Metabolic impact of redox perturbations on recombinant protein production in Komagataella phaffii under methanolic growth conditions

Recombinant protein extracellular production is a highly demanding process in terms of energy and redox cofactor regeneration. Yeasts such as Komagataella phaffii (aka. Pichia pastoris ) seem to adapt their metabolism by increasing ATP, NADH or NADPH regeneration to compensate for the metabolic burden exerted by such costly process. Earlier studies have proven that increased NADPH regeneration capacity has a positive effect on recombinant protein production when using glucose as sole carbon source, while counteracting increased NADH/NAD + ratios has also been proven beneficial. To evaluate the metabolic impact of increasing NADPH and/or NAD + regeneration caused by the introduction of a cytosolic NADH kinase and an alternative NADH oxidase, we initially performed in silico simulations based on the use of a genome-scale model. Next, we investigated the effect of overexpressing the S. cerevisiae ’s POS5 NADH kinase encoding gene (targeted to the cytosol) and an alternative oxidase ( AtOX ) in a K. phaffii strain producing extracellularly a Rhizopus oryzae lipase (Rol) under control of the alcohol oxidase 1 ( AOX1 ) promoter (P AOX1 ), thereby assessing the impact of such engineering strategies on recombinant protein production and cell performance under methanol growth conditions. Small-scale cultures allowed us to conclude that such genetic modifications increase protein production significantly under methanol growth conditions. The redox-engineered strains were further characterized in bioreactor chemostat cultures using a glucose/methanol mix (60/40%, w/w) as a carbon source to corroborate their performance. Thereby, up to a 36% increase in Rol specific productivity was observed when overexpressing two copies of POS5 , whereas AtOX overexpression did not improve production. Notably, combined two-copy POS5 and AtOX overexpression resulted in a 53% increase in lipase production compared to the reference strain, suggesting that perturbation of the NADH/NAD + ratio leads to widespread changes in metabolism ultimately affecting cell growth and product formation, resulting in the observed additive effect. An in-silico interpretation of the physiological macroscopic data from chemostat cultures using a genome-scale metabolic model allowed us to gain further insights on the metabolic impact of these modifications. Our study provides a DBTL cycle workflow generating valuable insights for a quantitative understanding and prediction of cellular response to redox- based perturbations and subsequent test bedding of metabolic engineering applications in K. phaffii .