Metabolic Analysis And Regulation Of Microorganism For The Applications Of Biomanufacturing

Microbes have inhabited the earth widespread for billions of years and may be the earliest life forms on our planet.Microbial metabolism is a series of biochemical reactions that includes numerous decomposed and synthesized chemical molecules.Microbial metabolism,as the center of microbial life,provides necessary energy and building blocks for cellular activities.Moreover,microbial metabolism performs critical functions in global elemental cycles,food processing,environmental conservation,pathological study and biomanufacturing.Currently,with the development of analytical chemistry and biotechnology,microbial metabolism has been widely applied in the production of high value-added chemicals including antimalarial drug artemisinin,anticancer drug paclitaxel,jet fuel farnesene and et al.Microbial cell factory is widely recognized as one of the most promising synthetic technique in the future.However,microbial biosynthesis efficiency is limited owning to the imbalanced intracellular metabolic pathways.For example,the lack of cofactors of NADH/NADPH and the accumulation of intermediates are major hurdles that limit the productivity of microbial cell factories.Integrative analysis of metabolite species,concentrations and fluxes is vital for the deep understanding of the kinetic and thermodynamics process of microbial metabolism,thus providing guidance for biomanufacturing pathways optimization to avoide the accumulation of toxic intermediates and to drive flux to the desired product.In this way,the production yield of microbial cell factories can be efficiently increased.Here,we make comprehensive descriptions and develop effective microorganism metabolism analysis methods in biomanufacturing.From simple in vitro metabolism model to complex single and co-culture microorganism metabolism models,this work illustrates the researches in microorganism metabolism analysis and regulation with detail.The main contents of this dissertation are as follows:（1）In view of the interconnected interactions between and within intricate metabolic pathways in microorganism,this work reconstructed the metabolism process with single enzyme in vitro to avoid the influences of other pathways and molecules for accurate component metabolism analysis.Specifically,the process of CO₂ bioreduction to formic acid is reconstructed in vitro and the component metabolism is analyzed to provide guidance for metabolic regulation.By reconstructing metabolism process in vitro,this work clearly figures out that the long-lived photo-induced electron can facilitate cofactor NADH regeneration and the NADH regeneration further improved the biosynthesis efficiency of formic acid.（2）Component metabolism regulation analyzed in vitro is further validated in vivo,providing guidance for microbial cell factory.Here,engineered farnesene producing E.coli with the mevalonate（MVA）pathway is chosen as the research model and the influence of long-live photo-induced electron is further analyzed.In this part,the cofactor NADPH regeneration metabolism are also analyzed both in vitro and in vivo and the results point out that the cofactor regeneration metabolism regulations are also suitable in E.coli.Further,based on the cofactor metabolism regulations,MVA pathway in E.coli is regulated by introducing long-lived electrons to facilitate NADPH regeneration and thus improving the farnesene production in engineered E.coli.（3）This work developed differentiated response time-resolved analysis method for the real-time and dynamic monitoring of complex Rhodopseudomonas palustris（R.palustris）-Shewanella putrefaciens（S.putrefaciens）co-culture metabolism.By establishing the relationship between Fe metabolism and the lycopene biosynthesis efficiency in R.palustris-S.putrefaciens,the microbial co-culture metabolism is optimized and the biosynthesis efficiency is improved.