Knock-in at GluA1 locus improves recombinant human serum albumin expression in rice grain

Improving recombinant protein expression is a perpetual goal for molecular pharming. However, over-transcription of recombinant protein induces ER stress, and causes protein degradation. Here, we describe a knock-in approach to integrate a human serum albumin expression cassette into the locus of the rice storage protein GluA1 by site-specific integration via the nonhomologous end joining (NHEJ) pathway. The expression level of OsrHSA in the knock-in (KI) lines was much higher than that of the random integration (RI) lines. ER stress in KI line endosperm cells was not significantly altered even after massive OsrHSA accumulation in rice endosperm cell. Instead, ER stress induced by high OsrHSA expression was alleviated in the KI line via the inositol-requiring enzyme 1 (IRE1)-mediated/OsbZIP50 pathway. Furthermore, improvement of OsrHSA expression in KI lines is likely due to reduction of contents of glutelin and globulin in rice endosperm cell. These results provide insight into an approach to improving recombinant protein accumulation by alleviating ER stress and protein trafficking.