Construction And Application Of Glms Ribozyme Biosensor Responsive To GlcN

Glucosamine(GlcN)is an important component of proteoglycan synthesis in human articular cartilage matrix.It plays an important role in the health of the body and the body's immune regulation.At present,the commercialization of GlcN is all shrimp,crab and other raw materials of chitosan hydrolysis at high temperature.This method is serious pollution,so it is necessary to fundamentally change the production method of GlcN.Glms riboswitch is only able to riboswitch regulation of sugar metabolism.It only exists in gram positive bacteria,it is located in the glmS mRNA encoding 5'UTR,it requires small molecules to activate GlcN-6P.It has been found in the ribozyme is very rare,less relevant research on current Glms riboswitch.The main contents of this paper are as follows: The biosensor was constructed by Glms riboswitch and enhanced green fluorescent protein.The present Word first realized the glucosamine synthase gene(gfa1)of Saccharomyces cerevisiae is linked to the Glucosamine acetyl transferase gene(gna1),And then connected with the Glms biosensor.Meanwhile the glucosamine synthase gene(glms)of Bacillus subtilis is linked to the Glucosamine acetyl transferase gene(gna1),And then connected with the Glms biosensor.(1)gfa1-gna1-biosensor was transformed into Bacillus subtilis WB800 N by Bacillus subtilis expression vector PHT01.(2)glms-gna1-biosensor was transformed into a strain of Bacillus subtilis WB800 N by expressing plasmid PHT01.(3)glms-gna1-biosensor was directly integrated into Bacillus subtilis WB800 N.The relationship between fluorescence intensity and glucosamine content was determined by comparing the fluorescence intensity and the content of glucosamine.Through the comparison of fluorescence intensity and glucosamine,the strains with high yield of glucosamine were screened.The results showed that the combination of GFA1 and GNA1 and the combination of GLMS and GNA1 could be expressed in two different ways by shuttle plasmid and integrated genome in the expression system of Bacillus subtilis.The expression of these genes can increase the content of intracellular GlcN and GlcNAc.By analyzing the relationship between GlcN content and fluorescence expression,it is known that the final fermentation result of WB800N-glms-gna1-Ribo-egfp is the best one.The final yield was 20.4g/L,which was 4 times of the original GlcN...