Frequently Asked Questions: Fermentation
Q: What is the difference between methanol utilizing + (Mut +), methanol utilizing S (Mut S) and methanol utilizing - (Mut -)?
A: The Pichia genome contains two alcohol oxidase genes, Alcohol Oxidase I (AOX1) and Alcohol Oxidase 2 (AOX2). In the case of a Mut +, both the AOX1 and AOX2 gene are present and functional. Methanol metabolism is identical to the wild-type strain. The Mut S (S represents “Slow”) has the AOX1 gene disrupted and Pichia must relay on the AOX2 gene for methanol metabolism, which results in a significantly slower growth rate, about 5% to 10% of the activity compared to AOX1. In the case of a Mut - strain, Pichia is unable to metabolize methanol.
Q: Is the expression of a recombinant protein in shake flask directly transferable to a fermentor?
A: As a general rule of thumb, using a “standard” fermentation condition, it is possible to increase expression by about 5 to 10 fold from shake flask. This is usually a result of increasing the cell mass, i.e. shake flask is 10 to 20 O.D. to 250 to 350 O.D. in a fermentor.
Q: Is it best to pick the best expressing clone based on shake flask results?
A: It depends on your objective. If your objective is to make small amounts of protein, i.e. 50 to 100 mg of protein, then in most cases picking the clone from shake flask studies is sufficient. If the objective is to determine the best clone for protein production, the first step is to screen clones based on shake flask selecting 10 to 15 clones with different copy numbers of the recombinant protein. These clones are evaluated under similar fermentation conditions to determine the best clone.
Q: How do you know what fermentation conditions to use for selecting the best clone?
A: It is best to perform a 4 to 6 fermentation optimization to determine fermentation conditions suitable for clone selection. Use the standard basal salts medium and evaluated induction at 3 different pHs, 3.5, 5.0 and 6.5. Use a methanol feed rate during induction that generates an exponential growth rate of 0.025 h-1. Also, run fermentations at the three same induction pHs, except keep the methanol concentration during induction to 1.5 ml/L. Pick the best condition to determine the best clone. Note: this fermentation condition is the starting point for fermentation optimization.
Q: What is the best way to transition from glycerol to methanol?
A: The approach we take is described by Zhang et al., (2000). After the glycerol from the batch phase is consumed, feed glycerol for 1 hour at 20 g glycerol/Liter of initial fermentation broth. After 1 hour, inject methanol to a final concentration of 1.5 ml MeOH/L. At the same time the bolus of MeOH is added, initiate a linear ramp of the glycerol feed rate from 20 g/L/h to 0 g/L/h over 3 hours. During this time, the transition from glycerol metabolism to methanol metabolism will occur. Monitor the MeOH concentration or the D.O. Once the methanol is consumed or the D.O. spikes, initiate the methanol feed profile.
Q: Is chilled water needed to run high-cell-density fermentations?
A: This depends on the temperature of your domestic cold water and the growth rate during methanol induction. For growth rates of approximately 0.025 h-1, which is usually 1/3 the maximum growth rate, a cold water temperature not exceeding 20oC for fermentors sizes less than 50 L total volume is sufficient. Our fermentation lab is set up with domestic water chilled to 15oC. We have no heat issues with fermentor less than 60 L and at maximum growth rate.
Q: Is pure oxygen required to run high-cell-density fermentations?
A: The addition of oxygen is essential to control the dissolved oxygen.
Q: What is the best way to control the dissolved oxygen during a high-cell-density fermentation?
A: Use a two-stage approach. Set the air flow rate at 1 volume of air/volume of liquid/minute (VVM). Control the dissolved oxygen (D.O.) by increasing the agitation. Once agitation has reached a maximum, control the D.O. by supplementing the inlet air stream with pure oxygen. It is critical to have an oxygen proportional valve that is capable of controlling the D.O. to within 1% to 2% of the set point. Typical set point is 40%.
Q: How important is it to maintain a steady growth rate/conditions during induction?
A: This is ABSOLUTELY critical. The D.O. set point during methanol induction should be rock solid and the D.O. should not vary within 2% to 3%. Acceptable levels of +/- 5%. The D.O. fluctuating between 40% and 0%-5% is very detrimental to the fermentation process. Maintaining fermentation conditions that insure a steady growth is essential for high-cell-density fermentation.
Q: What are the best ways to combat the effect of proteases during MeOH induction?
A: Nearly all of the proteases found in the fermentation supernatant are generated due to cell lysis. The best way to minimize the release of proteases is to maintain a steady growth rate. The next step is to look at the effect of pH during induction. Pichia has acidic, neutral and basic proteases. Varying pH during MeOH induction can reduce the effect of proteolysis on your product. Next, the temperature during induction can be reduced from 30oC to as low as 15oC. The next step is to add complex nutrients, i.e. soytone, casamino acids, etc. during the induction. The last step is to investigate protease deficient strains, such as SMD 1163, 1165 or 1168.
