High-level secretory production of recombinant single-chain variable fragment (scFv) in Corynebacterium glutamicum.

We describe the development of a new secretory production system for the enhanced production of a single-chain variable fragment (scFv) against the anthrax toxin in Corynebacterium glutamicum. For efficient secretory production of the antibody fragment, the following components were examined: (1) signal peptides, (2) codon usage of antibody fragment, (3) promoters, (4) 5' untranslated region (5' UTR) sequence, and (5) transcriptional terminator. Among all the systems examined, the use of a codon-optimized gene sequence, a Sec-dependent PorB signal peptide, and a fully synthetic H36 promoter, allowed the highest production of antibody fragments in a culture medium. For large-scale production, fed-batch cultivations were also conducted in a 5-L lab-scale bioreactor. When cells were cultivated in semi-defined media, cells could grow up to an OD600 of 179 for 32 h and an antibody fragment concentration as high as 68 mg/L could be obtained in a culture medium with high purity. From the culture medium, the secreted antibody was successfully purified using a simple purification procedure, with correct binding activity confirmed by enzyme-linked immunosorbent assay. To the best of our knowledge, this is the first report of a fed-batch cultivation for antibody fragment production in C. glutamicum.

Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial organism that is widely used in the production of amino acids, nucleotides and vitamins. To extend its product spectrum and improve productivity, C. glutamicum needs to undergo further engineering, including the development of applicable promoter system. Here, we isolated new promoters from the fully synthetic promoter library consisting of 70-bp random sequences in C. glutamicum. Using green fluorescent protein (GFP) as a reporter, highly fluorescent cells were screened from the library by fluorescent activated cell sorting (FACS). Twenty potential promoters of various strengths were isolated and characterized through extensive analysis of DNA sequences and mRNA transcripts. Among 20 promoters, 6 promoters which have different strengths were selected and their activities were successfully demonstrated using two model proteins (antibody fragment and endoxylanase). Finally, the strongest promoter (P(H36)) was employed for the secretory production of endoxylanase in fed-batch cultivation, achieving production levels of 746 mg/L in culture supernatant. This is the first report of synthetic promoters constructed in C. glutamicum, and our screening strategy together with the use of synthetic promoters of various strengths will contribute to the future engineering of C. glutamicum.

Isolation of a potential anchoring motif based on proteome analysis of Escherichia coli and its use for cell surface display.

For bacterial cell surface display, the target protein needs to be linked to an anchoring motif, and it is essential to choose an appropriate anchoring motif for efficient and stable display of the protein on the cell surface. To isolate a potential anchoring motif that would allow a stable and enhanced display of target proteins on the surface of an Escherichia coli host, we analyzed the outer membrane proteome of E. coli. On the basis of this proteomic analysis, the outer membrane protein X (OmpX), which has a small, monomeric ß-barrel structure and is highly expressed, was selected as a potential anchoring motif. The role of OmpX as an anchoring motif for cell surface display was demonstrated using three important industrial enzymes: endoxylanase, lipase, and alkaline phosphatase. Two different positions (Lys(122), Val(160)) in the extracellular loops of OmpX were examined for C-terminal fusion, and the biological activities and localization of the displayed enzymes were analyzed. All three enzymes examined were efficiently displayed on the E. coli cell surface with high activity. These results reveal that the use of OmpX as an anchoring motif is an efficient method to display functional enzymes on the surface of an E. coli host.

Development of a secretion system for the production of heterologous proteins in Corynebacterium glutamicum using the Porin B signal peptide.

Corynebacterium glutamicum is one of the useful hosts for the secretory production of heterologous proteins because of intrinsic attributes such as the presence of few endogenous proteins and proteases in culture medium. Here, we report the development of a new secretory system for the production of heterologous proteins by using the porin B (PorB) signal peptide in C. glutamicum. We examined two different endoxylanases and an antibody fragment (scFv) as model proteins for secretory production. In the flask cultivations, all the examined proteins were successfully produced as active forms into the culture medium with high efficiency. For the high-level production of endoxylanase, fed-batch cultivation was also performed in a lab-scale (5L) bioreactor, and the endoxylanases were efficiently secreted in the culture medium at levels as high as 615mg/L. From the culture supernatant, the secreted endoxylanases could be purified with high purity via one-step affinity column chromatography.