GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris.

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin-auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol-based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol-based processes lead to large proportions of biotin-blocked binding sites of streptavidin due to biotin-supplemented media. Targeting these problems, this paper provides strategies for the methanol-free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin-blocked streptavidin. The optimized, easily scalable fed-batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin-free streptavidin and a productivity of 57.8 nM h(-1) based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un-optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (µ ≈ D < 0.01 h(-1) ), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855-864, 2016.

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

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±166nM were reached in shake flasks at a productivity of 49.6nMh(-1). Optimization of conditions led to a successful transfer to the bioreactor, yielding a maximal concentration of 2608±169nM and a productivity of 65.2nMh(-1) in fed-batch operation. The proportion of biotin-blocked binding sites of 8.3±4.3% indicated a highly bioactive product.

Development of fed-batch strategies for the production of streptavidin by Streptomyces avidinii based on power input and oxygen supply studies.

Streptavidin is a tetrameric protein with an extremely high affinity to biotin and different biotin-like peptide-tags. This characteristic causes its widespread use in biotechnology. Streptavidin is produced by the fermentation of wild type Streptomyces avidinii or by recombinant Streptomyces lavendulae, Escherichia coli, and Bacillus subtilis strains. However, little is known about the influence of power input and oxygen supply as well as feeding strategies on the production of streptavidin by S. avidinii. This paper provides a systematic analysis of the effect of rotary frequency of the stirrer, leading to a plateau-like streptavidin formation behaviour between 400 and 700 min(-1). This plateau was characterized by specific power inputs between 79 and 107 W L(-1) and corresponding maximal product concentrations of 6.90 µM in 6 days. Lower as well as higher rotary frequencies were not beneficial. Subsequently, a linear fed-batch procedure could be established reproducibly yielding 39.20 µM streptavidin in 14 days, characterized by a constant productivity of 114 nM h(-1). Fed-batch procedures based on dissolved oxygen were less efficient. The linear feeding strategy presented in this paper led to the highest streptavidin concentration ever reported and exceeded the maximal product level given in the literature drastically by a factor of 8.5.