Establish A Gene Knock Down Tools Based On CRISPR-Cas System In Hyperthermophilic Archaea Pyrococcus Yayanosii And The Study Of Prolidase Enzyme Properties

Pyrococcus yayanosii A1,which grows anaerobically at 75–103°C(optimum 98°C)and at pressures of 0.1–120 MPa(optimum 52 MPa),was a laboratory mutant strain used in the studies of environmental adaptation mechanisms in a group of piezophilic hyperthermophilic archaea in Thermococcaceae.The multi-omics analyses of P.yayanosii showed the overexpression in translation,chemotaxis,and CRISPR systems,while the repression in energy metabolism(hydrogenases,sulfhydrogenase,formate metabolism)in unoptimun growth condditon.The genetic analysis of the essential genes in these pathways through gene expression knockdown tools is of great significance in exploring the stress adaptation mechanism of the hyperthermophilic piezoarchaea P.yayanosii A1.In this study,a new genetic tool was established for P.yayanosii A1,which could be used to knock down gene expression based on an endogenous active type III-B CRISPR-Cas system.An artificial mini-CRISPR cluster was inserted into the shuttle vector p LMOShhp,consisted of the repeat sequences from endogenous CRISPR arrays Group I and a protospacer from a putative amylase gene(PYCH?13690).The plasmid with simvastatin resistance transcribed the cr RNA matched the m RNA of target gene which guide the CRISPR ribonucleoprotein(cr RNP)complex to cleave it.The ratio of m RNA cleavage of a high hydrostatic pressure inducible promoter P_hhp was 33%at 52 MPa and 50%at 0.1 MPa,while the cleavage of constitutive promoter P_hmtb was 42%at 52 MPa and 76%at 0.1MPa.As a proof,the gene knockdown mutant degraded less starch than its parental strain A1 in vivo.Thus,a gene knockdown system based on type III-B CRISPR-Cas system has been successfully constructed in P.yayanosii,which could be used in the genetic studies targeting essential genes in piezophilic hyperthermophiles.Prolidase isolated from hyperthermophilic archaea has the ability to degrade the X-Pro dipeptide,which is thought to participate in proline recycling.The high hydrostatic pressure(HHP)-inducible expression of prolidase hhp C and constitutive prolidase pyy Prol gene were heterologous expression with 6×His tag in E.coli Rosetta.Then use Ni-NTA affinity chromatography column to purify protein.The Hhp C protein was pretreated by anaerobic conditions at 85°C when the cell supernatant was broken,to obtain a single protein with a size of 45 k Da,but it was inactive at 50-99.9°C.pyy Prol protein was added with double 6×His tags at the N-terminus and C-terminus to increase the binding force to obtain a 45 k Da band and a 25 k Da broken band.The optimum temperature for enzyme activity was 99.9°C and the optimum p H was 7.0.And it has high stability of temperature and p H.