Hi Tomáš

What Pichia pastoris strain are you using and how does your media looks like?  

What DNA construct did you rekombinate, did you use an auxotrophic selection and did you monitor the protein forming with SDS-Page?

The Methanol should not be a limiting factor, if you have sugar inside your media, the yeasts will produce enough of their own alcohol anyway....usually the growth rate of Pichias is a lot higher than E-Coli, specially in flasks...

Did you mean 50ml/L-day? could you tell us yeast OD when you start add the methanol?

I think methanol too less

The strain is X-33 and vector is pPICZalpha A with zeocin selection (used only for selection, not anymore).

I started with about 100ml per hour (2 litres per day in total) and now I got some activity. Unfortunately as they have starved for two or three days, the culture is already old and the cells seem to start to die (the pH is rather rising) so I'll try to get the best from it this time and next time I'll know I must add much more! Plus some more glycerol doesn't hurt :(

@Wen Shiang Lee  No, it was 50 ml in total (10 l total volume)

Hi Tomas,

There could be a lot of things gong wrong with a fermentation. If you are sure that you get expression in shake flask but none in fermentation then I would consider the following:

1.Get the O2 probe working. It is very hard to do a pichia fermentation using methanol induction without some measure of dO2, esspecially if you also cannot measure MeOH concentration directly. We also had a problem with O2 probe which tuned out to be due to precipitation in media after autoclave. When we reduced salt conc of media ppt and probe problems disappeared (see http://www.ncbi.nlm.nih.gov/pubmed/11722185 for media ref) .

2. When the probe is working you can see if the cells have used up the initial carbon source (glucose or glycerol) by observing an increase in dissolved O2. Only then switch to MeOH. I've used average rate of 2ml/L/hr quite sucessfully (lower at first and then increase rate with time). You want the cells to always have just enough MeOH but not much more. You can check by turning off MeOH and if you see a sudden spike in dO2 then it's about right. Your MeOH rate sounds quite low, especially if you have a healthy culture before induction  (OD600 at least 50 better if ~ 100).

Good luck

@ Fergal O'Meara  insightful views

@Fergal O'Meara

Thanks for your help.

It is true, that the YNB used precipitated for some reason. However, later when I started to add more MeOH, I've observed some O2 decrease so it probably somewhat worked after all. Next time I'll try those fully defined media, just few questions - the glycerol (BTW I used only 10 g/l of glycerol, so I obviously heavily underestimated the power of fermentation) is autoclaved together with the salts, right? What's the sodium hexametaphosphate? Na6(PO3)6? Is it possible to use normal ortho-phosphate?

Further about the pH. Everybody seems to use pH 4 or 5. However, my protein has pI 5.5, so I'd like to avoid the range 4.5 - 6.5 and I have actually never kept my protein at lower pH than 7 (or maybe 6, I don't remember). Now I have used pH of 6.7 (0.1 M K-Pi pH 7.0 plus the other stuff made it to 6.7) and the yeast grew. I will try how my protein holds at lower pH, but what would you recommend?

BTW after I started studied this more, I increased the feeding with MeOH (unfortunately after 3 days of starving) with something about 10 ml/l.hour (incl. the PTM) and I got some activity, but then the culture pH started to grow (from lysed cells?) so I ended it and will purify my enzyme. So I got at least something from the first fermentation.

@Fergal O'Meara

few more questions :)

They do not mention phosphoric acid in the media recipe in the article you linked. Is it possible they do not add it at all or did they just forget to write it into the publication?

And those amounts are per initial volume or final volume? Because I'll start e.g. with 5l of media and then I'll feed it with some glycerol and methanol, so I'll end up with e.g. 7l.

 What is the buffer in the fermentor? Or is there no need for buffer because it controls the pH? Right now I used phosphate buffer either with pH 7.0 or 6.0 (where the phosphate doesn't buffer anyway), what shall I use if I wanted to try first this low-pH (4-5) in flasks to see whether my protein can withstand that?

