Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production

Abstract

The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial β-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy.

Combined 13C-assisted metabolomics and metabolic flux analysis reveals the impacts of glutamate on the central metabolism of high β-galactosidase-producing Pichia pastoris

“…In P. pastoris , there are two routes for NADPH generation, the oxidative branch of PPP and the acetate formation pathway catalyzed by acetaldehyde dehydrogenase (AADH) (Nocon et al 2014), which can use NAD + and/or NADP + as cofactor (Blank et al 2005). Assuming that AADHs only used NADPH as cofactor, the NADPH generation rates from oxidative PPP and AADHs were calculated (Fig.…”

Cell Factory Engineering

“…systems 625 biology or system metabolic engineering), which are moving towards holistic 626 design of cell factories and biological networks (Nocon et al 2014). 627…”

Applications of genome-scale metabolic network model in metabolic engineering

“…The flux distribution of a mutant is determined by seeking the closest point in the flux space of the mutant to the optimal state of the wild-type strain and, in most of the cases, the predicted flux distribution in the mutant is sub-optimal for cell growth. MOMA was applied to identify gene deletion targets for the production of the cytosolic human superoxide dismutase (hSOD) in Pichia pastoris [61]. The alcohol dehydrogenase adh2 was identified as a deletion target and the production of hSOD was increased by 20 % in the adh2 knockout strain without compromising the cell growth.…”