Production Of Hydroxy-L-proline And Trigonelline By Recombinant Escherichia Coli BL21(DE)with Genome-scale Metabolic Network Analysis

ObjectiveIn order to observe the effects of different analytical methods on genome-scale metabolic network model and optimize the gene knockout strategy,this research combined the genome-scale metabolic network model with metabolic engineering（production of hydroxyl-L-proline and trigonelline with recombinant E.coli）by modifying the related reaction of the initial model.As laboratory study,the M9 medium was optimized for culturing Escherichia coli under the in silico analysis and attempted to producing trigonelline synthase in recombinant E.coli.Methods1.Download the metabolic network model of E.coli BL21（DE）:iB21₁397,and add the metabolic pathway of hydroxyl-L-proline and trigonelline synthesis to form two new models.2.By analysing the unmodified iB21₁397 model with FBA（flux balance analysis）and predicting the essential genes,the simulation guide the optimization of M9 medium.3.The cell growth phenotypes of the hydroxyl-L-proline and trigonelline models which are added the ralated metabolic pathway,were simulated.Gene knockout strategy simulation was performed by using a combination of different methods such as FVA,OptKnock,GDLS,IdealKnock.And the differences between the simulation results are compared.4.Four groups of modified M9 medium were designed according to the simulation results:glucose group,glycerol group,glucose-iron group,glucose-glycerol group and double-glucose group.After the preparation of the bacteria suspension,0.1 ml was inoculated to fresh M9 medium.With the purpose to study the difference of colonie,the cells were inoculated into LB agar medium for 12 h and counted the colonie after 18 h.With the purpose to study the difference colonies in the logarithmic growth phase,they were inoculated into the corresponding M9 agar medium after 12 h,18 h and 24 h,respectively,and counted the colonie after 72 h.5.According to the information of trigonelline synthase CTgS2（BAC43759.1）in the GeneBank,the gene fragment was synthesized and conect to the vector pET24a（+）by restriction endonuclease Nde I and Xho I.The transformants were amplified by transforming into Escherichia coli DH5 a.The plasmids were then extracted and restriction enzyme reaction were performed to identify the mutations.After that,the recombinant plasmid was transformed into Escherichia coli BL21（DE）.To produce the recombinant protein,under the same culture condition,IPTG at the final concentration of 0.3 mM,0.6 mM,1 mM and TNDA-1 protein promoter at the final concentration of 0.8 g/L was added.The cells were maintained for 6 h,8 h,10 h,respectively.After the induction,the bacterial body was broken and subjected to SDS-PAGE electrophoresis to observe the expression of recombinant protein.Results1.It’ s successful to implement the modification of metabolic network model and FBA analysis.The in silico analysis result was compared to the literature experimental data.2.It predicted a gene knockout strategies to overexpression of synthetic enzymes:by knocking out ketoglutarate dehydrogenase,fructose 6-phosphate aldolase,isocitrate lyase,Phosphate glyceride dehydrogenase.The strategy was compared with other analysis results and found that it has a certain degree of credibility.3.It predicted a gene knockout strategies to overexpression of trigonelline synthase:by knocking out aldehyde dehydrogenase,malosterase,pyruvate kinase and transhydrogenase.4.As for M9 medium optimization experiment,glucose can promote the growth of cells than glycero.Not only the addition of Fe2+ in the medium could promote the growth of the bacteria,but also increasing the concentration of the same carbon source.There was no significant difference between two methods in the effect of the promotion.The difference only occured after 24 h culturing.The experimental results were basically consistent with the simulation results.5.The recombinant plasmid was transformed into E.coli BL21（DE）by restriction enzyme after digestion and sequencing.The recombinant plasmids were induced by different concentrations of inducers and different induction time.SDS-PAGE electrophoresis results showed that the targered protein didn’ t be expressed.However,the understanding of recombinant protein technology and the knockledge of choosing the vecter has been improved.Conclusion1.Metabolic engineering can be easily combined with the genome scale metabolic network model in order to be simulated and analyzed in silico.FBA analysis can be a great guide on predicting the maximum production and the space that can be improved.2.Alternative knockout reactions screened with the IdealKnock method are more effective than the FVA method.It’ s more suitable for OptKnock for gene knockout prediction.3.OptKnock method has a long time to predict gene knockout strategies,but the success rate is higher than that of GDLS method,as long as the appropriate model reaction pretreatment method is combined.4.As for the recombinant E.coli to produce hydroxyl-L-proline,the simulation showed that knock-out ketoglutarate dehydrogenase（AKGDH）,fructose 6-phosphate aldolase（F6PA）,isocitrate Lyase（ICL）,phosphoglycerate dehydrogenase（PGCD）,may be beneficial for overexpression of proline 4-hydroxylase.5.As for the recombinant E.coli to produce trigonelline,the simulation showed that knock-out acetaldehyde dehydrogenase（ALDD2y）,malate enzyme（ME2）,pyruvate kinase（PYK）,NAD（P）transhydrogenase（THD2pp）may be beneficial for the overexpression of the trigonelline synthase.6.The simulation results of FBA analysis on metabolic network,has a strong guiding significance to the optimization of M9 medium.Adding appropriate amount of Fe2+ can effectively promote the growth,without increasing the concentration of glucose.Glucose is better than glycerol slightly in promoting cell growth.But if the production needs to use glycerol,the glycerol concentration need to be increased properly,or with adding glucose,in order to achieve growth which by using glucose alone.7.In this study,the gene fragment was ligated with the vector pET24a（+）by restriction endonuclease Nde I and Xho I according to the information of the gangrene synthase CTgS2（BAC43759.1）in the GeneBank.The recombinant plasmid pET24a-CTgS2 was transformed into Escherichia coli DH5 a for amligication and Escherichia coli BL21（DE）for expression of target protein.But the target protein was not successfully express.It was suggested that the failure could be related to the selection of the vector and the lack of perfection in the process of gene recombination.