Effect Of Phosphate Concentration On Rhamnolipid Production And Gene Expression Of Pseudomonas Aeruginosa PAO1

Rhamnolipid,one of the most important biosurfactants in recent years,becomes a potential alternative to synthesis surfactants,due to the characteristics of its widely range of application and environment friendliness.Howerver,its application in the industry is greatly restricted by its low yield and small scale and high-cost substrate.Pseudomonas aeruginosa is one of the well-known producers for rhamnolipid,yet the biosynthesis of rhamnolipids is rather complex since it is influenced by numerous factors at both genetic control and environmental/nutrational levels,among which the regulation of genes expression in the QS system such as rhl system,las system and pqs system is closely related to environmental/nutritional factors.To improve and optimize rhamnolipid production of P.aeruginosa PAO1 by anthrone colorimetry quantitative analysis,Plackett-Burman(PB)design and response surface methodology(RSM)using central composite design were adopted in culture conditions.The resluts of PB design showed that the concentration of phosphate concentration,pH value and C/N ratio were the three significant factors on rhamnolipid production,and the most significant factor was the phosphate concentration.Then the RSM was used to optimize the above critical internal factors.As a result,the optimum values of phosphate concentration and pH value and C/N ratio were 1.71g/L,6.0 and 15.5,respectively.Rhamnolipid production could be up to 20.72 g/L,which was in agreement with the predicted production at 21.35 g/L.Compared with the production of original 12.63 g/L,64.05%increment had been obtained.On this basis,different phosphate concentration as the key factor was selected to study the influence on rhamnolipids production of strain PAO1.The results showed that when the phosphate concentration was from 0.01 g/L to 0.5 g/L(phosphate limitation),the yields of rhamnolipids were higher than that in the optimum phosphate concentration,especially the production of rhamnolipid reached the maximum of 24.93 g/L when the phosphate concentration was 0.5 g/L.However,the yield of rhamnolipid decreased to 16.86 g/L when there was no phosphate in the culture medium(phosphate starvation).The blood plate experiments showed that the hemolysis circle was the largest under the phosphate limitation condition,which was consistent with the production of rhamnolipids.Meanwhile,expressed differential proteins of strain PAO1 at different phosphate concentration were detected by the SDS-PAGE gel electrophoresis.The relative expression of genes in rhl system and las system and the two-component system were determined at different phosphate concentration by qPCR.The results showed that the relative expression of rhll/rhlR?lasl?IasR?rsaL?phoB and phoR were changed,respectively.Thus,it followed that different phosphate concentration could affect the biosynthesis of rhamnolipids production and its regulation of genes expression in strain PAO1.In order to further investigate the gene expression of rhamnolipid production in Pseudomonas aeruginosa at different phosphate concentration,the rhl system mutant(?rhlI,?srhlR)and las system mutant(?lasI,?lasR and ?rsaL)were constructed,and the yields of rhamnolipids and expression of related genes were monitored in those mutant strains at different phosphate concentration.The results showed that the yields of rhamnolipids in Arhll and ArhlR were 18.35 g/L and 16.04 g/L,which was decreased by 26.39%and 35.66%respectively,compared to the yield of wild type PAO1 24.93 g/L under the phosphate limitation condition.There were unchanged in the mutant ?lasI and?lasR,particularly the yield of rhamnolipid in ?lasI was 23.3 g/L,which was almost the same as that of strain PAO1.While the yield of rhamnolipid in ArsaL was higher by 13.88%than that of strain PAO1.The relative expression of genes rhlA and rhlB were decreased of Arhll and ArhlR,but that of Alas1 and AlasR were little changed,and that of ArsaL was increased by qPCR.The relative expression of genes phoB and phoR of the rhl system mutants and ArsaL were decreased,while that of AlasI and AlasR were unchanged under the phosphate limitation condition.Under the phosphate starvation,the yields of rhamnolipids by strain ?rhlR and ?lasR were 12.86 g/L and 13.02 g/L,which was decreased by 15.79%and 22.78%respectively,compared with the yield of wild type PAO116.86 g/L.The yields of rhamnolipids by Arhll and ?lasI were consistent with that of PAO1.Nevertheless,the yield of ArsaL 20.12 g/L was higher by 19.34%than that of PAO1.The results of qPCR showed that the relative expression of genes rhlA and rhlB were down-regulated of ?rhlR and ?lasR,while that of ArsaL was up-regulated.The relative expression of gene phoB and phoR was almost unchanged in wild type PAO1 and mutant strains under the phosphate starvation condition.Therefore,the rhl system played a key role in the biosynthesis and regulation of rhamnolipid production,and the two-component regulatory system was also involved in the regulation of rhamnolipid production under the phosphate limitation condition.While the genes rh1R in rhl system and the lasR in las system played an important part in the biosynthesis of rhamnolipids and regulation of gene expression,and the two-component regulatory system was of no use in the regulation of rhamnolipid production under phosphate starvation condition.The gene rsaL,as an inhibitor in las system,could cut down the yield of rhamnolipid.In a word,the biosynthesis and the regulation of genes expression of rhamnolipids were influenced by the nurtritional factors,and the response mechanism of Pseudomonas aeruginosa was different,which could directly or indirectly affect the biosynthesis of rhamnolipids under different phosphate conditions.