Mechanism Of The Influence Of Key Molecules In The Outer Membrane Of Gram-Negative Bacteria On Global Regulation And Polyhydroxyalkanoates Production

The outer membrane of Gram-negative bacteria include lipopolysaccharide and a lot of flagella and fimbria structures,their biosynthesis involves dozens of genes and consume a lot of nutrients,and are not conducive to be applied in fermentation.In this study,genome-reduction systems were constructed in Escherichia coli K12 and Pseudomonas putida KT2442to remove their non-essential genes about LPS,and flagella and fimbrea,resulting membrane reduction strains.We further studied the global impacts on cells and analyzed the global regulatory mechanism,then applied them to facilitate PHA production.The main conclusions are as follows:?1?The LPS was truncated to the minimal structure,Kdo2-lipid A,and the Kdo2-lipid A production was further improved by engineering the LPS biosynthesis pathway in E.coli.In E.coli W3110,3-deoxy-d-manno-oct-2-ulosonic acid?Kdo?₂-lipid A is the conserved structure domain of lipopolysaccharide and is an important stimulator for studying the mechanism of the innate immune system and for developing bacterial vaccine adjuvants.Kdo₂-lipid A has not been chemically synthesized to date and could only be isolated from an Escherichia coli mutant strain,WBB06.WBB06 cells grow slowly and have to grow in the presence of tetracycline.In this study,a novel E.coli mutant strain,WJW00,that could synthesize Kdo₂-lipid A was constructed by deleting the rfaD gene from the genome of E.coli W3110.The rfaD gene encodes ADP-l-glycero-d-manno-heptose-6-epimerase RfaD.Based on the analysis by SDS-PAGE,thin layer chromatography?TLC?and electrospray ionization mass spectrometry?ESI/MS?,WJW00 could produce 1.31?M?2.94?g/mL?of Kdo₂-lipid A,similar levels to WBB06,but decreased 35.1%than W3110.WJW00 cells grow much better than WBB06 cells and do not need to add any antibiotics during growth.Compared with the wild-type strain W3110,WJW00 showed increased hydrophobicity,higher cell permeability,greater autoaggregation and decreased biofilm-forming ability.Therefore,WJW00 could be a more suitable strain than WBB06 for producing Kdo₂-lipid A and a good base strain for developing lipid A adjuvants.Furthermore,the production of Kdo₂-lipid A could be improved 93.2%,25.5%and 189.8%by removal of ptsO,overexpression of msbA and overexpression of lpxC,respectively.This suggested the overexpression of lpxC is the most efficient strategy to improve Kdo₂-lipid A production,and the LpxC is the key limited enzyme for Kdo₂-lipid A biosynthesis.?2?The global influence on cells and poly-3-hydroxybutyrate production by LPS truncation were studied through determining the metabolites changes with transcriptome analysis.The WJW00 strain was further systermly studied to better undersdand the global impacts of rfaD deletion on E.coli cells.The results showed that WJW00 cells were significantly different from W3110 cells on phospholipid composition,outer membrane protein expression,flagellar formation,cell shape,membrane thickness,and the intracellular levels of acetyl-CoA,organic acids,amino acids,and gamma-amino butyric acid.Transcriptome analysis further showed that the lipopolysaccharide simplification had a global effect on cell envelope and intracellular metabolism.These results suggest that simplifying lipopolysaccharide to Kdo₂-lipid A might increase the elasticity of cell envelope and the intracellular ratio of carbon to nitrogen in E.coli.Therefore,WJW00 may be applied into PHB accumulation.When the plasmid pDXW-8-phaCAB which harbors the gene cluster for PHB synthesis was introduced into WJW00 and W3110,WJW00/pDXW-8-phaCAB cells accumulated more PHB granules with much larer cell size than W3110/pDXW-8-phaCAB;and WJW00/pDXW-8-phaCAB cells produced 67.8%PHB,increased 3 times than the control.To confirm the universality,the gene rfaD was also removed from other E.coli species DH5?and JM109,resulting WJD00 and WJJ00,respectively.As expected,WJD00 and WJJ00 only synthesized Kdo₂-lipid A,and WJD00/pDXW-8-phaCAB and WJJ00/pDXW-8-phaCAB also efficiently produced PHB with enlarged cell size,and produced 78.6%and 84.8%PHB.These results demonstrate that simplification of lipopolysaccharide structure in E.coli is beneficial to produce PHB.?3?The deletion of 25 genes relevant to LPS biosynthesis causes global impact on E.coli cells and facilitates PHB production.Lipopolysaccharide,the major component in the outer membrane of Escherichia coli,is composed of Kdo₂-lipid A and a polysaccharide.In this study,25 genes involved in synthesizing the polysaccharide were deleted in E.coli