Establishment Of Efficient Expression Systems Of Recombinant Proteins In Corynebacterium Glutamicum

The existing recombinant protein expression systems are more or less deficient,which limits their application in the industrial production of recombinant protein.Corynebacterium glutamicum is an excellent host strain suitable for foreign protein expression.At present,the expression vectors use antibiotic-resistance as selection marker C.glutamicum,and most of them used in the production of recombinant protein of C.glutamicum are inducible expression vectors with TPTG as inducer,while IPTG is expensive and has certain toxicity.The expression vectors using IPTG as inducer have great limitations in the expression of recombinant protein,which limits the expression of recombinant protein related to food industry.Secretory production of recombinant proteins can simplify purification steps,decrease production cost.Therefore,it is very necessary to develop new and efficient expression systems of recombinant C.glutamicum protein suitable for industrial production.Besides an genetically modified host strains,an efficient expression systems requires an efficient expression vector,and an ideal inducible expression vector needs to use a "strong promoter-operator" heterozygous transcription regulatory region composed of strong promoter and negative regulatory system operator sequences.In this study,CRISPR-Cpfl gene editing technology was used to modify C.glutamicum,and construct a series of new high-efficiency secretory expression vectors in C.glutamicum,which together constitute high-efficiency secretory expression systems of recombinant protein in C.glutamicum.The practicability of the C.glutamicum high-efficiency secretory expression systems was verified by using amyF gene derived from G.stearothermophilus as reporter gene.The main results of this study are as follows:（1）A resistance marker intermediate vector pAU29K containing T7 RNA polymerase gene T7gene1 was constructed by using the basic shuttle vector pAU2 as the starting plasmid;（2）On the basis of pAU29K,a novel high-efficiency secretory constitutive expression vector pAU29KS carrying the synthetic clone/expression cassette S-Boxl containing tacM-T7 double promoter and Sac-type strong signal peptide cgR₂070 and a novel high-efficiency secretory constitutive expression vector pAU29KT carrying the synthetic clone/expression cassette T-Boxl containing tacM-T7 double promoter and Tat-type strong signal peptide cgR₀904 were constructed.Using amyF gene encoding amylase from Bacillus stearothermophilus as reporter gene,the feasibility of double transcription system based on tacM promoter-C.glutamicum RNA polymerase and T7 promoter-T7RNA polymerase interaction,Sac strong signal peptide cgR₂070 and Tat strong signal peptide cgR₀904 in C.glutamicum were proved;（3）Based on CRISPR-Cpfl gene editing technology,a mutant strain C.glutamicum Δalr∷araE was constructed.Alanine racemase gene alr was deleted and arabinose permease gene araE from Bacillus subtilis was inserted at the same time in C.glutamicum Δalr∷araE chromosome.It was found that there are certain limitations in the C.glutamicum transformants selection by using a vector with complementary alr screening markers.Furthermore,the glutamate racemase gene murI was deleted and a double deletion mutant strain C.glutamicum Δalr∷araE ΔmurI was constructed.The deletion of the murI gene optimized the screening of C.glutamicum transformants harbouring complementary alr screening marker vectors.（4）The alr complementary selection marker intermediate vector pAU29 containing T7 RNA polymerase gene T7 gene 1 was constructed by using the basic shuttle vector pAU28 as the starting plasmid,and then intermediate vector pAU30 containing the repressor protein AraR-encoding gene araR of L-arabinose operon negative regulation system from Bacillus subtilis was constructed;（5）On the basis of pAU29,a new type complementary selection marker and highly secretory constitutive expression vector pAU29CS carrying the synthetic clone/expression cassette S-Boxl was constructed;Another new type complementary selection marker and highly secretory constitutive expression vector pAU29CT carrying the synthetic clone/expression cassette T-Boxl was constructed;The amyF gene was used as reporter gene,which proved the practicability of the high-efficiency secretory constitutive expression systems in C.glutamicum（6）Based on pAU30,a novel alr complementary selection marker and highly secretory inducible expression vector pAU30S carrying the synthetic clone/expression cassette S-Box2 was constructed.S-Box2 sequence contains tacM-T7 double promoter,Sac-type strong signal peptide cgR₂070 and araO operator derived from arabinose operon negative regulation system of B.subtilis;another novel alr complementary selection marker complementary and highly secretory inducible expression vector pAU30T carrying the synthetic clone/expression cassette S-Box2 was constructed.T-Box2 sequence contains tacM-T7 double promoter,Tat-type strong signal peptide cgR₀904 and araO operator;The amyF gene was used as reporter gene,which proved the practicability of the C.glutamicum high-efficiency secretory induced expression systems.