Hi,
For months I've been trying to work with Pichia pink (all four strains from Invitrogen). Unfortunately the cells clump all the time and after one/two days of growth I have a big mess in my flasks (it looks almost like a cottage cheese instead in medium-some example on photo). Under the microscope the cells form extremely large colonies, although the cells picked from plate and dissolved in medium look fine (single cells).
I've exchanged the medium (I'm using the medium included in the Invitrogen kit), used different water, fresh flasks, flasks with agitators, different rpm, different temperatures. I tried to transform such a Pichia but there is no expression product.
Any suggestions? We also have Saccharomyces cerevisiae and this one grows ok.
Hello Marta,
To be frank, the word "clumped" looked strange to me, I've hardly heard it in yeast genetics world :)
The picture helps a lot. If I see correctly the picture shown, it looks to me a lot like a flocculation occurred.
I've attached to my answer two papers on flocculation in yeasts. the first, TiBTech 1993, is a general review on yeast flocculation and still very valuable to read. The second deals with applications in biotech of the flocculation.
In yeasts, not only cerevisiae but other yeasts, flocculation may result of two events: aggregation of cells by so-called zymolectins (or flocculins) that are glycosylated membrane proteins. If this is what happens in your hands, I woudl recommen to add MANNOSE at 100 mM in the culture medium and, if flocculins are involved, the "clumped" look will vanish!
Another possible reason of flocculation in yeasts are some cationic ions such as Ca2+. Would your culture medium contain some ? Hope this will help you!
Please tell me what you'll get... the easiest to do in my opinion is to add some mannose and see if the clumping still occur?
Best regards,
Prof Philippe Urban
Hello Marta,
To be frank, the word "clumped" looked strange to me, I've hardly heard it in yeast genetics world :)
The picture helps a lot. If I see correctly the picture shown, it looks to me a lot like a flocculation occurred.
I've attached to my answer two papers on flocculation in yeasts. the first, TiBTech 1993, is a general review on yeast flocculation and still very valuable to read. The second deals with applications in biotech of the flocculation.
In yeasts, not only cerevisiae but other yeasts, flocculation may result of two events: aggregation of cells by so-called zymolectins (or flocculins) that are glycosylated membrane proteins. If this is what happens in your hands, I woudl recommen to add MANNOSE at 100 mM in the culture medium and, if flocculins are involved, the "clumped" look will vanish!
Another possible reason of flocculation in yeasts are some cationic ions such as Ca2+. Would your culture medium contain some ? Hope this will help you!
Please tell me what you'll get... the easiest to do in my opinion is to add some mannose and see if the clumping still occur?
Best regards,
Prof Philippe Urban
you can add 5mM EDTA to your culture and check the flocculation . Tje flocs should dissaper after adding EDTA .
Best regards,
Santhosh
I'm having the same problem as Marta with the PichiaPink strain #4. Philippe is right, it is classic flocculation. But we can't add mannose, because that will tend to repress the AOX1 gene we use for heterologous protein expression in yeast. And I tried 5 mM EDTA as recommended by Santosh, but it didn't cause the flocs to disappear.
In Rebnegger et al. (Pichia pastoris exhibits high viability and low maintenance-energy requirement at near-zero specific growth rates. Appl Environ Microbiol. 2016) they deal with the flocculation by (1) using wet cell weight rather than OD to determine biomass concentration "on the fly" (one could use packed cell volume too), and (2) the knock out the P. pastoris FLO8 gene, which encodes a flocculin, and saw a reduction in clumping.
Also, Curvers et al. (Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation. Biotechnol Prog. 2001) reported flocculation by P. pastoris at pH 4 but not pH 3 or pH 5.
And Tanneberger et al. (A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris. J Biol Chem. 2007) have an interesting discussion on flocculation of P. pastoris as a function of growth phase and nutrition.
- Badges
- Science method