Optimized Expression Strains

Deletion of the DE3 prophage from E. coli expression strain BL21(DE3) and pgl gene knock-in

The popular T7 expression strain BL21(DE3) carries an inducible copy of the T7 RNA polymerase in a lysogenized DE3 prophage. While this allows for stable expression of the T7 RNA polymerase, the DE3 prophage remains functional and may spontaneously be induced to enter a lytic lifecycle.

To address this problem, 89% of the DE3 prophage sequence was removed from the genome of the E. coli expression strain BL21(DE3). The optimized strain T7E1 shows no phage activity yet maintains the consistent bacterial growth and heterologous protein expression productivity of its parent strain, BL21(DE3).

E. coli B strains are pgl-deficient, a mutation that can be traced back to a UV-induced deletion of the galM-ybhJ locus found in the parental strain E. coli B707. The pgl gene encodes the enzyme 6-phosphogluconolactonase (PGL), which catalyzes hydrolysis of 6-phosphogluconolactone to 6-phosphogluconate. Loss of PGL enzymatic activity negatively impacts E. coli metabolism and leads to non-specific protein gluconoylation. In the absence of PGL, its electrophilic substrate (6-phosphogluconolactone) accumulates, which leads to random protein gluconoylation. In wildtype BL21(DE3), 6.4% of recombinantly expressed protein was shown to have undesired gluconoylation. Thus, the pgl gene in T7E1 (see above) was repaired, creating the strain T7E2. In the optimized strain T7E2, recombinantly expression protein showed no undesired gluconoylation¹.

¹ Noll S, Reyelt J, Rysiok T, Kellner R, Güssow D, Jäkel S, Hager S and Kranz H, 2013, Gezielte Optimierung von Escherichia coli BL21(DE3), Biospektrum, 19, 211