Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

Abstract

Silk-elastin-like proteins (SELPs) are a family of genetically engineered recombinant protein polymers exhibiting mechanical and biological properties suited for a wide range of applications in the biomedicine and materials fields. They are being explored as the next generation of biomaterials but low productivities and use of antibiotics during production undermine their economic viability and safety. We have developed an industrially relevant, scalable, fed-batch process for the high level production of a novel SELP in E. coli in which the commonly used antibiotic selection marker of the expression vector is exchanged for a post segregational suicide system, the separate-component-stabilisation system (SCS). SCS significantly augments SELP productivity but also enhances the product safety profile and reduces process costs by eliminating the use of antibiotics. Plasmid content increased following induction but no significant differences in plasmid levels were discerned when using SCS or the antibiotic selection markers under the controlled fed-batch conditions employed. It is suggested that the absence of competing plasmid-free cells improves host cell viability and enables increased productivity with SCS. With the process developed, 12.8 g L−1 purified SELP was obtained, this is the highest SELP productivity reported to date and clearly demonstrates the commercial viability of these promising polymers.

Development and in silico evaluation of an expression platform based on E.coli for the production of a recombinant beta-glucosidase.

“…Values of total soluble proteins (obtained by the Bradford method), OD600 and dry cell weight for the same samples are gathered in Table 1212 as well, revealing that the SE1/BGL system produced a higher activity than the BL21/BGL system, in spite of similar levels of dry cell weight and total soluble proteins. These results concur with those of (189,264,266), who also observed higher expression levels for the SE1 toxin-antitoxin system than for the BL21 + antibiotic system. CodonPlus strain with a pET21a plasmid carrying the same BGL gene.…”

“…Successful applications of TA systems include the production of considerable levels of human interferon-β protein (188), silk-elastin-like protein (189) and BGL (190) in ccdA/ccdB systems based on E. coli, reaching 1.8 g/L in the case of silk-elastin-like protein and 1.0 g/L of protein in the case of BGL. Likewise, miniantibodies (191), astaxanthin (192) and poly-3-hydroxybutyrate (PHB) (193) were produced in E. coli with the hok/sok system; in the first case, a high titer of functional miniantibodies was obtained (3.3 g/L); in the second case, a level similar to that of the antibiotic system was achieved (385 mg/L); and in the last case, a very high level of PHB was attained (81 g/L).…”

“…Nonetheless, plasmid stability is still very high for E. coli BL21 in the beginning of the assay -of 97% for 12 h, 24 h and 36 h -and decreases moderately to 92% at t = 60 h. These results corroborate other works in the literature. (189,264,266), which found that the SE1+pStaby system preserves plasmid stability at approximately 100%, as opposed to the BL21(DE3) + antibiotic system, which suffers from plasmid loss over time. Moreover, they validate the assumption that the SE1 strain would be ideal for the proposed inoculum production scheme.…”

“…Although no direct comparison of the two strains grown on defined medium was found in the literature other than the aforementioned ref (264),. the few works that do compare SE1 and BL21(189,266) have not found significant differences between the strains in terms of viable cell count or dry cell weight. Provided that there are no modifications in the SE1 strain undisclosed by its manufacturer, we can only assume that the addition of antibiotics (ampicillin) to the BL21/BGL cells is the reason why they attain a lower value of maximum OD600, possibly by bringing on a heavier metabolic burden to the cell than the ccdA/ccdB…”

Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces

“…Using this system, stable and high proteins yields, without the addition of antibiotics into the production process, was achieved [ 22 ]. This SCS strategy has also been shown to be effective in E. coli [ 23 – 26 ].…”