Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Abstract

BackgroundSaccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain.ResultsOur results show that the concentrations of ACA in the feeding solution, corresponding to those routinely used in the literature, are limiting for the growth of S. cerevisiae BY4741 [PIR4-IL1β] in fed-batch reactor. Even in the presence of a proper ACA supplementation, S. cerevisiae BY4741 [PIR4-IL1β] did not achieve a high cell density. The Δyca1 deletion did not have a beneficial effect on the overall performance of the strain, but it had a clear effect on its viability, which was not impaired during fed-batch operations, as shown by the kd value (0.0045 h-1), negligible if compared to that of the parental strain (0.028 h-1). However, independently of their robustness, both the parental and the Δyca1 mutant ceased to grow early during fed-batch runs, both strains using most of the available carbon source for maintenance, rather than for further proliferation. The mathematical model used evidenced that the energy demand for maintenance was even higher in the case of the Δyca1 mutant, accounting for the growth arrest observed despite the fact that cell viability remained comparatively high.ConclusionsThe paper points out the relevance of a proper ACA formulation for the outcome of a fed-batch reactor growth carried out with S. cerevisiae BY4741 [PIR4-IL1β] strain and shows the sensitivity of this commonly used auxotrophic strain to aerated fed-batch operations. A Δyca1 disruption was able to reduce the loss of viability, but not to improve the overall performance of the process. A mathematical model has been developed that is able to describe the behaviour of both the parental and mutant producer strain during fed-batch runs, and evidence the role played by the energy demand for maintenance in the outcome of the process.

Carbon metabolism limits recombinant protein production in Pichia pastoris

“…In another study, the increase in the yield of product was obtained by the addition of auxotrophy‐complementing amino acid to an auxotrophic host, which caused increased strain viability (Paciello et al, 2009). In the present work, glutamine was most effective on productivity and was used as nitrogen and carbon source.…”

An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

“…Cell wall proteins have been used as fusion partners for incorporation of recombinant proteins into the yeast cell wall; however, most of the studies conducted to date have focused on the anchoring domain 14 32 . Two of our selected TFPs containing pre- and pro-peptides ( CIS3 and HSP150 ) have already been reported as fusion partners for the production of xylanase, β-lactamase, rat nerve growth factor receptor, the VP8 rotavirus antigen, lipase, human IL-1β, endoglucanase, glycosyltransferases, rat alpha 2,3-sialyltransferase and laccase 14 33 34 35 36 37 38 39 40 41 . These fusion partners were used to produce target proteins as fusion proteins, while our TFPs were removed at the Golgi complex via an artificially introduced KEX2 cleavage site, resulting intact proteins.…”

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.…”

“…Fed-batch cultures were started by feeding the reactor with an exponentially increasing flow rate after 15 h batch phase, when glucose was exhausted, and before ethanol produced in batch phase was completely oxidized. The feeding solution contained glucose (50 % w/v), salts, trace elements, glutamic acid and vitamins according to Paciello et al [14].…”