Discovery Of Heat-stress Tolerant Decvices And Molecular Mechanism Of Bacillus Coagulans 2-6

Creatures must survive and thrive under a variety of stress conditions.Among them,the change of temperature has the most direct effect on the organism.One of the most amazing aspects of the heat shock response is that it is triggered by a temperature increase of just a few degrees.A small increase in temperature could lead to protein depolymerization,entanglement and non-specific aggregation.Many morphological and phenotypic effects of heat stress could be explained by protein aggregation and imbalance of proteostatsis.For heat-resistance genetic devices,thermophilic microbial resources have great potential to be explored and discovered as a treasure trove.This study is based on the multi-omics data of Bacillus coagulans 2-6 which is isolated from the soil sample.We focused on the difference gene expression analysis between at 37 ? and 60 ?,than established a database related to temperature changes for screening genetic devices with potential function of heat-stress tolerance.In recent years,studies on the molecular chaperone function represented by heat shock protein and cold shock protein have revealed the response process of cells to external environmental stimuli at the molecular level.Heat shock proteins usually bind to the polypeptide chains,assist in protein assembly,and stabilize the spatial conformation to ensure that the protein performs normal physiological functions.Unlike heat shock proteins,CspA,a member of the cold shock protein family,was first discovered in the low-temperature response process of Escherichia coli.Numerous studies have shown that CspA is actually RNA chaperone whose main function is to stabilize RNA secondary structure and regulate gene expression during temperature downshift.Although this phenomenon has been discovered for a long time,related research and background knowledge are limited to the low temperature response level.In this study,we screened the corresponding high temperature response gene cspL,a new cold shock gene,in B.coagulans 2-6 based on multi-omics database.Unlike previous cognition,cspL significantly increased both temperature tolerance and biomass in the constructed validation strain E.coli DH5?.To further verify the universality of cspL function,we transferred the gene into eukaryotic yeast cells and Pseudomonas putida for heterologous expression.It was found that cspL can increase temperature tolerance as well as biomass accumulation in two different strains.The low-temperature response mechanism of cold shock proteins has been basically cleared,but studies on high-temperature response have rarely been reported.We expressed and purified the CspL protein and confirmed that CspL can bind RNA and single-stranded DNA fragments longer than 4 nt,and can bind to neither double-stranded DNA nor protein by using biolayer interferometry(BLI).Furthermore,we used E.coli DH5? strain as the host cell and heterologously expressed CspL.Then we used RNA-seq to reveal the effect of CspL on the transcription level of E.coli,more than 27% of genes in E.coli were significantly increased;the function of CspL was studied by RIP-seq.The result showed that 597 mRNA targets can be captured by CspL in vivo.Combining the two analysises,we found that the CspL has a global impact on the strain,and COG clustering showed that the CspL target genes were distributed in categories of ribosomal genes,protein transport,and RNA metabolism.Accordingly,cspL was transferred to the industrial microorganisms Bacillus licheniformis,Actinosynnema pretiosum and Micromonospora echinospora,respectively and the results showed that the yields of those three target compounds were significantly increased.In summary,we systematically analyzed the multi-omics database of Bacillus coagulans 2-6.Through screening and verification,it is proved that the novel cold shock protein CspL maintain the cell homeostasis under normal and even higher temperature conditions.Meanwhile,as an RNA molecular chaperone,CspL can play an "enhancer" role in genetic engineering for increasing the yield of specific compounds.We envision that once the CspL has been used in more industrial microorganism sucessfully,it will push the development of application of genetic principles and techniques to reform some special productive strains;subsequently,the development of corresponding engineering technology and industrial production may become possible.