11th World Congress on Biotechnology and Biotech Industries Meet
Univerite de Liege, Belgium
Title: Pichia process optimisation by co-feeding procedure : How to reduce oxygen uptake and heat generation without loss of pAOX1 promoter induction
Biography: Patrick Fickers
Recombinant protein production driven by AOX1 promoter (pAOX1) is challenged by a high oxygen demand and heat production, especially in large-scale bioreactor. A promising solution relies on a methanol/sorbitol co-feeding strategy during the induction phase. In this work, transient continuous cultures were first performed to quantitatively assess the benefits of a methanol/sorbitol co-feeding process with a P. pastoris Mut+ strain bearing a pAOX1-lacZ construct served as a reporter gene. Our results demonstrated that cell-specific oxygen consumption (qO2) could be reduced by decreasing the methanol fraction in the feeding media. Optimal pAOX1 induction was achieved and maintained in the range of 0.45~0.75 C-mol/C-mol of methanol fraction. In addition, the qO2 was reduced by 30% at most in those conditions. Based on a simplified metabolic network, metabolic flux analysis was performed to quantify intracellular metabolic flux distributions during the transient continuous cultures, which further shed light on the advantages of methanol/sorbitol co-feeding process. Secondly, chemostat cultures were performed to investigate the cell growth, metabolism and regulation of pAOX1 regarding co-feeding rate of optimized methanol/sorbitol mixture. Our results highlight that methanol/sorbitol co-feeding allowed cells to adapt to oxygen transient limitation that often occur at industrial scale with reduced effect on pAOX1 induction and cell viability. The optimal feeding rate tested here was 6.6 mmolC (DCW h)-1 at an oxygen transfer rate (OTR) of 8.28 gO2 (l.h)-1 with over five-fold pAOX1 induction (probably directly associated with target protein productivity) compared with previous work.