Biosynthesis Of R-3-hydroxybutric Acid In Expressing E. Coli Host With Duet Compatible Vectors

R-3-hydroxybutyric acid (R-3HB) is the key material in the synthesizing of many very important substances, such as antibiotics, biological polyester, beta polypeptide and etc. Biosynthesis of R-3HB becomes research focus with the development of synthetic biology. We constructed the metabolilc pathway of R-3HB in expressing E.coli host, and get R-3HB via the method of biosynthesis.Firstly, genes of enzymes realated to the metabolic pathway of R-3HBwere cloned and epressed actively. tesB gene and phaAB gene cluster were cloned from Escherichia coli genome and Wautersia eutropha HI6genome by PCR, and ybgC gene was obtained by total gene synthesis. The tesB、ybgC genes were insterted into vector PET30a(+) and got the reconstructed plasmid PT and PY, respectively. They were expressed in BL21(DE3) and the enzyme activities were22.1U/mL and4.32U/mL; the phaAB gene cluster was inserted into pBluescript SK(+) and got the plasmid PBHR69. It was expressed in DH5a, and the enzyme activity was5.57U/mL and2.57U/mL.Secondly, to realize the coexpression of phaA, phaB and thioesterase, the three genes were respectively inserted into Duet compatible vectors. The phaAB gene was inserted into ACYCDuet-1and RSFDuet-1vectors, and the SDS-PAGE result showed that the ACYCduet-1vector can express the phaAB genes better. When they were coxepressed with the third gene ybgC, results showed that the two-plasmids coexpression system was better than the single-plasmid coexpression system when the thioesterase was constructed on RSFDuet-1. Then this paper compared the different combinations of plasmids (ACYC+RSF) and (ACYC+PET30). The results showed that the combination of (ACYC+RSF) can express proteins more balance and yield R-3HB higher. Then this paper compared the effects of thioseterase tesB and exogenous thioesterase ybgC on the metabolic pathway. The results showed that when using tesB as a hydrolase, quantity of the R-3HB can be greatly enhanced, thus heighten the thioesterase activity can effectively enhance the yield of R-3HB. Finally, the construction system of the three gene phaAB and tesB was optimized. When phaAB was contructed on ACYCDuet-1and tesB on RSFduet-1, R-3HB yield can be further enhanced.We successfully constructed the metabolic pathway of R-3HB in expressing E. coli host, and get the engineering bacteria, the concentration of R-3HB by shake flask fermentation of which can reach up to1.8g/L.