RESUMO
Komagataella phaffii (aka Pichia pastoris) is a yeast able to grow in methanol as the sole carbon and energy source. This substrate is converted into formaldehyde, a toxic intermediary that can either be assimilated to biomass or dissimilated to CO2 through the enzymes formaldehyde dehydrogenase (FLD) and formate dehydrogenase, also producing energy in the form of NADH. The dissimilative pathway has been described as an energy producing and a detoxifying route, but conclusive evidence has not been provided for this. In order to elucidate this theory, we generated mutants lacking the FLD activity (Δfld1) and used flux analysis to evaluate the metabolic impact of this disrupted pathway. Unexpectedly, we found that the specific growth rate of the Δfld1 strain was only slightly lower (92%) than the control. In contrast, the sensitivity to formaldehyde pulses (up to 8mM) was significantly higher in the Δfld1 mutant strain and was associated with a higher maintenance energy. In addition, the intracellular flux estimation revealed a high metabolic flexibility of K. phaffii in response to the disrupted pathway. Our results suggest that the role of the dissimilative pathway is mainly to protect the cells from the harmful effect of formaldehyde, as they were able to compensate for the energy provided from this pathway when disrupted.
RESUMO
Pichia pastoris is a well-established cell factory for recombinant protein synthesis. Various optimization strategies of processes based on AOX1 promoter have been investigated, including methanol co-feeding with glycerol or sorbitol during the induction stage. Compared with carbon sources, comparatively little research has been devoted to the effects of nitrogen sources. Several reports have described the benefits of adding casamino acids (CA) to the recombinant protein production medium, however, without considering its effects at the gene expression level. Using enhanced green fluorescent protein as a reporter protein, monitored using flow cytometry, CA was shown to downregulate AOX1 promoter induction. Despite higher growth rates, cultures containing CA exhibited slower transition to the induced state, whereas metabolite analysis revealed that methanol consumption was reduced in the presence of CA compared with its absence. The repressive effect of CA was further confirmed by analysing the synthesis of extracellular recombinant Candida antarctica lipase under control of the AOX1 promoter. These findings highlight nitrogen source selection as an important consideration for AOX1-based protein production.