The Development And Application Of Dynamic Pathway Regulation In Escherichia Coli

Traditional metabolic engineering mainly adopts static regulation strategy to overexpress,weaken the expression or knock out the key genes.On the basis of maintaining cell homeostasis,it is difficult to balance the relationship between cell growth and production,and it is difficult to obtain high conversion of product synthesis,at the same time,it is difficult to efficiently accumulate products which inhibit the growth of cells.In order to solve the above problems to a certain extent,this study constructed a dynamic regulation strategy to regulate gene expression"on"and"off"at the same time.Because L-serine synthesis pathway is short and highly competitive with glycolysis pathway,and excess serine has strong toxicity to cells,the construction of L-serine cell factory is selected as the application mode.Therefore,starting from wild-type Escherichia coli W3110,a high-yield L-serine production strain was constructed by means of metabolic engineering,and then the dynamic regulation strategy was applied to the synthesis of L-serine.In this study,the dynamic regulation strategy integrates two systems:the gene strengthening system and the weakening system.Arabinose induction was determined as the best strategy after comparing several inducers.Arabinose induction system is a two plasmids expression system,in which pSTV28 plasmid contains the expression frame of arabinose induced T7RNA polymerase(ara C-P_BAD-T7RNAP),and pTRC99a plasmid contains the target gene expression frame driven by T7 promoter（taking the expression of green fluorescent protein egfp gene as an example）.The weakening system also includes a two plasmids expression system:pSTV28 plasmid expresses tetracycline repressor protein gene tetR through P_BAD promoter,and pTRC99a plasmid expresses the target gene by P_tetApromoter（taking red fluorescent protein gene mCherry as an example）.In this system,when arabinose is added to the culture medium,the cells turn up the expression of green fluorescent protein and turn down the expression of red fluorescent protein at the same time.L-serine belongs to nonessential amino acid,which is widely used in food,medicine,cosmetics and other fields,and has important commercial value.In this study,wild-type E.coli W3110 was modified in the following aspects:1.Release the feedback inhibition of PGDH enzyme;2.Knockout the degradation pathway of L-serine;3.Enhance the synthesis pathway of L-serine;4.Replenish the one carbon unit;5.Transform the cell morphology;6.Enhance the L-serine tolerance of the engineering strain;7.Overexpress the transporter of L-serine.Then selected the best constructed strain for fermentation test in a 5 L fermentor.The results show that stain Y15 produced 65.2 g/L L-serine with a yield of 29.6%in 48 h.In shaking flask,stain Y15 produced 16.7 g/L L-serine with a yield of 26.3%.Finally,the dynamic regulation system was applied to the engneering strain to produce L-serine.The target genes chosed to induce and weaken in the dynamic regulation system are serA^fbr and glyA.Taking the yield of L-serine produced by fermentation as the index,it was determined that the best induction time was fermentation 12 h,the best induction concentration was 30 mg/L,the yield of L-serine reached 25.7 g/L.Compared with static regulation,the dynamic regulation increased L-serine production by 53%.