| Colanic acid is a heteropolysaccharide with unique physiological activity.It can promote the lifespan of humans by regulating mitochondrial homeostasis,and has potential applications in the fields of cosmetics,health,medical treatment and scientific research.However,colanic acid is produced at low temperature,which is not conducive to the large-scale production of colanic acid.The molecular weight of colanic acid secreted naturally is relatively large.In addition,high molecular weight colanic acid also increases the viscosity of fermentation broth,which is not conducive to the further improvement of colanic acid production.In order to solve the above problems,in this study,Escherichia coli K-12 MG1655 was used as the original strain.The SRP-4 strain with high production of colanic acid was construted by relieving the RCS system regulation on colanic acid synthesis and enhancing the precursor supply.Finally,the high yield of low molecular weight colanic acid was successfully achieved by optimizing the culture medium and adding hydrolase.The main findings are as follows:（1）Using E.coli K-12 MG1655 as the original strain,the genes related to the regulation of colanic acid synthesis by the RCS system were knocked out,and the SR series recombinant strains were constructed.The colanic acid fermentation temperature of the SR series recombined strains was increased from 20°C to 30°C.SR-7 had the highest titer of colanic acid,which was 971 mg·L-1.The results of real-time quantitative PCR showed that the transcription level of the wza gene cluster of SR-7 was 3.61 times that of E.coli K-12,which confirmed that the regulation of the RCS system on the synthesis of colanic acid had been relieved successfully.Furthermore,by changing the medium to TB and adding 20 g·L-1 of glucose,the colanic acid titer of SR-7 was increased to 6.4 g·L-1.（2）By overexpressing the galu,gale,gmd,manc and manb gene sequentially in E.coli K-12 MG1655,the colanic acid precursors supply of UDP-glucose,UDP-galactose and GDP-fucose was enhanced.At 30°C,the colanic acid titer of the SP-5 reached 146 mg·L-1,which was 7.3-fold of the wild-type strain.However,the colanic acid titer of SP-5 was much lower than that of SR-7.Therefore,the strain SRP-4 was constructed by knocking out hns and lon genes and overexpressing the rcsa gene in the strain SP-5.The colanic acid titer of SRP-4reached 7.5 g·L-1,which was 1.17-fold of the SP-5.（3）In this study,colanic acid hydrolase derived from the NST1 bacteriophage was purified,and it was used to hydrolyzed colanic acid.The hydrolyzate was detected by ion liquid chromatography,thin layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The hydrolysate was the repeating unit of colanic acid with a molecular weight of 1148 Da,which proved that the hydrolase can reduce the molecular weight of colanic acid.（4）The high concentration colanic acid with high molecular weight increased the viscosity of the fermentation broth.After staining with nigrosine and observing under a microscope,a large amount of colanic acid was aggregated together,and capsuled strains inside.After detection,the colanic acid capsule inhibited the strain growth and glucose acquisition,and it also hindered the further increase of colanic acid titer.After adding 4000 U·L-1 colanic acid hydrolase to disrupt the colanic acid capsule layer,the colanic acid titer of strain SRP-4 reached17.04 g·L-1 in shake flasks.In the 3-L bioreactor,the colanic acid titer of the control group without adding colanic acid hydrolase was 12.12 g·L-1.After adding 4000 U·L-1 colanic acid hydrolase,the colanic acid titer reached 24.99 g·L-1,which was 2.06-fold of the control group.In addition,the molecular weight of colanic acid decreased by 98.7%compared with the control group（8.71 MDa）to 106.854 k Da. |
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