PUB - Publikationen an der Universität Bielefeld

The biotin-binding protein streptavidin (SAV) is applied in a large variety of methods due to its extremely high affinity to the vitamin biotin, ranging from protein purification and use as a bioinsecticide to tumor staining. However, common processes for the production and purification of the protein show a diverse range of serious deficiencies like low productivities and concentrations of product, the application of the toxic and flammable organic solvent methanol in heterologous production, and labile, expensive gel materials used for affinity chromatography of SAV. Hence, this project focused on the development of advanced strategies for the production and purification of the protein, targeting an economical and sustainable supply of SAV for common applications.

Optimization of process conditions for the natural producer Streptomyces avidinii led to a highly ecological bioreactor fed-batch process based on the constant feeding of glucose. Reproducibly yielding 39.2 µM of SAV in 14 days (114 nM/h), this strategy surpassed previously reported concentrations for this host by the factor 12.7. Continuous cultivation indicated that even higher productivities can be achieved at dilution rates in the range of 0.2 1/h. A systematic variation of the rotary frequency of the stirrer revealed shear-sensitive properties of the streptomycete. Moreover, shake flask studies led to a selection of efficient strategies for the control of morphology.

Heterologous expression of the SAV gene was analyzed applying three hosts: the Gram-negative bacterium Escherichia coli and the yeasts Pichia pastoris and Hansenula polymorpha.

For E. coli, broad optimization of process conditions allowed a more holistic view of the production of SAV by this biotechnological model bacterium. Application of the periplasmic ’leaky mutant’ JW1667-5 (Δlpp-752::kan), the constitutive β-lactamase promoter from E. coli, the bglA-leader peptide from Bacillus amyloliquefaciens, and addition of the non-ionic surfactant Triton led to the secretion of more than 90 % of SAV to the medium. Bioreactor fed-batch fermentation at 30 ° C resulted in 2.6 ±0.2 µM of highly bioactive SAV in 40 h (65.2 nM/h), exceeding all reference concentrations for the soluble, secretory production of full-length SAV by E. coli.

For P. pastoris, a new strategy for the methanol-free production of a shortened form of SAV based on the constitutive GAP promoter was developed. This study demonstrates for the first time that SAV can be produced in a growth-associated manner by the biotin auxotrophic yeast, obviating the need for an induction by methanol. Productivity was greatly enhanced by successive cooling and acidification throughout the cultivation. Model-based evaluation of the optimized conditions in the bioreactor revealed that the majority of product accumulated in the late phase of fermentation at diminishing growth rates. This is atypical for GAP promoter-based production processes, since the activity of PGAP is usually known to be positively correlated to the growth rate of the host. The final fed-batch process led to 4.2±0.1 µM of SAV in 72 h (57.8 nM/h). Compared to literature, the proportion of biotin-blocked binding sites Qblocked was lowered from 20 % to 0±2 %.

The yeast H. polymorpha has not been used for the production of SAV prior to this project. Establishing this host for the production of SAV included genetic aspects, process development, and up-scaling to the bioreactor. Like observed for E. coli, cultivation at 30 instead of 37 ° C turned out to be beneficial, resembling typical conditions for the natural producer S. avidinii. Application of the FMD promoter and a full-length SAV gene allowed the accumulation of SAV in the absence of methanol (’derepression’). However, production was enhanced upon induction by the organic solvent. A three-stage process, consisting of a batch phase and two phases of DO-stat feeding of glucose and methanol, respectively, yielded 11.4±0.2 µM of SAV in 216 h (52.5 nM/h). These properties resemble reference results reported for the methanol-based expression of a full-length SAV gene using P. pastoris. Remarkably, Qblocked was as low as 1.1±3.8 %.

In addition to these studies, a novel fluorometric assay was developed for the rapid detection of biotin-blocked binding sites of SAV based on the heat-based displacement of biotin from the binding pocket. Model-based evaluation of association and dissociation courses led to conditions allowing the detection of all biotin-blocked binding sites in a sample independent of the degree of biotin saturation. This new strategy facilitates analysis of the bioactivity and host toxicity of SAV during heterologous production. Furthermore, it may simplify the development of methods for the non-denaturing separation of SAV and biotin by revealing changes of the overall concentration of SAV rather than the concentration of biotin-free binding sites.

To demonstrate the latter feature, the assay was used for the development of methods for the partial recovery of biotin-blocked binding sites of SAV by hydrogen peroxide treatment and UV radiation. Incubation in the presence of hydrogen peroxide at elevated temperatures resulted in the recovery of up to 30 % of biotin-blocked binding sites of a completely biotin-saturated sample of SAV without major effects on the overall bioactivity of SAV.

Purification of SAV was studied by aqueous two-phase extraction, thermal inactivation of contaminating proteins, diafiltration, dialysis, and ammonium sulfate precipitation. The efficiency of the methods was assessed by SDS-PAGE analysis and determination of total protein. The results indicate that for many applications the direct use of crude supernatants or SAV purified by these simple and inexpensive procedures may be sufficient, whereas purification by chromatography may be necessary if high product purities are required.