Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

Abstract

As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (q p) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l−1) and at a growth rate close to μ max (0.15 h−1). Between 0.017 and 0.115 h−1 in glycerol-limited fedbatch cultures, basal levels of q p > 0.4 mg g−1 h−1 CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h−1, an elevated q p occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the q p(μ) relationship (kinetics of product formation), the entire μ range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, μ values were repeatedly shifted from 0.004 to 0.074 h−1 and vice versa. Changes in q p were related to changes in μ. A rough estimation of μ range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.

Control of specific growth rate for the enhanced production of human interferon α2b in glycoengineered Pichia pastoris: process analytical technology guided approach

“…Dynamic variation of μ sp to different set values in a single control experiment could be an attractive option from an operational perspective, but would in turn exert dynamic pressure on AOX machinery of Pichia pastoris . This would lead to various levels of stress (transient adaptation of cells to different methanol concentrations) and complicate the interpretation of the protein expression level.…”

Recent Advances in Pichia pastoris as Host for Heterologous Expression System for Lipases: A Review

“…Important efforts have been made to reduce the time and the number of experiments to get the relationship between specific growth and production rates. In only a single fed-batch experiment the kinetics of product formation has been obtained in a novel synthetic variant of the AOX1 promoter (31).…”

“…Recently Candida antarctica lipase B (CALB), one of the most applied lipase in biotransformations, has been expressed under alternative promoters. A novel synthetic PAOX1 variant has been characterized physiologically to demonstrate its functionality and to study the dynamic conditions for switching in order to control these modified promoters using appropriate process strategy (31). CALB has also been successfully expressed under the constitutive PGK1 promoter.…”

“…The relationship between specific CALB secretion rate and specific growth rate was stablished for P. pastoris growing on glycerol under limited substrate availability (< 1 g L -1 ) in fed-batch cultures. With this new approach from synthetic biology and bioprocess engineering, the results suggesting that the secretion kinetics observed are not determined by promoter regulation but instead by production/secretion capacity (31).…”

Increase of Candida antarctica lipase B production under PGK promoter in Pichia pastoris: effect of multicopies

“…However, the use of methanol may be undesirable in large-scale fermentation processes because of its flammability and toxicity. Variants of P AOX1 are being developed, in order to regulate the production without the need for methanol, but the production rates are not as well studied as the classic P AOX1 [11]. Constitutive expression systems represent an attractive alternative, with the advantage that different carbon sources can be employed, including crude glycerol, a by-product of the biodiesel industry [12].…”