Construction And Optimization Of Pseudomonas Protegens For Producing Rhamnolipid Biosurfactant

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suita-ble for future industrial development.The current research focused on developing a relatively safe and efficient microbial strain for the production of rhamnolipid biosurfactant via genetic engineering.In this research,We knock outed the pha gene which encode the synthesis of hydroxyl fatty acids via recombineering system for Pseudomonas.The competitive inhibition of rhamnolipid synthesis was eliminated and the production of rhamnolipid was enhanced.At the same time,the rhamnosyltransferase gene?rhlAB?from P.aeru-ginosa PAO1 was transferred in Pseudomonas protegens Pf-5 by a-mphipathic conjugation.The recombinant expression vector pBBR1-genta-RhlAB containing the rhamnosyltransferase gene was modified via Red/ET homologous recombineering.Then,three recombinant e-xpression vectors pBBR1-genta-P_apra-RhlAB,pBBR1-kan-P_tac-RhlAB and pBBR1-kan-P_rhaB-RhlAB with three different constitutive promot-ers(P_apra,P_tac and P_rhaB)were constructed by subcloning technology,and electrotransformated the resultant expression vectors into P.pro-tegens Pf-5.When the flask in LB medium was fermented to 42 h,the rhamnolipid concentration of Pf-5/pBBR1-genta-RhlAB supernat-ant was 17.56 mg/L,with that of Pf-5/pBBR1-genta-P_apra-RhlAB,Pf-5/pBBR1-kan-P_tac-RhlAB and Pf-5/pBBR1-kan-P_rhaB-RhlAB supernat-ant were 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.When the flask in LB medium with 1%glucose as the carbon sou-rce was fermented to 42 h,the rhamnolipid titer of Pf-5/pBBR1-g-enta-RhlAB supernatant was 18.778 mg/L,with that of Pf-5/pBBR1-genta-P_apra-RhlAB,Pf-5/pBBR1-kan-P_tac-RhlAB and Pf-5/p-BBR1-kan-P_rhaB-RhlAB supernatant were 38.859 mg/L,375.742 mg/L and 418.551 mg/L.When the flask in LB medium with 1%olive oil as the carbon source was fermented to 42h,the rhamnolipid titer of Pf-5/pBBR1-genta-RhlAB supernatant was 30.208 mg/L,with that of Pf-5/pBBR1-genta-P_apra-RhlAB,Pf-5/pBBR1-kan-P_tac-RhlABand Pf-5/pB-BR1-kan-P_rhaB-RhlAB supernatant were 55.011 mg/L,937.887 mg/L and 844.517 mg/L.When the flask in PG medium with 1%olive oil as the carbon source was fermented to 42 h,the rhamnolipid ti-ter of Pf-5/pBBR1-kan-P_tac-RhlAB and Pf-5/pBBR1-kan-P_rhaB-RhlAB supernatant were 403.27 mg/L,522.034 mg/L.When the flask inMSP medium with 1%olive oil as the carbon source was ferment-ed to 42h,the rhamnolipid titer of Pf-5/pBBR1-kan-P_tac-RhlAB and Pf-5/pBBR1-kan-P_rhaB-RhlAB supernatant were 410.022 mg/L,586.079 mg/L.The best engineered strain Pf-5/pBBR1-kan-P_tac-RhlAB was obtained through the optimization of culture medium,and the titer reached the highest 937.887 mg/L within 42 h.High performance l-iquid chromatography-mass spectrometry analysis showed that there were total 4 rhamnolipid derivatives in the Pf-5 engineering bacteria.Furthermore,the rhlAB gene expression was later examined by qRT-PCR with the result showed that the expression level of rhlAB ge-ne after P_apra,P_tac and P_rhaB promoter were 0.93,2.16 and 2.77 fold higher than that of the original one.In conclusion,we first studied the regulation function of rhlAB gene and successfully constructed the high-yield rhamnolipid strain by directional genetic modification in P.protegens Pf-5.Our resear-ch not only laid the foundation for the study of rhamnolipid biosy-nthetic regulatory networks,but also proved that the use of gene r-ecombination technology was feasible and effective to increase the yield of rhamnolipid in P.protegens Pf-5...