title: Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield.
abstract: 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 OD₅₉₅⁻¹) 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 (k(L)a) 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.
authors.affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Routledge
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hewitt
authors.forename: Nagamani
authors.initials: N
authors.lastname: Bora
authors.forename: Roslyn M
authors.initials: RM
authors.lastname: Bill
mesh_terms: D000934:Antifoaming Agents / Q000494:pharmacology*; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D003470:Culture Media / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011108:Polymers / Q000494:pharmacology; D011409:Propylene Glycols / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000934:Antifoaming Agents; D003470:Culture Media; D011108:Polymers; D011409:Propylene Glycols; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C012504:polypropylene glycol