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Abstract

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 fivefold pAOX1 induction (probably directly associated with target protein productivity) compared with previous work.

Biography

Patrick Fickers has completed his PhD in Biochemistry from Universite de Liege, Belgium and from Institut National Agronomique, Paris, France. In 2004, he was at Polytech’ Lille, France as a Post doctorate. In 2005, he has joined the Centre of Protein Engineering, Liege, Belgium as FNRS fellow. He was an Associate Professor at Universite libre de Bruxelles and the Head of the Biotechnology and Bioprocess Unit from 2009 to 2014. Since 2015, he is Professor at Gembloux Agro BioTech, University of Liege in the Microbial Processes and Interactions. He is also a Scientific Collaborator at University of Camaguey, Cuba and Adjunct Professor at TDT University (Ho Chi Minh City, Vietnam). He has published more than 40 research papers in peer-reviewed journals, 6 book chapters and several review articles. His researches focus on the development of yeast and bacterial strains by metabolic engineering. He is also interested in process development in bioreactor for the production of valuable compounds.

Email: pfickers@ulg.ac.be