Towards improved membrane protein production in Pichia pastoris: General and specific transcriptional response to membrane protein overexpression

Membrane proteins are the largest group of human drug targets and are also used as biocatalysts. However, due to their complexity, efficient expression remains a bottleneck for high level production. In recent years, the methylotrophic yeast Pichia pastoris has emerged as one of the most commonly used expression systems for membrane protein production. Here, we have analysed the transcriptomes of P. pastoris strains producing different classes of membrane proteins (mitochondrial, ER/Golgi and plasma membrane localized) to understand the cellular response and to identify targets to engineer P. pastoris towards an improved chassis for membrane protein production. Microarray experiments revealed varying transcriptional responses depending on the enzymatic activity, subcellular localization and physiological role of the membrane proteins. While an alternative oxidase evoked primarily a response within the mitochondria, the overexpression of transporters entering the secretory pathway had a wide effect on lipid metabolism and induced the upregulation of the UPR (unfolded protein response) transcription factor Hac1p. Coexpression of P. pastoris endogenous HAC1 increased the levels of ER-resident membrane proteins 1.5- to 2.1-fold. Subsequent transcriptome analysis of HAC1 coexpression revealed an upregulation of the folding machinery correlating with an expansion of the ER membrane capacity, thus boosting membrane protein production. Hence, our study has helped to elucidate the cellular response of P. pastoris to the expression of different classes of membrane proteins and led specifically to new insights into the effect of PpHac1p on membrane proteins entering the secretory pathway.