Pichia growth in MGY/MGYH media - (Jul/10/2012 )

Hi all,

I've been working with the GS115 strain of P. pastoris for the past month. I'm using Invitrogen's EasySelect kit, and I did not have any issues with the cloning or selection (all clones are zeocin-resistant and show desired inserts). In the beginning I was working with the X33 strain and had no issues with growth or expression, then my cells suddenly wouldn't grow well in MGY media, only YPD. Even when I inoculated MGY with a strong YPD culture of the yeast the growth was weak. I switched strains to GS115, proceeding from scratch, and I see the same issue. It's been 4 days since I picked colonies from zeocin plates into MGYH media, and the cultures are very weak and smell sour. I've tried varying glycerol, YNB, and biotin sources to make sure I wasn't using a bad reagent, and I keep the shaker set at 220rpm, 28 degrees. Is it an issue with pH or some other factor?

Any advice? I need strong MGY/MGYH cultures to get our fermenter going. Thanks!

Update: for whatever reason, we figured out that our Pichia strains (X33 and GS115) would not grow in generic YNB, only Invitrogen's YNB. Very frustrating! Unfortunately, the cultures are still not very strong, but I plan to try a few other conditions to improve their strength.