Salt Alkali Heat Adaptation Mechanism Of Natranaerobius Thermophilus And Expression Of Glycine Betaine Synthase Gene

Natranaerobius thermophilus DSM 18059^T is an anaerobic,halophilic,alkaliphilic and thermophilic Gram-positive bacterium.The optimum conditions for its growth are 3.9 mol/L Na⁺,p H^55?9.5 and temperature 53?.Its unique living environment implies that it must have a special mechanism of life activities,so it is of great significance to expand the theory of extremophiles.Glycine betaine is an important compatible solute.It can stabilize the cell structure,maintain the activity of enzyme,protein complexes and the integrity of cell membrane under external environmental stress conditions.Studying its function under multi extreme conditions is helpful to analyze the unique adaptive mechanism of poly-extremophile and provide new gene/protein resources for the potential application of biotechnology.In this study,we used the i TRAQ(isobaric tags for relative and absolute quantification)proteomics analysis technology to explore the adaptation mechanism of strain in three extreme conditions:salt,alkali,and temperature.The average number of proteins detected by i TRAQ under three extreme conditions was about 2000,including 717 significant differentially expressed proteins(DEPs)in salinity,842 DEPs in alkalinity,and 444 DEPs in temperature.Their isoelectric points were widely distributed,with a relatively concentrated distribution between 3 and 6,accounting for about 64%.The significant differentially expressed proteins detected under the three extreme conditions were mainly distributed in energy conversion,amino acid synthesis and transport,signal transduction and membrane transport,accounting for about 65%.Among the differentially expressed proteins,46 proteins which related to salt alkali heat related genes were detected,15 proteins were up regulated in three extreme conditions.Among them,glycine betaine transporter was up-regulated in salinity,alkalinity and temperature conditions,and the maximum up-regulation fold was 4.8,while glycine betaine synthase(GSMT and SDMT)was up-regulated significantly under salinity and alkalinity conditions(up-regulation fold>1.5).It suggests that glycine betaine,as an important compatible solute,plays an important role in the adaptation of bacteria to various extreme environmental stresses.At the same time,we used the droplet digital PCR technology to verify the transcriptional level of up-regulated genes related to salt alkali heat and the genes up-regulated by three kinds of proteomics.More than 90%of the gene were consistent with the proteomics results,indicating that the experimental results were accurate and reliable.For the key proteins GSMT and SDMT(Nther?0676,Nther?0677).The transcriptome and proteome were up-regulated by more than 1.5-fold in salinity and alkaline environment,and the gene expression in alkaline environment was about 10 times that in proteome,indicating that glycine betaine de novo synthase gene plays an important role in the adaptation of Natranaerobius thermophilus to strong alkaline environment.The number of extremophiles that synthesize glycine betaine by de novo glycine synthesis pathway is small,and the specificity of its function has not been fully confirmed.In this project,GSMT and SDMT were heterologous expressed in E.coli by point mutation,and GSMT+SDMT protein were co-expressed.Using a variety of bioinformatics software to predict the protein structure and key amino acid sites of GSMT and SDMT is helpful to explore the special functions of enzyme proteins,and then construct genetic engineering strains that can tolerate a variety of extreme environmental stresses.In conclusion,glycine betaine de novo synthetase has more advantages and potential application prospects in the construction of genetically engineered strains or plants with anti reversion genes.