Cloning And Characterization Of Escherichia Coli K5 Promoters Pgnt,Pgat And Pmp In Response To Heparosan Synthesis

Glycosaminoglycans?GAGs?are linear polysaccharides composed of uronic acid and N-acetyl glucosamine repeat disaccharide unit,which are widely used in biomedicines and materials.With the assistance of a variety of protein receptors and chemokines,GAGs play important roles in many physiological processes,such as anti-tumor,anticoagulation,antivirus and immunomodulation.As an important type of GAGs,heparin?HP?is widely used in clinic because of its anticoagulant activity.However,the raw materials of HP drugs are all from animal tissues?such as porcine intestinal mucosa?,which results in that the polysaccharides are structurally heterogeneous and easily contaminated.These shortcomings limit the development of HP drugs.In the past few years,people have become more and more interested in developing methods for the production of non-animal HP and chemical synthesis,chemoenzymatic and metabolic engineering are among the effective synthesis strategies.The capsular polysaccharide of Escherichia coli O10:K5?L?:H5?ATCC23506,abbreviated as E.coli K5?,heparosan,is consistent with the HP skeleton structure that is not sulfated and isomerized.Previous studies have shown that heparosan can be used to synthesize bioengineered HP and sulfated HP analogues.Because heparosan is a kind of capsular polysaccharide produced by bacteria,its preparation process is simple and not easy to be contaminated.This provides a broad prospect for the development of the production methods of non-animal HP.In addition,as a natural polysaccharide,heparosan is also an excellent drug-carrying material and has good medicinal value.However,the wild strain E.coli K5 has a low ability to synthesize heparosan??100 mg/L?.Transformation of natural strains through synthetic biology,or de novo construction of engineered strains with high yield of heparosan through introduction of heparosan synthetic pathway into chassis cells has become effective ways to prepare heparosan.Therefore,heparosan synthesis pathways,regulatory mechanisms and domestication of high-yield strains have become research hotspots.In addition,with the development of in vitro enzymatic synthesis system of heparin oligosaccharides,obtaining more efficient tool enzyme molecules through the directed evolution of heparosan synthase and other heparin skeleton synthesis-related enzyme molecules has also been increasingly valued by the scientific community.Through systematic analysis,the promoter elements excavated from E.coli K5 and preliminary analyzed in response to the synthetic ability of heparosan are the research content of this project.The main contents,results and conclusions are as follows:?1?Based on the fact that E.coli K5 strain can naturally synthesize heparosan,through gene editing or protein recombination technology system,a series of E.coli K5 engineering strains with different heparosan synthesis ability were constructed.A set of E.coli K5 gene editing technology was established based on CRISPR/Cas9 gene editing system.By using the gene editing technology,the heparosan synthase coding gene KfiA was deleted from the wild type E.coli K5 genome and the engineering strain E.coli K5NK with complete loss of heparosan synthesis ability was obtained,and the engineering strain E.coli K5NE with the change of heparosan synthesis ability was obtained from the wild type E.coli K5 genome by knocking out the heparosan cleavage related gene ElmA.At the same time,a plasmid overexpressing KfiA was constructed and transformed into E.coli K5,and a strain E.coli K5 with strong ability to synthesize heparosan?E.coli K5OK?was obtained.?2?The transcriptome analysis of E.coli K5NK,E.coli K5NE and E.coli K5 with significant differences in heparosan synthesis ability showed that the transcriptional levels of Pgnt,Pgat and Pmp were significantly correlated with the heparosan synthesis levels.According to the credibility and statistical difference analysis of transcriptome data,the transcriptional levels of 45 genes in E.coli K5NK with complete loss of heparosan synthesis ability were significantly up-regulated compared with wild-type E.coli K5.The transcriptional levels of 226 genes were significantly down-regulated.Combined with the changes in the transcription levels and the functional analysis of the protein encoded by the genes,it was determined that the transcription levels of the membrane protein gene?coded as ECK5₇980?,glucuronosyltransferase gene?coded as ECK5₂2940?and N-acetylglucosamine transferase gene?coded as ECK5₂2960?were significantly related to the heparosan synthesis levels.The promoters Pgnt,Pgat and Pmp were cloned and determined.Using bioinformatics methods,the promoter information of the above three protein-coding genes was determined from the whole genome database of E.coli K5,respectively,named Pgnt,Pgat and Pmp.Using green fluorescent protein?GFP?as a reporter gene,the sequences of Pgnt,Pgat and Pmp were successfully cloned and determined.?3?It was confirmed that the promoters,Pgnt,Pgat and Pmp,could respond to the biosynthesis of heparosan in E.coli K5.Based on the analysis of the expression of green fluorescent protein?GFP?in E.coli K5NK,E.coli K5 and E.coli K50K under the control of the promoters mentioned above,it was found that the fluorescence values of the strains containing Pgnt,Pgat and Pmp promoters were enhanced with the enhancement of heparosan synthesis.Among them,the transcriptional level of Pgat promoter was most obviously regulated by the biosynthesis ability of heparosan.In summary,we successfully determined that the transcriptional level of promoter Pgat was positively correlated with the biosynthesis ability of heparosan in E.coli K5.The findings of this study is not only of theoretical values for the regulation and control of the synthesis of bacterial capsular polysaccharides,the promoter sequence but also can be used to construct the original biosensor for the synthesis of heparosan.The findings can also be used to construct the intracellular orientation platform of GTs such as KfiA,and the application value is also outstanding.