利用Duet系列共表达载体构建工程菌生物合成3-羟基丁酸(3HB)

通过PCR获得β-酮基硫解酶(PhaA)、乙酰乙酰辅酶A还原酶(PhaB)和硫酯酶(TesB)的基因phaA,phaB和tesB。以pACYCDuet-1、pRSFDuet-1、PET30a(+)为载体,以大肠杆菌表达型宿主E.coli BL21(DE3)为宿主茵,分别构建了两株表达体系不同的工程茵,建立了R-3-羟基丁酸(3HB)的代谢途径,实现3HB的生物合成。将两株工程茵分别进行诱导表达及发酵,结果表明,三个基因均可在表达型宿主BL21(DE3)中进行活性表达,通过三个酶的连续催化生物合成3HB,并且三个基因的均衡表达更有利于3HB的生物合成。将phaAB基因簇构建在ACYCDuet-1上,tesB基因构建在RSFDuet-1上,获得的工程茵BL21(AAB+RB),3HB产量可达到1.8 g/L。

Biosynthesis of R-3-hydroxybutric acid （3HB） by constructing engineering bacteria with Duet compatible vectors

The phaA ,phaB and tesB genes of β-ketothiolase （ PhaA）, acetoacetyl CoA reductase （PhaB） and thioesterase Ⅱ （TesB） were cloned by PCR. Then these genes were coexpressed in E. coli BL21（ DE3 ） with plasmid pACYCDuet-1, pRSFDuet-1 and PET30a（ ＋ ）. Two engineering bacteria with different expressing systems were constructed, and R-3- hydroxybutric acid （3HB） was successfully biosynthesized with the construction of metabolic pathway of 3HB. Compared with the detection results after the induction and fermentation of the two engineering bacteria, it was found that the three genes could all be actively expressed in expressing host cell BL21 （DE3）. The balanced expression of the three genes was more beneficial to the production of 3HB. The engineering bacteria BL21 （AAB ＋ RB）were obtained by integrating phaAB genes into ACYCDuet-1 and tesB gene into RSFDuet-1 with the production of 3 HB as high as 1.8 g,/L.