Antifoam addition to shake ask cultures of recombinant Pichia pastoris increases yield

Background: Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast. Results: Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD595 ) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (kLa) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium. Conclusions: We show that addition of a range of antifoaming agents to shake flask cultures of P. pastoris increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture. Background The laboratory-scale production of recombinant proteins using P. pastoris requires that cells are cultured either in large shake flasks or in continuously stirred tank bioreactors. In these vessels, the formation of foam is an issue that requires intervention. This is in contrast to the situation in the small vessels typically used in the initial stages of protein production experiments where foaming is minimal [1]. Foaming can lead to reduced yields since bursting bubbles can damage proteins [2] and can also result in a loss of sterility if the foam escapes [3]. In bioreactors, foaming can lead to over-pressure if a foam-out blocks an exit filter. To prevent the formation of foam, mechanical foam breakers, ultrasound or, most often, the addition of chemical antifoaming agents (or “antifoams”) [3] are routinely employed. There is a well-established literature on antifoams [3]. One useful classification categorizes them as either * Correspondence: [email protected] School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK Full list of author information is available at the end of the article Routledge et al. Microbial Cell Factories 2011, 10:17 http://www.microbialcellfactories.com/content/10/1/17 © 2011 Routledge et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hydrophobic solids dispersed in carrier oil, aqueous suspensions/emulsions, liquid single components or solids [4-6]. Several mechanisms of action for these agents have been suggested which include bridging-dewetting, spreading fluid entrainment and bridging-stretching [7]. Many are commercially-available, with 19 being sold by Sigma-Aldrich alone. While little information is routinely given about their composition, their specific antifoam properties have been thoroughly investigated. These include their effects on foam height with time, their influence on the volumetric oxygen mass transfer coefficient (kLa) of the system, their gas hold-up characteristics and their globule size and distribution in relation to their action upon foams [3,5,7-11]. Such studies have been performed in various growth media in both the absence and presence of cultures of prokaryotic and eukaryotic microbes. In contrast, literature on the biological effects of antifoams on recombinant protein yields from microbial cell factories is more limited. Additional file 1: Table S1 shows an analysis of representative examples of this body of work including previous studies on four bacterial hosts and one yeast species. In some cases, the additives tested are not antifoams sensu stricto. It is also noteworthy that the yeast, Schizosaccharomyces pombe, is not widely used in biotechnology applications and that there have been no prior studies on the biological effects of antifoam addition to recombinant P. pastoris cultures. A recent review stated that in the last 15 years, 80% of all recombinant genes reported in the literature were expressed in either Escherichia coli or P. pastoris [12]. In this study, we therefore examined five antifoams that are widely used in controlling the foaming of recombinant P. pastoris cultures [13-16] in order to analyze effects over and above that of their de-foaming action. We looked at polypropylene glycol (PPG) P2000 that is analogous to previously-examined liquid single components of the PPG-type [11] as well as examples from other categories such as Antifoam A and Antifoam C, which are silicone polymers, SB2121, which is a polyalkylene glycol, and J673A, which is an alkoxylated fatty acid ester on a vegetable base and has not previously been documented in this context: for all antifoams examined, this was the first report of their effect on the yield of recombinant GFP secreted from shake-flask cultures of P. pastoris. Results We wanted to establish whether antifoams affect recombinant protein yield in P. pastoris X33 cultures, and if so to investigate the underlying mechanisms. To examine this we chose an experimental system, under the control of the methanol-inducible AOX1 promoter, comprising GFP secreted from 20 mL cultures in shake flasks in the presence of five different antifoams at a range of concentrations from 0-1% v/v. These concentrations are higher than the 0.1% routinely used for de-foaming purposes. The total amount of GFP in these 20 mL cultures (the total yield) was measured by fluorimetry 48 h post-induction. Antifoam addition affects total GFP yield in shake flasks The total yield of GFP as a function of Antifoam A addition rose significantly at concentrations of 0.6% and above (Figure 1A) with no further increases above 1% (data not shown). A similar but more pronounced trend was observed for Antifoam C (Figure 1B), which is unsurprising since Antifoam C is a 30% emulsion of the same antifoam concentrate as Antifoam A, but with different non-ionic emulsifiers [17]. Figure 1C shows that addition of 1% J673A almost doubled the total yield of GFP compared to the control without antifoam, representing one of the largest effects of the antifoams evaluated. At concentrations above 1%, the total yield of GFP decreased (data not shown). Addition of P2000 (Figure 1D) also resulted in a significant increase in total yield at or above 0.6%, while addition of SB2121 (Figure 1E) increased total yield at concentrations above 0.4%. In both cases the largest improvement was obtained on addition of 1% of the antifoam, again almost doubling the yield. Overall, the five antifoams tested all increased the total yield of GFP at concentrations in the range of 0.4-1% v/v. The highest yield was achieved by adding 1% P2000 (422 μg GFP) followed by 1% SB2121 (396 μg GFP), 1% J673A (394 μg GFP), 0.6% Antifoam A (373 μg GFP) and 0.8% Antifoam C (348 μg GFP). All five yields were significantly higher than the corresponding yields from the 0% control, as shown in Figure 1. The effects of antifoam addition are due to changes in culture density for P2000 and SB2121 To account for any changes in the growth characteristics of the cells on addition of the antifoams, we normalized the total yield to the optical density of the cultures to obtain the specific yield (μg OD595 ). OD595 was demonstrated to be a reliable measure of cell density in these experiments by comparing the number of cells at a given OD595 in the absence and presence of a range of concentrations of the different antifoams used in our study: there was no statistically significant difference in cell number between cells harvested at a given OD595 in the absence or presence of all antifoam concentrations tested. Typical values were 4.8 × 10 cells/ mL at an OD595 of 20.5 in the absence and presence of 0.5% SB2121. For Antifoam A, Antifoam C and J673A, the specific yield data were similar in trend to the total yield data Routledge et al. 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