Heterologous Protein Expression System Of Corynebacterium Glutamicum Using 3A Assembly Technology

Corynebactrium glutamicum(C.glutamicum),a gram-positive bacterium with high GC content,is a potential host for the production of recombinant proteins due to its excellent characteristics,including not producing endotoxins,lower activity of protease in the culture supernatant and powerful protein secretion system.To date,a few research process has been developed and used for the production of recombinant proteins in C.glutamicum.However,there are some problems,such as limited available expression elements,few high-efficiency chassis cells,and no standardized element library,compared with the classical recombinant protein expression system Escherichia coli(E.coli)and Bacillus subtilis(B.subtilis).The proposal of BioBrick-based biological element library has greatly improved the redesign and research of life system by increasing the efficiency of engineering construction.But it’s not suitable for C.glutamicum.To alleviate this problem,it is necessary to establish a standardized element library to fulfill the production of recombinant proteins.And the yield of Rhv3(hirudin variant III)was improved by host modification and the multi copy gene strategy based on the standardized element libraries in C.glutamicum.In this study,the 3A(Three antibiotics)assembly system was developed based on C.glutamicum CGMCC1.15647 as host,and the strandard element library of promoters and signal peptides was established to express the recombinant protein efficiently.By analyzing the genome sequence of C.glutamicum ATCC13032,molecular chaperone system was identified and developed,and with the help of this system,the expression of Single chain variable(Scfv),Human interferon(Hifn)and Rhv3,was improved.Finally,the strategy of multi-copy of genes based on 3A assembly and response surface design was used to optimize the production of recombinant proteins.The major conclusions are as follows:(1)The 3A assembly system of efficient and fast was established to facilitate the rapid assembly of plasmids.The four standardized plasmids with different resistance genes were constructed by modifying the p19-0 plasmid.Through the transcriptome,proteome,and references reported,different strength of promotes and different secretory pathway of signal peptides were screened and inserted into the standardized plasmid to form a BioBrick library of promoters and signal peptides.The different promoters were assembled with enhanced green fluorescent protein gene(e GFP),respectively,using the 3A assembly technology.Three strong promoters P2252,Podhi and Pywe A were screened by analyzed of promoter intensity.Meanwhile,the secretory expression cassette of recombinant hirudin variant III(Rhv3)controlled by Ptac was constructed.Results showed that all 5 signal peptides could mediate the secretion expression of Rhv3.Finally,the combinatorial optimization of promoters and signal peptides was used to optimize the secretion expression of Rhv3,and the highest Rhv3reached(1.21±0.09)g/L in the shake flask,which was 1.82-fold higher than strain harboring the plasmid p3A-Ptac-CspA-Rhv3((0.66±0.02)g/L).Our research provides a powerful tool for gene manipulation of recombinant protein in C.glutamicum.(2)The recombinant protein-assisted chaperone system was developed and applied to optimize recombinant protein synthesis.To further improve the production of recombinant protein,seven molecular chaperone genes were identified from the genome sequence of C.glutamicum ATCC13032 based on NCBI database.Four chaperone plasmids were constructed to improve the expression of recombinant protein,and applied for the expression of Scfv.The soluble fraction and total amount of Scfv level was increased by co-expression of molecular chaperone.Furthermore,about(70.5±3.56)mg soluble Scfv was purified from 1 L culture,which is higher than 12.5 mg reported previously.Then,Hifn and Rhv3 were used as model proteins to confirm that molecular chaperones could improve the expression of recombinant protein,and results showed that different molecular chaperones had different effects on the synthesis of recombinant protein.The Rhv3 activity after co-expression DJ2 EK was 1.87 folds higher than without chaperone.The establishment of a chaperone-assisted protein expression system and the successful expression of Scfv,Hifn,and Rhv3 has an important reference significance of C.glutamicum as a platform cell of recombinant protein expression.(3)The synthesis of recombinant protein was improved by multi-copy gene strategy in C.glutamicum.Universal ribosome binding site(RBS)primers were developed based on the specific sequences of BioBrick,and the strongest RBS sequence was screened using egfp as the reporter gene.To improve the secretory production of Rhv3,we constructed the Rhv3 expression plasmids with different signal peptides and copy numbers.Results showed that the highest yield of Rhv3 reached(1.57±0.06)g/L when the copy number is 6,which was2.41-fold greater than single copy of Rhv3.The production level of Rhv3 in a flask level achieved(1.89±0.13)g/L after culture medium optimization,which was 1.2-fold higher than pre-optimization.The method used here has an important significance on further promoting the clinical application of Rhv3 and the research of recombinant protein production using C.glutamicum.