Enhancement of protein secretion by transcription factor HAC1 in two Komagataella phaffii strains metabolic modified.

Komagataella phaffii (Pichia pastoris) is a widely recognized yeast strain to produce recombinant proteins. The efficiency of protein production and secretion is influenced by various factors, namely the caracteristics of signal peptide used, the typo of protein being produced, the operational parameters and the manipulation of genes to achieve optimal expression levels.
Particularly, the secretory pathway plays a key role in the folding protein process, and its study has been a subject of interest to improve the production and secretion of recombinant proteins.
The transcription factor HAC1 gene has been investigated for its implications in the unfolded protein response. On the other hand, methanol pathway engineering has been studied to improve carbon source assimilation.
The complementation of these two research focuses has been carried out in the present investigation, where the impact of the constitutive overexpression of the transcription factor HAC1 under the control of two constitutive promoters (i.e., pMDH3 and pGAP) in strains in which the assimilative and dissimilative route of methanol has been modified through the overexpression or deletion of genes involved in the methanolcatabolism. The lipase CalB was used as a model of recombinant protein, and its production yield was investigated.
Herein, the focus was directed towards expressing a key transcription factor gene HAC1, involved in the unfolded protein response in two different genotypes in a MUT+ strain modified in the dissimilative pathway and assimilative pathway to have a metabolic enhancement. The expression plasmids were constructed using the GoldenPICS toolbox expression vectors and CalB as a reporter gene. The comparison of CalB production and secretion in three different genotypes, namely the control, weak promoter pMDH3 and strong promoter GAP constitutive revealed intriguing findings in the different genotypes. Remarkably, the genotype based on the weak promoter pMDH3 for CalB expression has demonstrated significantly higher CalB activity (30 %) as compared to the control conditions.