Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli ‘leaky mutant’

• Streptavidin (SAV) production by an E. coli ‘leaky mutant’ was established.
• Product secretion was improved by cultivation at 30 °C and Triton® supplementation.
• bglA leader, constitutive bla promoter and full-length SAV gene were beneficial.
• A bioreactor fed-batch process led to 2.6 μM of tetrameric SAV in 40 h (65.2 nM/h).
• Less than 10% of biotin-blocked binding sites indicate a highly bioactive product.

Due to its various applications the protein streptavidin is a highly interesting target for heterologous production. This study focuses on different Escherichia coli-based constructs targeting a high-level expression and secretion of streptavidin to the medium. The effect of various promoters, variants of the target gene, leader sequences and host strains on expression and secretion into the culture broth was analyzed. Constitutive production of full-length streptavidin fused with the leader sequence of the bglA gene from Bacillus amyloliquefaciens by the periplasmic ‘leaky mutant’ E. coli JW1667-5 (Δlpp-752:kan) at 30 °C generated the highest yield of the conditions tested, surpassing the extracellular concentration of a conventional T7-based expression system. Supplementation of the medium by the non-ionic surfactants Triton® X-100 and X-45 led to an improved secretion of the protein to the culture supernatant. Tetrameric concentrations of streptavidin of 2790 ± 166 nM were reached in shake flasks at a productivity of 49.6 nM h−1. Optimization of conditions led to a successful transfer to the bioreactor, yielding a maximal concentration of 2608 ± 169 nM and a productivity of 65.2 nM h−1 in fed-batch operation. The proportion of biotin-blocked binding sites of 8.3 ± 4.3% indicated a highly bioactive product.