Engineering Of Escherichia Coli To Produce Monophosphoryl-lipid A And Pertussis Vaccine Oligosaccharide Antigen

Lipopolysaccharide（LPS）is located in the outer membrane of gram-negative bacteria.It is an important virulence factor and protective antigen.LPS is generally composed of lipid A,core oligosaccharide and O-antigen repeats,and its biosynthesis is a complex process involving dozens of genes.The oligosaccharide was the key portion of LPS,can induce the production of bactericidal antibodies.The particular lipid A can produce appropriate amount of cytokines and assist the host to eliminate invasive microorganisms.In this study,the molecular structure of E.coli lipopolysaccharide was precise edited by genetic engineering,and a series of recombinant E.coli strains were constructed.The excellent E.coli mutants were selected for the efficient industrial production of Bordetella pertussis oligosaccharide or MPL.（1）Interrupting the polysaccharide synthesis and improving the phospholipid content in the outer membrane,chromosome integration,or plasmid expression of lipid A modified genes in MG1655 to efficiently produce MPL.Fnlpx E encoding the 1-phosphate phosphatase in Francisella novicida,Se Pag P encoding the hexadecanoyl transferase in Salmonella,and Se Pag L encoding the deacylase in Salmonella were integrated on MG1655 chromosome,resulting in MW004.Compared to MG1655,the growth of MW004 was not restricted,but the structure of lipid A in MW004 changed,with all lipid A changed to monophosphate lipid A.However,there are four main types of monophosphate lipid A structure of MW004,which are 2-acyloxyacyl-monophosphoryl-lipid A（P-MPLA）,3-deacyl-2-acyloxyacyl-4?-monophosphoryl-lipid A（MPL）,4?-monophosphoryl-lipid A（MPLA）,and 3-deacyl-4?-monophosphoryl-lipid A（D-MPLA）.Then,58 genes of MG1655,including O-antigen,core oligosaccharide,ECA,and colanic acid biosynthesis were deleted,then,another 3 genes including pld A,mla A,and mla C were further deleted,resulting in MW012.TLC and LC-MS analysisshowed that about 50%of the lipid A structure of MW012 was palmitoylated.Fnlpx E,Sepag P,and Sepag L were expressed in MW012,resulting in MW012/p WEPL.Lipid A isolated from MW012/p WEPL with addition of 25 m M EDTA,and approximately 75%of that was MPL.MW012/p WEPL can produce 14.20 mg/L total lipid A（10.65 mg/L MPL）when grown in the fermentation medium.Compared to MG655,all lipid A synthesized in MW012/p WEPL were monophosphate lipid A,and75%of that were MPL.However,the growth of MW012/p WEPL was restricted.（2）Regulating the molecular composition of the outer membrane of MG1655and optimizing the glucose transport system to improve the production of MPL.126genes involved in synthesizing the fimbriae,flagella,ECA were deleted in E.coli MG1655,then another 4 genes involved the phospholipid transport and glucose transport were further deleted,resulting in MW019.TLC and LC-MS analysis showed that about 50%of the lipid A structure of MW019 was palmitoylated.However,the growth of MW019 was not hindered.MW019 was grown in the LB and fermentation medium,and the highest OD₆₀₀values were 4.29 and 32.11,respectively.Fnlpx E,Sepag P,and Sepag L were expressed in MW019,resulting in MW019/p WEPL.MW019/p WEPL with addition of 25 m M EDTA can produced mainly two lipid A species,one is MPL,and the other is D-MPLA.Subsequently,gad Y was expressed in the MW019/p WEPL,resulting in MW019/p WEPLp Bgad Y.MW019/p WEPLp Bgad Y was grown in the LB medium,and the highest OD₆₀₀values was 8.10,and can also efficiently produce MPL.However,in the fermentation medium,the overexpression of gad Y did not significantly improve the growth performance of the strain.（3）Transferring the essential gene fab I from chromosome to plasmid can improve the stability of the MPL producing recombinant without adding antibiotics.Fnlpx E,Sepag P,and Sepag L were inserted in plasmid p FT24,resulting in p TEPL.p TEPL was transferred in MW019,resulting in MW019/p TEPL.Finally,ept A and fab I were deleted in MW019/p TEPL,resulting in MW021/p TEPL.MW021/p TEPL does not need to add any additional antibiotics and chemical reagents during the growth process.MW021/p TEPL was grown in the fermentation medium,and the highest OD₆₀₀values were 32.12.MW021/p TEPL was grown in a 2-L fermenter containing 1 L of culture,and the highest OD₆₀₀values were 38.54,and can produce 13.84 g/L dried cells and 88.99 mg/L total lipid A.TLC and LC-MS analysis showed that the total lipid A of MW021/p TEPL contains mainly two lipid A species,one is MPL,and the other is D-MPLA.（4）Engineering E.coli MG1655 to produce Bordetella pertussis oligosaccharide with multiple trisaccharide units.26 genes involved in synthesizing the polysaccharide were deleted in E.coli MG1655,resulting in MDCO001.MDCO001 cells only synthesized Kdo₂-lipid A analysed by SDS-PAGE and LC-MS.p W contains the 13genes associated with core oligosaccharide biosynthesis and p B contains the 11 genes associated with LOS terminal trisaccharide synthesis in B.pertussis were transferring into MDCO001,resulting in MDCO001/p Wp B.MDCO001/p Wp B was grown in the LB medium,and the highest OD₆₀₀values was 7.73.SDS-PAGE analysis showed that MDCO001/p Wp B had two LPS structures similar to those of B.pertussis CS.Then,another 32 genes involved in synthesizing the ECA and colanic acid biosynthesis and one gene met J encoding a methyltransferase were deleted from MDCO001,resulting in MDCO020.p D5 was constructed,which can express the 3 genes related to the repeating trisaccharide unit,and then transferred into MDCO020,resulting in MDCO020/p Wp Bp D5.MDCO020/p Wp Bp D5 was grown in the LB medium,and the highest OD₆₀₀values was 3.69,but more B.pertussis oligosaccharide containing repeating trisaccharide units was produced.MDCO020/p Wp Bp D5 was grown in a 30-L fermenter containing 15 L of culture for 40 h,and the highest OD600 values was 28.00,can produce 490 g of bacteria and 2 g of LPS.More importantly,the structure and the immune reactivity of B.pertussis oligosaccharide produced by MDCO020/p Wp Bp D5were similar with that of the B.pertussis CS.