Abstract:In this research, the recombinant expression vector pBBR1gentaRhlAB containing the rhamnosyltransferase gene (RhlAB)was modified via Red/ET homologous recombineering. Then, three recombinant expression vectors pBBR1gentaPapraRhlAB,pBBR1kanPtacRhlAB and pBBR1kanPrhaBRhlAB with three different constitutive promoters (Papra、Ptac and PrhaB)were constructed by subcloning technology,and then we electrotransformated the resultant expression vectors into P.protegens Pf5.When the flask in LB medium was fermented to 42 h, the rhamnolipid concentration of Pf5/pBBR1gentaRhlAB supernatant was 17.56 mg/L, with that of Pf5/pBBR1gentaPapraRhlAB, Pf5/pBBR1kanPtacRhlAB and Pf5/pBBR1kanPrhaBRhlAB supernatant being 11.135 mg/L, 441.135 mg/L and 557.764 mg/L, resulting in 0.63,25.12 and 31.76 fold variation after promoter optimization. High performance liquid chromatographymass spectrometry (LCMS/MS)analysis showed that there were total 4 rhamnolipid derivatives in the Pf5 engineering bacteria. Furthermore, the RhlAB gene expression was later examined by qRTPCR with the result showed that the expression level of RhlAB gene after Ptac and PrhaB promoter was 2.16 and 2.77 fold higher than that of the original one. Generally, this study preliminarily realized the expression of RhlAB gene in Pf5, and found that the constitutive promoter Ptac and PrhaB was more efficient than the original promoter which could provide a significant reference with significance for heterologous biosynthesis of rhamnolipid.