Expression of human interleukin-1β in Saccharomyces cerevisiae using PIR4 as fusion partner and production in aerated fed-batch reactor

Abstract

To circumvent cell wall retention commonly associated to Saccharomyces cerevisiae when used as a host for heterologous protein production, we have created a translational fusion of human interleukin-1β (IL-1β) to the Pir4 cell wall protein, so as to drive the secretion of the recombinant product to the growth medium. The auxotrophic S. cerevisiae BY4741 was used as host to express the Pir4-IL1β fusion protein. Once it was ascertained that the fusion protein was secreted to the culture medium and behaved as a growth-linked product, S. cerevisiae BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor to achieve high cell density and, consequently, high product concentration in the medium. Two cultivation media were employed, a rich complex and a defined mineral medium, the latter suitably supplemented with bacto-casamino acids as ACA (auxotrophy-complementing amino acid) source. The rich complex medium allowed a good performance of the producer strain only during batch growth, but was revealed to be inadequate for long-term fed-batch operations. The defined mineral medium ensured a better performance, even though not yet satisfactory in spite of a proper ACA supplementation. The behaviour of BY4741 was attributed to an intrinsic sensitivity of the producer strain to long-term aerated fed-batch operations.

High cell density culture with S. cerevisiae CEN.PK113-5D for IL-1β production: optimization, modeling, and physiological aspects

“…The rationale behind this optimization study, resided on the possibility of reducing the working time by acting on the operative conditions, since other factors of implementation such as selection of suitable host with a high specific growth rate [6], efficient expression system [9], and proper medium composition [14] had already been investigated.…”

“…The fusion had been obtained subcloning the amplified IL-1b coding sequence in the BglII-SalI sites of PIR4 [9], with the loss of the carboxyterminal fragment of subunit II of Pir4 that contains three of the four highly conserved cysteine residues that are responsible for cell wall retention of Pir4 through disulphide bridges [8].…”

“…In the present work, to discriminate the effects given by a heterologous metabolic burden over those related to the cultural conditions, we have compared the growth parameters of CEN.PK113-5D expressing an heterologous product against those of the non-transformed strain. The recombinant strain carries, on the episomal pIA1 derivative [7], the coding sequence for human interleukin-1b (IL-1b) gene fused with the regulatory sequences of PIR4 gene [8] that promotes IL-1b secretion to the growth medium [9].…”