Development And Application Of Highly Efficient Bicistronic Expression System For The Expression Of High-value Proteins In Corynebacterium Glutamicum

With the rapid growth of the recombinant protein expression market,there is a need for more efficient and safer protein expression hosts to meet market demand.Although Escherichia coli is the earliest and most widely used prokaryotic host,its secretion ability is poor and it has the problem of producing endotoxins as byproducts,which limits its application to some extent.Therefore,the development of emerging protein expression hosts has become an inevitable trend.Corynebacterium glutamicum has gained widespread attention due to its non-endotoxic,generally recognized as safe（GRAS）status,low extracellular protease activity,and strong secretion ability.Over the past decades,researchers have demonstrated the remarkable potential of C.glutamicum in the secretion expression of recombinant proteins,and have developed numerous expression elements and genetic tools.While these advancements have laid a solid foundation for the efficient,low-cost biomanufacturing of recombinant proteins in C.glutamicum,several challenges still need to be addressed to fully realize this goal.For example,many existing expression systems exhibit poor compatibility and stability when facing with different gene sequences.This can cause variation into the recombinant protein expression process,making it difficult to choose the appropriate expression system.The efficiency of the existing expression systems also need to be further improved.Limitations in expression efficiency could lead to inadequate yields in large-scale production,thereby increasing production costs and reducing efficiency.In addition,there is still lack of a universal and systematic development plan for protein production processes.Despite the development of numerous expression elements,ensuring that each step from gene cloning to protein production is fully optimized to achieve the highest production efficiency and product quality remains an unresolved issue.This study aims to address these challenges by developing a proprietary,high-efficiency C.glutamicum protein expression system and systematic process development framework,further enhancing the compatibility of the expression system and achieving ultra-high-level production of exogenous proteins.It is expected to provide a safer,more efficient,cost-effective,and universal solution for the production of recombinant proteins,especially pharmaceutical and food proteins.Moreover,in order to promote the synthetic biology applications and metabolic engineering of C.glutamicum,it is also necessary to develop new tools for gene expression regulation,enabling more precise and reliable control of gene expression.（1）Enhanced P_btac expression systems were constructed based on the fore-cistron sequence screening strategy,and their efficient performance in recombinant protein expression was confirmed at multiple levels including translation efficiency,induction efficiency,and sequence compatibility.The E.coli derived P_tac promoter combined with 24endogenous fore-cistron sequences were constructed in a bicistronic manner in C.glutamicum.Three strong P_btac expression vectors were isolated and demonstrated higher expression effciency under different culture conditions.The good compatibility of these bicistronic vectors was further validated using six model proteins-aldehyde dehydrogenase（ALDH）,alcohol dehydrogenase（ADH）,Ram A（regulator of acetate metabolism）,bovine interferon-α（Bo IFN-α）,glycoprotein D protein（g D）of infectious bovine rhinotracheitis virus（IBRV）and procollagen typeΙN-terminal peptide（PΙNP）.All examined proteins were highly expressed compared with the original vector with a monocistronic tac promoter.The strongest vector,pbtac-HT-11,was utilized for the large-scale production of PINP,achieving the highest yield of 1.2 g/L in a 5 L bioreactor under fed-batch cultivation.These results demonstrate that the established P_btac expression system can better meet the high-level gene expression demand,especially for efficient production of recombinant proteins,in C.glutamicum.（2）Leveraging the P_btac expression system and the previously developed high-throughput cultivation platform,we established a holistic process development framework from gene cloning to protein production in C.glutamicum.This framework has effectively enhanced the secretion of outer membrane lipoprotein A（Oml A）,a vaccine antigen against porcine contagious pleuropneumonia（PCP）.The expression patterns,expression element combinations,medium composition,and induction conditions were comprehensively screened or optimized in microwell plates（MWPs）using the design of experiment（Do E）strategy,followed by fermentation parameter optimization in a 4×1 L parallel fermentation system（CUBER4）.Oml A yield gradually increased.An unprecedented yield of 1.01 g/L Oml A was ultimately achieved in a 5-L bioreactor following the scaling-up strategy of fixed oxygen mass transfer coefficient（k _La）,and the produced Oml A antigen showed well-protective immunity against Actinobacillus pleuropneumoniae challenge.The process development framework established in this study significantly increased the yield of Oml A,and the cost of Oml A production medium was reduced from 0.019 USD/mg to 0.006 USD/mg.This chapter provides a rapid and reliable pipeline for the hyper-production of Oml A,and possibly other recombinant vaccines,in C.glutamicum.（3）The application of the bicistronic strategy was expanded to leaderless promoters lacking a 5’UTR.This design enhances the orthogonality of the expression elements and provides a new tool for the precise and reliable contro of gene expression in C.glutamicum.The bicistronic strategy was first applied to exogenous(P_H36 and P_H30)and endogenous(P_cg0124)promoters to verify its applicability in leaderless promoters.Three protein models-green fluorescent protein（GFP）,variable domain of heavy-chain antibody（VHH）,and beta-galactosidase（Lac Z）,were then utilized to demonstrated the excellent stability of leaderless BCD toward different target gene sequences.Compared to traditional BCD expression cassettes constructed based on promoters with a 5’UTR(P_tac,P_aph,and P_tuf),the genetic elements in the leaderless BCD exhibited significantly improved orthogonality across different genes of interest.Mutations in the fore-cistron start codon and Shine-Dalgarno（SD）motif resulted in a reduction in the expression levels of the leaderless BCD,highlighting the importance of the fore-cistron and SD motif in maintaining the strength of leaderless BCDs.Additionally,we established a library containing 55,901 fore-cistrons and demonstrated that the regulatory range of gene expression in leaderless BCDs can be broader by modifying the fore-cistron sequence.High,medium,and low fluorescence intensity sub-libraries were then obtained through flow sorting,and the key features of the fore-cistron sequences required for different gene expression levels were identified through next generation sequencing（NGS）.This chapter provides a novel synthetic biology tool based on leaderless BCD for fine-tuning gene expression in C.glutamicum using fore-cistrons.Moreover,the strategy developed here can also be applied to improve the performance of other leaderless promoters in other bacteria.