Study On Heterologous Expression Of Streptomyces Phospholipase D By Brevibacillus Choshinensis

Phospholipase D(PLD,EC 3.1.4.4)introduces functional polar heads into common phospholipids by transphosphatidyl reaction to synthesize some new medicinal bioactive phospholipids,but its application in industry is severely limited for its high price and low output.Streptomyces PLD has attracted much attention in recent years because of its highest transphosphatidyl activity and the production of Streptomyces PLD was increased by genetic engineering in many researches.As a nonpathogenic Gram-positive bacterium,Brevibacillus choshinensis(B.choshinensis)could strongly secrete extracellular proteins and hardly secrete proteases to the outside,which is beneficial to keep the stability of the target protein,and to promote the formation of disulfide bonds to help the folding of foreign proteins.Therefore,B.choshinensis was selected as the host strain to heterologously express Streptomyces PLD in this study.Firstly,the constitutive weak promoter P5 was used to regulate the expression of PLD.The results showed that B.choshinensis could express Streptomyces PLD under P5 regulation,but the PLD activity was very low(0.026 U/mL).Then,the constitutive strong promoter P2 was used to regulate the expression of PLD.It’s found in the presence of Mg2+,the expression of extracellular PLD enzyme was increased by 67 times compared to the P5 promoter,reaching 2 U/mL under P2 regulation.After the promoter was chosen,screening medium,optimizing medium composition and imposing Mg2+ were conducted to improve the extracellular PLD activity,which finally increased to 9.3 U/mL.And the Specific yield is 0.19 U/mL/h,which is 4 times higher than that of Streptomyces antibioticus(0.048 U/mL/h).On the other hand,this work studued the regulation mechanism of Mg2+increasing PLD activity and improving the growth status of B.choshinensis.By measuring the transcription levels of related genes,it found that PLD could accumulate in the cell with the expression of PLD in B.choshinensis under strong promotor regulationin the absence of Mg2+.When the accumulated PLD reached a certain level,the growth of B.choshinensis was inhibited.Meanwhile,the strong expression of PLD could impact its correctfold.The presence of Mg2+ inhibited the strong regulation of P2 promotor,and maintained a relatively low transcriptional level of PLD gene during growth,which matched the secretion efficiency of B.choshinensis.It enabled PLD to be secreted in time while ensuring that PLD was folly folded,which thereby increased extracellular PLD activity.In addition,the intracellular Ca2+concentration increased when B.choshinensis expressed PLD,which will result in a series of adverse physiological metabolic changes to cause cell damage and even death.When Mg2+ was imposed,the intracellular Ca2+concentration significantly reduced and intracellular ions were redistributed,which improved the growth status of B.choshinensis and facilitated further PLD expression.In this study,the PLD production of B.choshinensis was greatly improved by optimizing the promoter,changing the composition of the medium,and imposing salt stress,which was the first study to express Streptomyces PLD in B.choshinensis.The extracellular secretion of PLD by B.choshinensis is conducive to downstream isolation and purification,laying a foundation for the industrial production of PLD and providing the broad prospects in the pharmaceutical and food industries due to its nonpathogenicity.