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

<p>Abstract</p> <p>Background</p> <p><it>Pichia pastoris </it>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.</p> <p>Results</p> <p>Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of <it>P. pastoris </it>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 (<it>k_La</it>) 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.</p> <p>Conclusions</p> <p>We show that addition of a range of antifoaming agents to shake flask cultures of <it>P. pastoris </it>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.</p

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.Published versio

Understanding the yeast host cell response to recombinant membrane protein production

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes

Smear Ripened Cheeses

International audienceCheeses are produced at local and industrial scales generating over 1,000 varieties of cheese in Europe. Most cheeses have a complex successional microbial flora through milk fermentation, curd maturation and storage, to cheese maturation. Microorganisms are introduced with raw materials, by deliberate inoculation and from the environment. Microorganisms are responsible for most of the changes which produce cheese, give it its organoleptic properties and contribute to its preservation, but they may include pathogens, of which Listeria monocytogenes is the most common. The activity of aminotransferase, enzymatic degradation of L-methionine and the subsequent formation of volatile sulphur compounds leads to the development of the typical flavour in smear cheese. The characteristic features of cheese evolve from complex interaction of the metabolic activities of the smear cheese flora. So it is essential to define and identify surface microflora to enable the selection of strains that generate the colour, aroma and organoleptic qualities of specific cheeses, and to screen for anti-listerial activity

The role of inorganic-phosphate, potassium and magnesium in yeast-flavour formation

Inorganic-phosphate, potassium, and magnesium are key-minerals required for yeast growth, metabolism, and survival, the present work investigated its impact in yeast-flavour formation using a multi-factor experimental design, which was used to generate a range of phosphorous-potassium-magnesium resulting in a 28-point D-optimal design. Samples were evaluated using HPLC (ethanol), GC–MS (aroma), and CountStar (total yeast cell). Results revealed that yeast requires a minimal amount of inorganic-phosphate, potassium, and magnesium (250, 500, and 70 mg/L, respectively) to support yeast-growth and ethanol/flavour formation. Inorganic-phosphate was important for fatty acid esters formation/short chain fatty acid (SCFA) reduction. Potassium was important in the formation of acetate esters/higher alcohols. Magnesium was the most important inorganic element for ester formation/SCFA reduction; furthermore, ethanol production is magnesium-dependent. In conclusion, inorganic phosphate, potassium and magnesium play an important role in yeast-growth, esters and higher alcohols formation; and SCFA reduction. Ethanol formation is Mg-dependent

Mycetocola reblochoni sp. nov., isolated from the surface microbial flora of Reblochon cheese

Four Gram-positive, aerobic, non-sporulating, rod-shaped bacteria isolated from the surface microflora of Reblochon cheese at the late stage of ripening had chemotaxonomic properties characteristic of members of the family Microbacteriaceae. The isolates had virtually identical SDS-PAGE whole-organism protein patterns, shared many chemical and phenotypic characteristics and formed an independent branch in the Microbacteriaceae 16S rRNA gene tree that was most closely related to the type strains of Mycetocola species. The new isolates had chemotaxonomic properties consistent with their classification in the genus Mycetocola but were readily distinguished from recognized members of this taxon based on DNA–DNA relatedness, whole-organism protein and phenotypic data. The combined genotypic and phenotypic data indicate that the isolates should be classified in the genus Mycetocola as members of a novel species, for which the name Mycetocola reblochoni sp. nov. is proposed. The type strain is LMG 22367T (=R-20377T =BRB-1L41T =DSM 18580T)