| As an important pyrimidine metabolism intermediate,uridine 5?-monophosphate(UMP)has been widely used for oligosaccharide synthesis and production of anti-cancer drugs as food additives anddrug intermediates.In particular,UMP is short supply on the market because of its favorable potential commercial value andcomplex production processes.Saccharomyces cerevisiae has shown great potential in terms of UMP production due to its advantages of rapid growth and reproduction,safe and non-toxic products,and clear background of genome.In order to clarify the metabolic flux of S.cerevisiae recombinant strain produced UMP,the following experiment was carried out in this paper.Firstly,as to achieve the maximum biomass of S.cerevisiae,the synthetic medium composition that glucose as the sole carbon source and fermentation conditions of pYX212-URA5/YPH499(?URA6)were investigated and optimized by single-factor analysis and response surface methodology.Secondly,Metabolic network model of UMP synthesis by recombinant S.cerevisiae was constructed by Matlab software and Cell Net Analyzer.Metabolite accumulation or depletion rate would calculated of of batch fermentation steady state,then resulting in metabolic flux distribution with metabolic network model.On the other hand,ideal metabolic flux distribution of UMP maximize was calculated with linear programming of Matlab.Compare metabolic flux distribution between the actual fermentation process and the ideal condition to identify potential regulatory mechanisms of promoting the UMP yield.Thirdly,the influence of different dissolved oxygen and initial glucose concentrations on metabolic flux distributions of UMP synthesis were investigated with metabolic network model.Compare differences of key nodes to get conclusion.Finally,the fermentation process were regulated according to the conclusion of metabolic flux analysis.The regulatory approach is add glycolytic inhibitor and overexpressed zwf1 gene that coding gene of glucose 6-phosphate dehydrogenase.The cell concentration(OD600)of S.cerevisiae was reached to 26.8 by optimized test,increased 5.2-fold than pre-optimized,which is providing basis of biomass for subsequent metabolic flux analysis.According to the metabolic flux analysis,the conclusion is as follows:1.In practical batch fermentation,98.32% of the carbon flow into the glycolytic pathway(EMP),1.22% of the carbon flow into the pentose phosphate pathway(HMP),the yield of UMP accounts for 0.62 percent ofthe theoretical maximum yield.The ratio of flow into the EMP and HMP was close to2: 1 in ideal metabolic flux.Compared with the ideal flow distribution,proper control of dissolved oxygen,increasing and decreasing the metabolic flux of HMP and TCA respectively will help improve the flow of UMP synthesis,while decreasing the generation of by-products organic acids can reduce the waste of carbon source.2.Analysis of the flux distribution of different dissolved oxygen(DO)shown that,the flow into HMP increased by 20% with super-low DO,but only 25% of this part of flow were used to synthesis of UMP.About 38% of carbon flow were used to synthetic ethanol under anaerobic condition,resulting in waste.DO maintain 1% was the most beneficial to the accumulation of UMP,higher than the anaerobic fermentation,which is related to the synthesis of ATP.In the metabolic networks,pyruvate was flexible node,glucose-6-phosphate,oxaloacetate and ?-ketoglutarate are rigid nodes.Node analysis shown that inhibition of alcohol dehydrogenase activity can indirectly increase the synthesis of glutamate and aspartate,but not significant with low DO,so orotate that the precursor of UMP should be added.Moreover,improve the activity of key enzyme from glucose-6-phosphate into HMP in favor of the synthesis of glutamate,aspartate and ribose 5-phosphate.3.To investigate the effects of different initial glucose to metabolic flux distribution,the results showed that the difference of flow distribution between EMP and HMP was small when the concentration of initial glucose less than 40g/L.When the concentration of glucose higher than 40g/L,the flow into HMP reduced by about 30%,only about 20% of this part flow used for the synthesis of UMP,so the non-oxidative route of HMP should be suppressed.The concentration of glucose was 40g/L were most conducive to the accumulation of UMP due to higher flow of glutamate and aspartate.In the metabolic network,the variation of flow of each node key at different concentration of glucose was not significant,which both were rigid nodes.According to metabolic flux analysis,the first regulatory approach shown that 0.5%(v/v)methylbenzene and 0.18 mg/L sodium fluoride were added at fermentation 48 h,the UMP yield reach to 55mg/L after 30 h reaction,increasing2.75 times more than before.The second regulatory approach constructed transformed strain pYX212-URA5/YPH499(?URA6)-zwf1 overexpressed zwf1 gene that coding gene of glucose 6-phosphate dehydrogenase(G6PDH).Theenzyme activity of G6 PDH increased 1.2-fold,and the yield of UMP was raised to80.59mg/L,42% higher than before. |
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