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

Abstract

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.

Prospective bacterial and fungal sources of hyaluronic acid: A review

“…Pichia pastoris is a methylotrophic yeast that is in contrast to H. polymorpha not thermotolerant. P. pastoris , as well as S. cerevisiae , is extensively used as an expression system for recombinant protein production [404] , in particular for biosynthesis of intricate bioactive molecules such as parasin I peptide [405] and streptavidin [406] . It was previously reported by Farinha that P. pastoris could be used as a source of valuable polysaccharides, e.g.…”

Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability

“…These drawbacks have prompted the use of methanol-independent promoters such as the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) from central carbon metabolism [29]. Despite the constitutive nature of PGAP, its strong expression capacity depends on the carbon source and growth rate of the particular culture [30,31]. Although the expression of ROL has been already reported under this constitutive promoter, w-proROL-gene, which encodes both the prosequence and the mature sequence, is needed to alleviate the adverse effects of producing the mature sequence of ROL under PGAP [32], as this lipase form is harmful to the host cell [10].…”

Hybrid fed‐batch bioreactor operation design: control of substrate uptake enhances recombinant protein production in high‐cell‐density fermentations

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