E. Coli Mevalonate Pathway And Regulation,

Mevalonic acid, produced in the mevalonate pathway (MVA pathway) of most eukaryotes and higher plants, is a very important organic acid. Mevalonic acid can be used as an effective substrate added to the medium to synthesize functional terpenoids. It also has potential functions in the chemical industry. These have caused a great need on mevalonic acid production, especially natural 3R-mevalonate.MVA pathway is a classical biological pathway employed by most eukaryotes and high plants to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the precursors of terpenoids synthesis. Having the same function with MVA pathway, a newly found pathway is 1 -deoxy-D-xylulose-5-phosphate pathway (DXP pathway), which exists in most prokaryocytes and higher plants.Genes constitute the mevalonate pathway from Enterococcus faecalis were cloned into Escherichia coli in two separate compatable vectors. Two new pasmids are pZYSE and pZYKDK, which carry upstream and downstream genes of MVA pathway, respectively. Through site-directed mutagenesis, the Ala110 residue of 3-hydroxy-3-methyl-glutaryl-CoA synthase (HMGS) was mutated into Gly110, which was reported to improve the activity of HMGS to 140-fold. As 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) was cloned into the same vector (formed pZYSE), a new peak emerged in GC chromatograph map, which was subsequently proved to be mevalonic acid. Quantitative experiments on DH5αΔptsG/pZYSE fermentation obtained a mevalonate production of 168 mg/L. Metabolic flow was further regulated by gene knock-out technique. Genes encoding enzymes which convert acetyl-CoA, pyruvate into acetate, and ptsG in phosphate transport system were interrupted in the strain MG1655ΔptsGΔpoxBΔpta/pZYSE. Fermentation of this strain reached a mevalonate production of 284 mg/L. When 0.3% glycerol was added into the fermentation medium, mevalonate production was elevated to 747 mg/L. Through gene knock-out, the initiators of MVA pathway were accumulated to enhance the desired metabolic flow, which was proved by mevalonate production. With the addition of plasmid pZYKDK containing mvaK1, mvaD, and mvaK2, the complete MVA pathway was constructed into E. coli in addition to DXP pathway. This engineered strain can also be used as a universal valuable terpenoids synthesis platform. When cloned different terpenoids synthetic genes into this strain, different terpenoids can be synthesized.