W3110,resulting in the mutant WJW02.WJW02 cells only synthesized Kdo₂-lipid A and showed significant differences on cell envelope and intracellular metabolism from W3110 cells,including phospholipid composition,outer membrane protein expression,flagellar assembly,cell shape,membrane thickness and cell-cell interactions.The intracellular levels of acetyl-CoA,organic acids,amino acids,and gamma-amino butyric acid were also significantly changed in WJW02cells.Transcriptome analysis indicates that 4139 genes involved in cell envelope and intracellular metabolism were differentially regulated in WJW02 cells.The global impact of lipopolysaccharide truncation in WJW02 was further confirmed by poly-3-hydroxybutyrate production.When the plasmid pBHR68 which harboring genes for the biosynthesis of poly-3-hydroxybutyrate production was introduced,WJW02 cells were fully filled with poly-3-hydroxybutyrate,and the cell volume was enlarged 25 times,comparing to the wild type control;and produced 82.4%PHB.This suggests that lipopolysaccharide truncation in E.coli could increase the elasticity of cell envelope as well as the intracellular ratio of carbon to nitrogen and global regulatory systerm.This study may open a new avenue for improving production efficiency in industry Gram-negative bacterial strains.?4?The deletion of 59 genes about flagella and 9 genes about fimbria improves the cell characteristics and contributes to PHB synthesis.In E.coli,flagella and fimbrea are distributed in the outer membrane and consume 2%of energy of whole cell.We deleted 59genes relevant to the flagella,and 9 genes relevant to the fimbrea by CRISPR/Cas9 system,resulting WJW010 and WJW011,respectively.Compared with the wild-type strain W3110,WJW010 and WJW011 showed decreased hydrophobicityand decreased biofilm-forming ability,WJW010 decreased 10.2%and 45%,respectively;and WJW011 decreased 18.4%and82.2%,respectively.While,the above deletion had not effects on the other membrane structure biosynthesis and membrane phenotypes.In addition,the intracellular metabolism were also affected,compared with the wild-type W3110,WJW010 showed faster growth,less glucose consumpriton,and produced 10.0%less Ala but 72.0%more GABA;WJW011 showed faster growth,less glucose consumpriton,and produced 10.0%less Ala but 100%much GABA.When the plasmid pBHR68 was introduced,comparing to the wild type control W3110/pBHR68,WJW010/pBHR68 produced 68.1%more dry cell weight?DCW?and produced 18.7%PHB;WJW011/pBHR68 produced 34.4%more DCW while a little increase of PHB.The results suggested that the removal of 59 genes relevant to flagella could facilitate the PHB biosynthesis.The further deletion of 68 genes relevant to flagella and fimbria in WJW02 did not cause further improvement of the PHB production.?5?Two efficient genome-reduction systems were constructed,and further used to delete 76 genes relevant to flagella and pili to improve cell characteristics and polyhydroxyalkanoate synthesis in P.putida.P.putida KT2442,a natural producer of PHA,spends a lot of energy and carbon sources to form flagella and pili;therefore,deleting the genes involved in the biosynthesis and assembly of flagella and pili might improve the PHA productivity.In this study,two novel deletion systems were constructed in order to efficiently remove the 76 genes involved in the biosynthesis and assembly of flagella and pili in P.putida KT2442.Both systems combine suicide-plasmid-based homologous recombination and mutant lox site-specific recombination,and involve three plasmids.The first includes pK18mobsacB,pWJW101 and pWJW102,and the second includes pZJD29c,pDTW202 and pWJW103.These newly-constructed systems were successfully used to remove different gene clusters in P.putida KT2442 and showed a high deletion efficiency?above 90%?whether for the second-round or the third-round recombination.Both systems could efficiently delete the gene PP4378 encoding flagellin in putida KT2442,resulting in mutant strain WJPP01.The second system was used to remove the pili-forming gene cluster PP2357-PP2363 in putida KT2442,resulting in the mutant strain WJPP02,and also used to remove the flagella-forming gene cluster PP4329-PP4397 in WJPP02,resulting in the mutant strain WJPP03.Compared with the wild-type KT2442,the 1.2%genome-reduction mutant WJPP03 grew faster,lacked flagella and motility,showed sharply decreased biofilm and 3',5'-cyclic diguanylic acid?c-di-GMP?,but accumulated more PHA.The biomass,PHA yield and content of WJPP03 increased 19.1%,73.4%and 45.6%respectively with sodium hexanoate supplementation;also increased 11.4%,53.6%,and 37.9%respectively with lauric acid supplementation.