Recombinant Expression Of Phosphalipase D And Its Application In Biosynthesis Of Phosphatidylserine

Phospholipase D?PLD;EC 3.1.4.4? can use phosphatidylcholine as substrate to generate rare phospholipids in nature,which can be used in pharmaceutical and food industry.PLDs from microorganism have higher transphosphatidylation activity than that from animal and plant.The traditional way to obtain PLD is screen strains from soil,which is difficult to obtain high-yield strains.With the rapid development of molecular biology and genetic engineering technology,it is urgent to achieve high PLD expression through molecular engineering strategies.In this study,the PLD was heterologously expressed in different hosts for screening of optimal expression host.Then,a series genetic modification work were carried out to improve the expression level of PLD and the application of PLD in the preparation of phosphatidylserine was studied.?1?In our previous study,the sspld gene which encoding for PLD with high transphosphatidylation ability was obtained by gene mining.Herein,we use the sspld gene to construct recombinant strains Bacillus subtilis/pMA5-pld,Pichia pastoris/pPIC3.5K-pld and Corynebacterium glutamicum/pDXW-pld.The heterologous expression of PLD was realized in three hosts of B.subtilis,P.pastoris,and C.glutamicum.The PLD activity was 8.4 U/mL,13.2U/mL and 15.0 U/mL,respectively.Finally,C.glutamicum expression system was selected for further study.?2?In order to simplify the downstream preparation process and relieve the effect of enzyme on the growth of strain,six secretory recombinant plasmids were constructed in C.glutamicum by using signal peptide,and the secretive expression of PLD was successfully realized.A maximum PLD activity of 54.6 U/mL was achieved in the fermentation broth by the recombinant strain with WapA signal peptide.The ribosome binding site can determine the speed of protein synthesis.Through site analysis,a 15 bp ribosome binding site sequence AGAAGGAGATATACC was added before the signal peptide to further increase the recombinant enzyme activity to 63.6 U/mL.The promoter can control gene activity by binding to transcription factors.We conducted promoter engineering to enhance the expression level and application potential of PLD.The original promoter was replaced with the other twelve promoters,and a maximum PLD activity of 78.0 U/mL was achieved with pWapA promoter.?3?The enzymatic properties of PLD were studied.The optimal reaction temperature of PLD was 60°C,and the optimum pH was 7.5.5 mM Ca²⁺,K⁺,Mg²⁺and Li⁺could promote PLD activity.And 50 mM Ca²⁺can still promote PLD activity,while 5 mM Na⁺and Ag⁺can softly inhibit PLD activity.Chemical reagents such as DTT,SDS and EDTA showed inhibitory effects on enzyme activity.?4?In order to investigate the conversion ability of PLD towards phosphatidylcholine and serine,the qualitative analysis of the initially prepared product was carried out by thin layer chromatography.In order to explore out the optimal conditions for preparation of phosphatidylserine by PLD,the type of organic solvent,the ratio of organic phase to aqueous phase,the molar ratio of serine to phosphatidylcholine,conversion time,conversion temperature and calcium ion concentration were systematically optimized,and the yield of the product was determined by high performance liquid chromatography.The phosphatidylserine yield was up to 1.94 g/L,corresponding to a yield of 48.6%after optimization.This study provided a promising phospholipase D-producing candidate for the biocatalytic synthesis of phosphatidylserine.