Effect of an exponential feeding regime on the production ofRhodotorula araucariaeepoxide hydrolase inYarrowia lipolytica

Abstract

Aims: To evaluate the effect of and exponential feeding regime on the production of epoxide hydrolase (EH) enzyme in recombinant Yarrowia lipolytica in comparison to a constant feed strategy. Methods and Results: An exponential feed model was developed and fermentations were fed at six different exponential rates. A twofold increase in EH productivity and a 15% increase in volumetric EH activity was obtained by applying exponential glucose feed rates in fed‐batch cultivation. These responses were modelled to obtain a theoretical optimum feed rate that was validated in duplicate fermentations. The model optimum of 0·06 h−1 resulted in a volumetric EH activity of c. 5500 U l−1 h−1 and a maximum activity of 206 000 U l−1. This correlated well with model predictions, with a variance of <10%. Conclusions: The use of an exponential feed strategy at a rate of 0·06 h−1 yielded best results for all key responses which show a clear improvement over a constant feed strategy. Significance and Impact of the Study: The study was the first evaluation of an exponential feed strategy on recombinant Y. lipolytica for the production of EH enzyme. The results suggest a strategy for the commercial production of a valuable pharmaceutical enzyme.

Bioreactor-Scale Strategies for the Production of Recombinant Protein in the Yeast Yarrowia lipolytica

“…A second, narrower category concerns the medical and pharmaceutical fields. Heterologous enzymes produced in Y. lipolytica can serve to obtain enantiopure molecules suited for drug synthesis [21,22,23]. Hyperglycosylation, commonly observed in S. cerevisiae , is not critical in Y. lipolytica [24,25], rendering this host more adapted for the production of therapeutic proteins such as virus antigen [6], glucocerebrosidase [26], and lysosomal enzyme [20].…”

“…Improvement of secretion efficiency [45] and synthesis of pro-proteins [11,19] will further help the cells to tolerate overall expression pressure. Determination of an optimal growth rate—not always corresponding to the maximum growth rate µ max —during recombinant protein production promotes balance between growth and protein synthesis, especially for constitutive promoters [23]. At the process scale, inducible promoters exploited in a fed-batch mode allow biomass to grow at a high growth rate (close to µ max ) prior to the initiation of the production phase.…”

“…The transition from batch to fed-batch mode drives the process further. Biomass concentration values in the range of 50–100 g dry cell weight (DCW)·L −1 are commonly attained with fed-batch processes [13,15,21,22,23,36,37,53,112], while protein yields and productivity thrive from 2–3-fold [37,113] to 10-fold [22,53].…”

Development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast, Yarrowia lipolytica

“…Only a few reports have focused on the scale-up of recombinant protein production in this yeast [8,9]. On the other hand most of the reported proteins employed either XPR2 or hp4d promoter expression system.…”

“…However, the use of these systems remains limited and may not be compatible with some heterologous proteins or with industrial culture conditions. The inducible XPR2 promoter is active only at pH above 6 and its full induction requires high levels of peptones in the culture medium whereas the second promoter is constitutive; this can be problematic when the product being expressed is toxic to the host [6,9]. To overcome these limitations efforts have been made to develop new alternative promoters.…”