CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain.

Michael Panting,Inger Baeksted Holme,Yingxin Zhong,Jón Már Björnsson,Henrik Brinch-Pedersen

doi:10.3389/fpls.2021.755788

Abstract

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.

Highlights

The production of recombinant proteins in plants are of growing interest, but the yield from plants is mostly not competitive to traditional host systems like bacteria, yeast, and CHO cells
Commercial LIF recombinant protein is traditionally expressed in Escherichia coli (E. coli), but there is recombinant human LIF on the market produced in rice (Merck), barley (ORF Genetics), and recombinant mouse LIF produced in barley (ORF Genetics)
The knockout lines were named with Golden Promise (GP) followed by the knockout gene in superscript small letters (GPhsp70, GPhsp26, GPhsp16.9, GPgst, GPcrt, GPipi, and GPpdi)

Summary

INTRODUCTION

The production of recombinant proteins in plants are of growing interest, but the yield from plants is mostly not competitive to traditional host systems like bacteria, yeast, and CHO cells. The seven candidate genes selected for this study had an increased translation when aleurone layers were incubated with Giberillic acid (GA3) and TN (Barba-Espin et al, 2014) These were Protein disulfide isomerase (PDI), Calreticulin (CRT), heat shock proteins (Hsps) Hsp, Hsp16.9 kDA, Hsp26kDa, Glutathione-Stransferase (GST), and Isopentenyl diphosphate delta isomerase (IPI). First we wanted to study barley candidate genes for their involvement in UPR through inducible ER-stress This was done with an assay on germinating seeds, introducing TN during germination to induce ER-stress and initiate the UPR, mimmicking the high protein synthesis situation of grain maturation. EGF is a small peptide with three disulfide bonds and no glycosylation, while mLIF has three disuplhide bonds, but hosts several predicted glycosylation sites

MATERIALS AND METHODS

RESULTS

DISCUSSION

DATA AVAILABILITY STATEMENT