Construction of Corynebacterium glutamicum for efficient secretion and production of recombinant proteins using the technology of ARTP mutagenesis and fluorescen activated droplet microfluidic screening

In order to increase the secretion and expression of Corynebacterium glutamicum (C. glutamicum) as microbial cell factory, Actinobacillus porcine pleuropneumoniae outer membrane protein 7 (Omp7) was used as the target protein and a total of four mutant strains with efficient secretion and expression including CT1-109, CT2-9, CT3-28 and CT4-42 were obtained by the four rounds of Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and fluorescence-activated droplet sorting (FADS) technology. The mutant strain CT3-28 was verificated and showed the highest fluorescence value after mutagenesis saturation. Four recombinant proteins including Actinobacillus pleuropneumonia RTX, procollagen type I N-terminal peptide, hirudin variants III and variable domain of heavy chin of heavy-chin antibody, were expressed in CT1, CT2 and CT3 strains, respectively. The results showed that the mutant strains were able to increase the secreted expression of other recombinant proteins. The amplified fermentation experiment of the mutant strain CT3-28 was performed by using a 5 L fermenter with fed-batch, and the yield of Omp7 was about 211.3 mg·L-1, which was 82.6% higher than that of WT. A total of 91 single-nucleotide polymorphisms (SNPs) were identified by resequencing the genomes of C. glutamicum CGMCC1.15647 and the mutagenized strains CT1, CT2, and CT3, of which the optimal strain CT3 included four related genes. Further overexpression and knockout of the SNP genes indicated that the genes RS02840 and RS02495 had a significant effect on the secretory expression of recombinant proteins, and provides target genes and strategies for modifying chassis cells to increase secretion expression in subsequent studies.

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In order to increase the secretion and expression of Corynebacterium glutamicum (C.

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