Influence of methanol/sorbitol co-feeding rate on pAOX1 induction in a Pichia pastoris Mut+ strain in bioreactor with limited oxygen transfer rate

Abstract

High Pichia pastoris biomass density could be obtained using high co-feeding rate of methanol and sorbitol in a fed-batch or continuous culture, while further higher feeding rate finally leads to oxygen limitation in bioreactor. In the literature, there is lack of report about AOX1 promoter regulation with regard to dissolved oxygen level (DO). Therefore, in this work, chemostat cultures were performed to investigate the cell growth, metabolism and regulation of the AOX1 promoter (pAOX1) regarding co-feeding rate of optimized methanol/sorbitol mixture (methanol fraction 0.60 C-mol/C-mol) using a P. pastoris Mut+/pAOX1-lacZ strain. The oxygen transfer rates (OTR) in bioreactor were kept in the range of typical values of large bioreactor, i.e., 4-8 g/(L h) if DO equals 30 % saturation or 5-10 g/(L h) if DO nears zero. For DO >0, an increase of the carbon fed led to an increase of pAOX1 induction. By contrast, when dissolved oxygen was completely depleted, methanol accumulated, causing a 30 % decrease of pAOX1 induction. However, this decrease is more likely to be lined to methanol accumulation than to low level of dissolved oxygen (<4 % DO). Methanol/sorbitol co-feeding allowed cells to adapt to oxygen transient limitations that often occur at industrial scale with reduced effect on pAOX1 induction. The optimal feeding rate tested here was 6.6 mmol C (DCW h)(-1) at an OTR of 8.28 g O2(L h)(-1) with over fivefold pAOX1 induction (probably directly associated with target protein productivity) compared with previous work.

Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B

“…Glycerol was consumed at a rate of 0.05 g.g DCW −1 .h −1 within 12 h. P. pastoris strain RIY311 (pAOX1-αMF-pro-CalB) grew somewhat slower, with a specific growth rate of 0.27 h −1 during the exponential growth phase, and reached a maximum biomass of 4.8 g DCW .L −1 by the end of the culture. As previously highlighted, P. pastoris is able to co-metabolize sorbitol and methanol 18 . Here, it occurred with a higher rate for sorbitol (0.07 g.g DCW −1…”

“…After one hour of incubation at room temperature, the lipase activity was determined. Compared to control (i.e., sample diluted in PBS buffer), no significant reduction of lipase activity could be observed (18,197 ± 790 vs 20,057 ± 2596 U.ml −1 ; Supplementary Fig. 3).…”

“…Analytical methods. Cell growth was monitored either by optical density at 600 nm (OD 600 ) or dry cell weight (DCW) as previously described 18 . Methanol, sorbitol, glycerol, erythritol and erythrulose concentrations were determined by isocratic RID-HPLC (Agilent 1100 series equipped with UV and RID detector, Agilent Technologies, Santa Clara, CA, USA) using an Aminex HPX-87H ion-exclusion column (300 × 7.8 mm Bio-Rad, Hercules, CA, USA) with 5 mM H 2 SO 4 as mobile phase at a flow rate of 0.5 mL.min −1 at 65 °C.…”

“…The latter is an important consideration since the use of methanol on large-scale presents drawbacks such as toxicity and flammability, high oxygen consumption and high heat release. Another solution to minimize methanol addition involves different co-feeding processes that have been successfully developed, where part of the methanol used as inducer and energy source has been replaced by glycerol 15 or sorbitol 18 .…”

Cultivation of Pichia pastoris carrying the scFv anti LDL (−) antibody fragment. Effect of preculture carbon source

“…As soon as the carbon source for growth (glycerol, sorbitol, xylose, ribose, glucose)36, 46, 47, 48 has been exhausted, the step of induction with methanol begins 12 . Some carbon sources, maily glucose, were reported to inhibit the promoter expression, 36 while others such as sorbitol and glycerol were described as not inhibitory 49 . For this reason, the type of carbon source before the induction is of paramount significance for protein expression 50 …”