Study On Methanol Utilization Of Genetically Engineered Escherichia Coli

Methanol is a saturated one-carbon alcohol and acts as an important chemical raw material.Methanol production capacity of China increased fast and showed a trend of excess.Development of methanol economy needs a series of technologies that can transform methanol into value-added products.Methanol is cheaper and more reduced than sugar,making it an attractive substrate in bioproduction of biochemicals and biofuels.Biological utilization of methanol includes assimilation and dissimilation.Methanol assimilation can provide energy and carbon skeleton for cell growth and metabolism.While the dissimilation pathway can completely oxidize methanol to CO2 and provide large amounts of reducing power for biosynthesis.Native methanol-utilizing microorganisms are more difficult to engineer than genetically tractable hosts.Giving platform microorganisms such as Escherichia coli the ability to utilize methanol is an important route for methanol bioconversion.In this study,methanol assimilation and dissimilation were investigated.On one hand,E.coli that can assimilate methanol was constructed.On the other hand,methanol dissimilation pathway was constructed to provide reducing prower for biosynthesis.This study enriched the patterns of methanol bioconversion.Fristly,this thesis tried to construct E.coli strains by combining metabolic engineering and adaptive evolution.A series of methanol utilization genes were selected to construct two methanol assimilation pathways.The formaldehyde condensation pathway consisted of a methanol dehydrogenase and a formolase.The ribulose monophosphate pathway consisted of a methanol dehydrogenase,a hexulose-6-phosphate synthase and a 6-phospho-3-hexuloisomerase.Afterwards,a proofreading-defective element of the DNA polymerase of E.coli(? subunit encoded by dnaQ gene)was recruited to perform genome replication engineering assisted continuous evolution.Several mutants that can utilize methanol were screened.Crude enzyme activity assays showed that the activities of key enzymes involved in methanol assimilation pathway were improved in the mutants.By using 13C-labeled methanol as a substrate and mass spectrometry analysis of proteinogenic amino acids,this article confirmed that methanol was assimilated into metabolic pathway and used to synthesize biomass.Secondly,three key enzymes of methanol dissimilation pathway,methanol dehydrogenase,formaldehyde dehydrogenase and formate dehydrogenase,were heterologously expressed and purified.The purified enzymes were used to construct methanol oxidization pathway in vitro.NADH generated from methanol dissimilation pathway was used to drive the conversion of pyruvic acid to D-lactic acid by coupling with a D-lactic acid dehydrogenase,demonstrating a biosynthetic pathway that was driven by the reducing power generated by methanol dissimilation.D-Lactic acid production rate was almost triple that of methanol consumption rate,suggesting that three moleculars of NADH generated from the complete oxidization of one molecular of methanol were all used for D-lactic acid formation.This study investigated the use of methanol assimilation and dissimilation in methanol bioconversion.By combing metabolic engineering and adaptive evolution,E.coli mutants that can utilize methanol to synthetize biomass were obtained.Besides,enzymatic catalysis was performed to show that the reducing power produced by methanol oxidization can be used to drive biosynthesis.The results of this study provided some application examples of methanol bioconversion.