consensus metabolic model PpuQY1140 with 1140 genes,1171 reactions and 1104 metabolites was reconstructed.To deal with the unlimited ATP generation problem found in the pathway-consensus process,a new method aimed at improving GEM quality was proposed here by checking ATP generation pathways.Among 36 GEMs published from 2008,21GEMs had the infinite ATP generation problem.Errors leading to unlimited ATP generation were categorized into three groups and then detail methods to revise these errors were put forward.This method was used to improve the PGM model,which was reconstructed based on iJO1366 by integrating information from multiple species.Heterogenous reactions of E.coli were added manually and automatically,and then a series quality control and optimization procedures were used to improve the quality of PGM.Finally,a high-quality PGM model PgmQY3592 with 3592 reactions was built.A new PHB synthetic pathway,the threonine cycle,was predicted by using PpuQY1140.Compared with acetyl-Co A synthesis from pyruvate catalyzed by pyruvate dehydrogenase with one molecule CO₂ losing,threonine cycle converts pyruvate to acetyl-Co A by fixing one molecule CO₂.The calculated yield of PHB was improved from 0.67 to 0.78 C-mol/C-mol glucose.The calculated result from Escherichia coli iJO1366 model showed that the yield of PHB could be further increased to 0.8 C-mol/C-mol glucose using threonine cycle in Escherichia coli.Experimental result showed that the yield of PHB increased from 0.2 to 0.4 C-mol/C-mol glucose after activating threonine cycle.Four high-yield PHB synthetic pathways were predicted from PGM:non-oxidative glycolysis pathway,xylulose monophosphate pathway,glycerol absorption pathway and 4-hydroxybutyrate cycle pathway.The calculated PHB yields reached 0.88 C-mol/C-mol glucose by using the four pathways.Among those,the effect of non-oxidative glycolysis pathway was experimentally verified with an increased PHB yield from 0.26 to 0.43 C-mol/C-mol glucose.