Outer membrane lipoprotein A (OmlA) is a vaccine antigen against porcine contagious pleuropneumonia (PCP), a disease severely affecting the swine industry. Here, we aimed to systematically potentiate the secretory production of OmlA in Corynebacterium glutamicum (C. glutamicum), a widely used microorganism in the food industry, by establishing a holistic development process based on our high-throughput culture platform. The expression patterns, expression element combinations, medium composition, and induction conditions were comprehensively screened or optimized in microwell plates (MWPs), followed by fermentation parameter optimization in a 4×1 L parallel fermentation system (CUBER4). An unprecedented yield of 1.01 g/L OmlA was ultimately achieved in a 5-L bioreactor following the scaling-up strategy of fixed oxygen mass transfer coefficient (kLa), and the produced OmlA antigen showed well-protective immunity against Actinobacillus pleuropneumoniae challenge. This result provides a rapid and reliable pipeline to achieve the hyper-production of OmlA, and possibly other recombinant vaccines, in C. glutamicum.
Background: Corynebacterium glutamicum is a traditional food-grade industrial microorganism in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantages of low recombinant protein expression levels need to be solved. Here, according to the bacteria-specific polycistronic feature, trials have been made of inserting a leading peptide upstream of target genes as an expression enhancer, and we found it improving the expression level of proteins under the control of inducible tac promoter in C. glutamicum CGMCC1.15647 .Results: In this research, the Escherichia coli ( E. coli ) tac promoter combined with 24 different forecistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic vectors were isolated and exhibited higher strength under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins-aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-a (BoIFN-a), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type I N-terminal peptide (PINP). All examined proteins were highly expressed compared with the original vector of tac promoter. Large-scale production of PINP was also performed in fed-batch cultivation, and the highest PINP production level was 1.2 g/L.Conclusions: In this study, we improved the strength of the inducible promoter tac promoter for C. glutamicum by screening and inserting fore-cistron in front of the target genes. Those vectors with bicistronic expression pattern have strong compatibility for expressing various heterogeneous proteins in high level. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.
Background: Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. Results: In this research, the Escherichia coli ( E. coli ) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-a (BoIFN-a), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type I N-terminal peptide (PINP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PINP was also performed in fed-batch cultivation, and the highest PINP production level was 1.2 g/L. Conclusion: In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.
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