Construction Of Acid And Hot-resistant Sulfolobus Islandicus REY15A Chassis Cells Based On CRISPR-Cas Gene Editing And Identification Of Sulfolobus Membrane Anchored Protease Function

Sulfolobus islandicus REY15A is an extremophilic archaeon isolated from an acidic hot spring in Iceland.Its optimal growth temperature is 76℃,the optimal growth pH is 3,the whole genome size is 2.5Mb,and the generation time is 6～7h,which is one of the few archaea with a complete set of genetic tools,This thesis employs the crenarchaeal model to carry out two aspects of research:（a）construction of acidophilic and thermophilic chassis cells using a gene editing method based on endogenous CRISPR-Cas systems,and（b）functional characterization of a protease encoded in this crenarchaeon.First,extremophilic organisms are of especially interests to biomanufacturing since they show strong potential to facilitate the processes of industrial biotechnology most of which are under harsh conditions.The thermoacidophilic Sulfolobales thrive both at high temperatures（75-80℃）and low pH（pH 2-5）,which makes them as good candidates for relevant industrial applications.However,their applications are generally hampered by low rates of cell growth and low yields of biomass.To construct fast-growing chassis cells with Sulfolobus islandicus,a crenarchaeal model,genome comparisons were conducted for S.islandicus REY15A and M.16.4 strain and this revealed 6 nonessential genomic segments（NEGSs）.Attempts were made to delete the identified NEGSs using a classical CRISPR-based genome editing method but failed for most of them.Then,a novel method,named "Revealing and deleting nonessential genomic segments with tandem homologous arms",was developed based on the classical CRISPR method.The application of the innovative genome editing method facilitated the construction of the first-generation chassis cells for the thermoacidophile.Ten genome reduction strains were obtained in the genome reduction work.Among them,the maximum reduction about genome of DN6 strain is nearly 12%.Secondly,a membrane-anchored protease of Sulfolobus islandicus was characterized..The protease is encoded by SiRe₀965.Bioinformatic predictions suggested that the encoded protein could be a membrane-anchored protease with two transmembrane helix regions and this is confirmed by biochemical studies of the protease.Transcriptome data of ΔSiRe₀965 strain and qPCR experimental data showed that flagella-related genes were upregulated after protease deletion.The protease which are anchored on the surface of the cell membrane and degrade small peptides in the environment and then absorbed into the body.In summary,this protease is a nonspecific membrane-anchored protease and may be associated with nutrient uptake of this archaeon.