|Other Abstract||The energy crisis and environmental pollution have become the serious problems that need to be addressed urgently since the twentieth century. Biological method is attracted highly attention due to the renewable materials and few pollution. In this study, we constructed a platform cell factory of Escherichia coli for the production of acetyl-CoA-derived chemicals through metabolic engineering of global regulation, multi-level and multi-path. This platform cell factory is versatile, high efficiency and has a great market value.
We chose five genetic modifications of arcA knockout, iclR knockout, csrB overexpression, ackA knockout and acs overexpression in this study. Firstly, The two chemicals, phloroglucinol (PG) and 3-hydroxypropionate (3HP) both derived from acetyl-CoA, were used to evaluate the effects of arcA and iclR konckout on acetyl-CoA-derived chemicals production. The mutants significantly increased the glucose utilization and cell yield than the corresponding controls. The production of PG and 3HP was increased by more than 2 times. In additon, the mutants showed surprising efficiency to overcome acetate excretion.
The effects of arcA knockout, iclR knockout, csrB overexpression, ackA knockout and acs overexpression on PG produciton were compared and discussed. The five single genetic modificaion improved PG production in different degrees. The arcA mutant showed the best result for improving PG production by 2.5 folds. Then, the combined regulation strains were constructed between arcA and other genetic modifications. However, the PG produciton of combined regulation strains was lower than that of the arcA single mutant. So the platform cell factory was constructed without containing arcA konckout. The different recombinant strains were constructed with iclR knockout, csrB overexpression, ackA knockout and acs overexpression. The recombinant strains of Q2637,Q2628,Q2140 and Q2627 increased PG production by more than 3.5 folds. The combined regulation of csrB, ackA and acs improved PG production by 5 folds, from 0.41±0.02g/l to 2.12±0.09g/l. and 3HP production was improved by 2 folds, from1.75± 0.11 g/l to 3.49± 0.33 g/l. Considering all these, the combined regulation of csrB, ackA and acs can be used to constructed a platform cell factory to improve the acetyl-CoA-derived chemicals prodution.
The cell growth, glucose utilizaiton and fermentation products of the platform cell factory were also discussed during the production of PG and 3HP. The platform cell factory showd similar cell growth and glucose consumption curve to the control strains. However, the cell yield of recombinant strains was higher than the control strains. The platform cell factory can not only increase the production of PG and 3HP,but also overcome acetate excretion. The acetate concentration was decreased by more than 90%. In conlusion, the platform cell factory that was engineered of csrB, ackA and acs would be a good host for acetyl-CoA-derived chemicals production. It would be widely used in industry because of the higher glucose utilization, lower acetate excretion and lower production cost.|