Study On Phosphatidylserine Production Using Phospholipase D

Phosphatidylserine（PS）has been widely used in the fields of food and medicine,among others,because of its unique chemical structure and health benefits.However,the extraction of PS from animals and plants is expensive,complicated,and results in low yields.Thus,phospholipase D（PLD）-mediated bioenzymatic synthesis of PS has attracted more attention because of its simple operation,mild reaction condition,and eco-friendly properties.However,the production of PS using PLD remains a challenge due to the low transphosphatidylation activity.Therefore,in our study,first,the MBP tag and PLD co-expression method was designed to achieve the soluble expression of PLD in Escherichia coli（E.coli）.Then,a"reconstruct substrate pocket"strategy was proposed based on catalytic mechanism and molecular dynamics simulation,which expanded the substrate pocket,manipulated the coordination of L-Serine（L-Ser）in the active site,thus improving the transphosphatidylation activity.The main contents of this paper are as follows:（1）Screening and soluble expression of PLD.Firstly,PLD from Streptomyces racemochromogenes（SrPLD）was selected as the parent enzyme based on the transphosphatidylation activity.Then,to increase soluble expression of SrPLD,adding fusion tags at the expression vector stage was adopted.Four PLD expression plasmids with different fusion tags were constructed.As a result,pET-28a-Sr_MBPPLD exhibited the highest soluble expression level with activity of 18.57 U·mL^-1 under the optimal enzyme-producing conditions,which was 25.09-fold higher than SrPLD.Finally,PS was prepared using a two-phase system,and the parameter r_tp/r_h（0.77）was determined to the evaluate transphosphatidylation activity.（2）Increasing Sr_MBPPLD transphosphatidylation activity by protein engineering.Based on the catalytic mechanism,molecular docking and MD simulation of Sr_MBPPLD,the substrate-binding pocket appears to be too small for the PC.And the-OH group（L-Ser）was oriented away from H436,thus it was unfavorable for the deprotonation of H436.In order to enhance the transphosphatidylation activity,a"reconstruct substrate pocket"strategy was proposed by saturated mutation of 20 residues near PC,H166,and H436.Finally,the best mutant Sr_MBPPLD^{Mu6（L86E/W164Y/Y189R/D377G/Y379L）}was obtained,in which the r_tp/r_h ratio increased by2.04-fold compared to that in the WT and the k_cat/K_Mvalues increased by 28.69-fold.At the same time,the PS titer increased to 44.12 g·L^-1 with a 59.21%conversion.By analyzing the structure of Sr_MBPPLD^Mu6 and docking with ligands（PC and L-Ser）,mutation in Mu₆ introduced non-polar and small side chain amino acids,increasing the space and hydrophobicity of the active site,which facilitate the flexible binding conformation.A new hydrogen bond network is formed to manipulate the coordination of L-Ser in the active site,which improves the specificity of H436 to obtain protons fromL-Ser.（3）Optimization of PS production conditions.To evaluate the ability of SrMBPPLD^Mu6 to catalyze PS synthesis,PS production was carried out at 10 mL-scale under optimized conditions(20 g·L^-1 of whole cells,cyclopentyl methyl ether,pH 6.0,40°C,V_{（organic phase）}:V_{（aqueous phase）}=3:1,PC to L-Ser concentration ratio of 1:4,for 12 h).As a result,58.63 g·L^-1 PS were obtained with a 78.69%conversion.And,we successfully scaled up the 10 mL reaction system to a 3 L fermenter for PS production,the titer of PS reached 59.69 g·L^-1 in 12 h,which was 135.74%higher than observed for the WT(25.32 g·L^-1)under the optimized conditions.As a result,we obtained a high STY of 4.97 g·L^-1·h^-1.