Pathway Engineering Of Genome-scale Dynamic Metabolic Network Model And Metabolic Regulated Targets In FK506 Producing Strain

In this work,two parts of research work has been carried out for FK506high-efficiency production based on the method of systems biotechnologies.On the one hand,the searching,identifying and modifying of the potential targets in FK506biosynthesis were performed based on the constructed genome-scale dynamic metabolic network model.On the other hand,under the above engineered strain,the potential targets of metabolism regulation were investigated in FK506 biosynthesis using the chemical elicitors as the starting point.In the first aspect,pathway engineering had been performed based on the genome-scale dynamic metabolic network model.?1?In order to construct the genome-scale dynamic metabolic network model of S.tsukubaensis NRRL18488,a great quantity of work had been implemented to upgrade and correct the genome-scale pseudo-steady state metabolic network model constructed by our group before,including correcting the metabolic reactions direction,analyzing and filling the identified gaps,adding the exchange and spontaneous reactions and so on.Finally,the upgraded genome-scale pseudo-steady state metabolic network model was constructed successfully,which was composed of625 metabolites and 1042 reactions.?2?Based on the above metabolic model,the genome-scale dynamic metabolic network model was constructed by the precise designing of dynamic model framework,dynamic constraints,ordinary differential equations and so on.And the analysis of model sensitivity and the simulation accuracy were carried out.Subsequently,lots of targets for improving FK506 production was predicted successfully with help of MOMA and DFBA algorithms,including 52 genetic knockout targets and 29 genetic overexpression targets.?3?In order to validate the prediction of dynamic model,the overexpression targtes of tktB,ask and msdh,and the knockout target of gcdh had been investigated respectively according to the results of the targets ranking(f_PH values).The engineered strains of HT-?gcdh?HT-tktB?HT-ask and HT-msdh were constructed successfully,led to 77.49±5.69 mg/L,73.21±3.96 mg/L?65.17±4.56 mg/L and71.2±5.69 mg/L of FK506,1.41-,1.32-,1.18-,and 1.29-fold the value of the parent strain,respectively.Additionally,the combined effect of the genetic modifications had also been evaluated.The combined overexpressed strain of HT-tktB/msdh/ask owned showed 103.32±7.59 mg/L of FK506.And the strain HT-?gcdh-tktB/msdh/ask could produce 126.61±4.66 mg/L of FK506,229.8%higher than that of the parent strain.In the second aspect,the potential targets of metabolic regulation in FK506biosynthesis were analyzed under chemical elicitor treatment.?4?Using the above engineered strain,nine chemical elicitors and their combinations were firstly selected and evaluated for their effects on FK506overproduction.The results showed DMSO and DMSO&La combination showed the higher effects on FK506 accumulation,which could produce 263.87±9.54 mg/L and 303.6±24.76 mg/L of FK506,respectively.Subsequently,the time-serious response mechanism of intracellular metabolism had been investigated for the all effective chemical elicitors using a weighted correlation network analysis approach.The network analysis uncovered 13 metabolic modules and 16 hub metabolites significantly relevant to the selected chemical elicitor treatments.Finally,the RNA-Seq-bansed transcriptomics analysis within DMSO treatment had been identified several differentially expressed genes.Based on the analysis of these identified genes,the genes within the related biosynthetic pathways of secondary metabolites?i.e.,type-I polyketide synthase gene,PKS?showed the significant up-regulated tendancy,and the genes of SigE and AraC family regulators were down-regulated obviously.These above differentially expressed genes?i.e.,PKS,SigE and AraC family regulators?maybe the main metabolic regulated targets in DMSO treatment for FK506 overproduction.