Enhanced production of stem bromelain in Pichia pastoris by coexpression of unfolded protein response activator gene...

Expression of heterologous proteins and metabolites at high titers mounts several stress responses on the recombinant host. Stem Bromelain is a cysteine protease enzyme present in the stem and fruit of the pineapple plant Ananas comosus. The enzyme has a broad range of industrial application ranging from food, nutraceutical, cosmetic and pharmaceutical. The current work aims to study the effect of unfolded protein response regulator (UPR) Hac1 on folding and secretion of recombinant stem bromelain in Pichia pastoris. Stem bromelain gene (BL) from Ananas comosus was codon optimized and expressed in the Pichia pastoris X-33 host using constitutive and inducible promoters. To fold the misfolded protein aggregates in Endoplasmic Reticulum (ER), UPR regulator, Hac1p was co-expressed with bromelain under constitutive expression by GAP promoter. Shake flask studies resulted in a 2-fold increase in the protease activity of 4 U/mL when HAC1 was co-expressed with stem bromelain (BL). Fed batch studies were performed for both pGAPαBL1 and pGAPBLHAC1 clones in 3.7 L KLF bioreactor under glycerol limited condition and highest activity of 8 U/mL and 54 U/mL were obtained respectively. Gene expression studies of the major genes in folding and secretion pathway has shown that the activation of UPR has resulted in upregulation of major chaperones like Kar2p, Sec 63, Pdi, Cne1. The stem bromelain activity of 54 U/mL is the highest activity reported so far in the literature. The current work signifies Pichia pastoris as a robust platform to produce stem bromelain for various industrial applications.