Hi Tomas,

Sorry for the slow reply. To answer your questions in order:

1. Yes you can autoclave glycerol (4% w/v concentration) with the salts.

2. It should be fine to use ortho-phosphate - worked for me. In the paper they use the Na hexametaphosphate as the source of P

3. Pichia can grow fine at pH 7 (even up to 8 and down as low as pH 3 apparently). I've routinely used pH6. You can adjust it after autoclaving by addition of ammonium hydroxide soln.

4. There is no need for a buffer if you have pH control on your fermentation system. Typically use ammonium hydroxide to prevent culture getting too acidic (if pH is increasing there is something wrong with the culture).

5. If you started feeding them 10ml/L/hour MeOH after 3 days starving that was probably too much and MeOH built up to toxic levels.

6. See 2 above

7. Amounts are per initial volume.

8. If you want to try a shake flask at lower pH use and unbuffered medium. Cells will lower the pH (see Invitrogen manual), or use any suitable biology buffer (not familiar with one myself as never needed it).

Good luck

Thanks for your help, it's very helpful.

3. so you think there's no need to try lower pH? The growth is not impaired or so? Also, I was thinking, if the problem with the probe could be due to precipitation, it could just be formation of hydroxides of Me2+ which start to form around pH 6 and up. Maybe that could be a reason to try lower pH.

6. right, I forgot it's there :)

The culture itself should be fine at pH 6-7, but your protein maybe very susceptible to proteases at that pH. That maybe a reason to try it at a lower pH. Another alternative is to include Casamino acids in your medium. This will reduce protease activity.

You say you are using a pPICZalpha expression plasmid so presumably your protein is secreted out of the cell. This means that when you have a lot of cell lysis (as you probably had when starving the cells) you will get a lot of proteases released and therefore degradation of your protein. You should read the Invitrogen manual it will help with a lot of this.

So the proteases are more active at higher pH? Because in one of the articles they wrote they used pH 5 to limit the protease activity. I somehow assumed they meant they kept so high pH to limit proteases. But it's the other way? It would make sense I guess, since the yeast are not so acidic inside. However, my protein was usually pretty stable, no matter what.

Hi Tomás

The optima of fungal peptidase can vary between pH 4 to 9, so principally it is not so easy to exclude all kind of protease activity by switching the value within this range 

You are using a Yeast Eucariotic expression systems, usually yeasts have no problem to grow in acidic environments down to pH 4 or lower, so yeast proteases should not have activity problems at a pH of 5. You have Proteases like Pepsin with optima at pH2 and others like Alcalases for washing applications adapting to the high pH of this mixtures at ph 9 or above. Although  having possibility for Peptidase with neutral Optimum,  the suggestion of higher proteases activity at lower pH is the more realistic one. Even the "standard" procariotic Bacillus Protease can work down to a pH o 5.

Proteases inhibotrs are mostly expensive for bigger scale usage, usually it  is a help if you have enough other proteins that can be cut too, that is one of the mayor advantages (beside of better growth rates and more stable processing) of complex media comparing to synthetic, so if your lysis is high, it is not always a bad thing. You cannot ferment this secretory proteins unlimited anyway like you could with cytosolic expressed, therfore you have the eaiser workup and better activities with this systems....so a time limit for you expression can be helpful too

Hi Thomas

Is it secretary protein or intracellular? if it is secretary protein and your product pI is 5.5 and you do not want to take it to lower pH you set the fermenter pH to 6.5. There will be no problem for experssion of your target protein at 6.5pH. There are some proteins like human serum albumin are not stable at lower pH like 5.0, increasing the fermenter pH to 6.5 reducing the proteolytic activity and the product is stable.  In some cases like trypsin it will autolyse at 8.0 and at lower pH 3.0 it is stable. So it dipends on your product stablity and where the proteolytic activity is more on your product, based on that you can select the lower pH or neutral pH for expressiong your target protein.

You can also try adding nitrogen sources like ammonium chloride, ammonium sulphate, peptone or arginine to keep the possitive nitrogen metabolism, in some cases lack of nitrogen sources also leads to proteolytic activity.

You can reduce the temperature to 20 degree C to prevent proteolysis during induction.

If you want to find out what kind of proteolysis happening on your product you need to add six different types of protease inhibitors to confirm the type of proteolysis.

GOOD luck.