| Cell surface engineering is an effective strategy to improve the synthesis level of target product,which also plays an important role in efficient production of target protein.In this research,through optimizing the composition of phospholipids and blocking D-alanylacylation of lipoteichoic acid,a B.licheniformis host strain,DW2?dlt ABCD?pss A?cls A(DW2?DPC),was constructed for protein production,which significantly improved the yields of nattokinase and ?-amylase.Phospholipids are the main components of cell membrane in B.licheniformis,and the changes of composition and content play an important role in adapting cell to the changing environment and protein secretion.In this study,through deletion and overexpression of CDP-diacylglycerolserine O-phosphatidyltransferase gene pss A and cardiolipin synthase gene cls A,the synthesis pathways of phosphatidylethanolamine and cardiac phosphatidyl synthase were regulated,and the influence of phosphatidyl components changes on protein expression was investigated.Our results confirmed that deletions of CDP-diacylglycerolserine O-phosphatidyltransferase gene pss A and cardiolipin synthase gene cls A were not conducive to cell growth,and the biomass of gene deletion strains were decreased by 17.55% and14.17%,while the concentrations of total extracellular proteins were increased 33.76% and21.04%,respectively.The biomass of gene overexpression strains were enhanced,however,it was not conducive to protein secretion.Subsequently,through detecting the net negative extracellular charges of gene deletion strains,our results implied that the net negative charges of DW2?pss A and DW2?cls A were increased 31.97% and 26.15%,and the membrane permeability were increased by 20.18% and 10.03%,respectively.The net negative charge and membrane permeability of gene overexpressed strains were lower than that of control.Collectively,genes pss A and cls A are closely related to cell growth,and gene deletion can improve the extracellular net negative charge and cell membrane permeability,which resulted in a significant increases in protein secretion.While,overexpression of these genes were not conducive to protein secretion.In addition,nattokinase and ?-amylase expression plasmids p PRBS6 NK and p P43 SAT were respectively transferred into the gene deletion strains and control.Our results showed that nattokinase activities of DW2?pss A/p PRBS6 NK and DW2?cls A/p PRBS6 NK reached 32.54 FU/m L and 29.65 FU/m L increased by 27.59% and16.45% compared with the control strain DW2/p PRBS6NK(25.34 FU/m L),respectively.The?-amylase activities of DW2?pss A/p P43 SAT and DW2?cls A/p P43 SAT reached 270.10 U/m L and 223.10 U/m L,74.25% and 43.93% higher than that of DW2/p P43SAT(?-amylase activity were 155 U/m L),respectively,thus,our results indicated that deletions of genes pss A and cls A were beneficial to production of heterologous proteins.The Dlt operon(dlt ABCD)mediates the D-alanylation of cell wall lipoteichoic acid(LTA),which is responsible for inserting D-alanine into LTA,thus,and it may paly an important roles in the net negative charge of the cell wall and heterologous protein secretion.In this study,based on Bacillus licheniformis,dlt operon gene(dlt A,dlt B,dlt C,dlt D)single deletion strains were constructed.The results showed that the biomass of the gene-deleted strains were decreased by 11%,12.38%,9.17%,and 10.09%,respectively,compared with the control strain,however,the extracellular protein concentrations were all increased,especially for dlt B deficiency strain,the net extracellular negative charge of which increased by43.83%,and the extracellular protein concentration increased by 53.47%.Subsequently,a dlt operon multiple deletion strain DW2?dlt ABCD was constructed.Our results showed that,the biomass of strain DW2?dlt ABCD decreased by 15%,while its extracellular net negative charge increased by 55.57%,and the extracellular protein concentration increased by 55.58%,compared with those of control strain DW2.Therefore,our results indicated that the dlt operon multiple gene deletion strain have a stronger effect on promoting protein secretion,compared with single gene deletion strains.Therefore,heteroprotein expression assaies was conducted in DW2?dlt ABCD.The nattokinase and ?-amylase expression plasmid were transferred into DW2?dlt ABCD.Fermentation results showed that the nattokinase activity of DW2?dlt ABCD/p PRBS6 NK reached 31.02 FU/m L,increased by 21.13% compared to DW2/p PRBS6 NK,and the ?-amylase activity of DW2?dlt ABCD/p P43 SAT reached 250.10U/m L,increased by 60.84% compared with that of DW2/p P43 SAT.Collectively,all these results indicated that deletion of the dlt operon is beneficial for the production of heterologous proteins.Subsequently,a multi-gene deletion strain DW2?dlt ABCD?pss A?cls A(DW2?DPC)was constructed,using a combination of membrane wall gene breeding strategies of Bacillus licheniformis,and then transferred into the nattokinase and ?-amylase expression plasmids,respectively.The fermentation results showed that the nattokinase activity of DW2?DPC/p PRBS6 NK reached 37.15 FU/m L,increased by 46.08% compared to DW2/p PRBS6 NK,and ?-amylase activity produced by DW2?DPC/p P43 SAT was305.80U/m L,96.34% higher than that of DW2/p P43 SAT,and the yields of nattokinase and?-amylase activities were all higher than those of single gene deletion strains.Finally,the fermentation curves of strains were conducted.Our results showed that the cell biomass of DW2?DPC protein were lower than those of the control throughout the fermentation process,and the maximum biomass was decreased by 18.80%.While,the nattokinase produced by per gram cell reached 7.63 FU/g DCW,increased by 79.92% compared with DW2/p PRBS6NK(4.242 FU/g DCW).In addition,the ?-amylase produced by per gram cell reached 117.85 U/g DCW,increased by 141.32% compared with DW2/p P43SAT(49.24 U/g DCW).The results showed that the research on the membrane wall engineering of Bacillus licheniformis effectively promoted the production of heterologous protein,and provided an effective strategy for the heterologous protein expression system of Bacillus licheniformis. |
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