The type B Arabidopsis Response Regulators (ARRs) of Arabidopsis thaliana are transcription factors that act as positive regulators in the two-component cytokinin signaling pathway. We employed a mutant-based approach to perform a detailed characterization of the roles of ARR1, ARR10, and ARR12 in plant growth and development. The most pronounced phenotype was found in the arr1-3 arr10-5 arr12-1 triple loss-of-function mutant, which showed almost complete insensitivity to high levels of exogenously applied cytokinins. The triple mutant exhibited reduced stature due to decreased cell division in the shoot, enhanced seed size, increased sensitivity to light, altered chlorophyll and anthocyanin concentrations, and an aborted primary root with protoxylem but no metaxylem. Microarray analysis revealed that expression of the majority of cytokinin-regulated genes requires the function of ARR1, ARR10, and ARR12. Characterization of double mutants revealed differing contributions of the type B ARRs to mutant phenotypes. Our results support a model in which cytokinin regulates a wide array of downstream responses through the action of a multistep phosphorelay that culminates in transcriptional regulation by ARR1, ARR10, and ARR12.

bWith a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors. Since the approval of the first biopharmaceutical product (recombinant insulin) in 1982, biopharmaceuticals, as a prescription drug class, have enjoyed the highest growth rate within the pharmaceutical industry (1). With more than 230 approved products currently on the market, biopharmaceuticals are playing vital roles in the prevention and treatment of a wide variety of diseases, ranging across infectious diseases, inflammatory disorders, metabolic diseases, and cancer. Most biopharmaceuticals are manufactured from one of three different expression host systems: mammalian cells, yeasts, and bacteria (2). Bacterial systems (e.g., Escherichia coli) are the industry standard expression hosts for proteins that do not require posttranslational modifications (e.g., insulin, growth hormone, granulocyte colony-stimulating factor [G-CSF]). To date, mammalian cells (e.g., CHO) have been the preferred expression system for glycoproteins and monoclonal antibodies (MAbs), primarily due to their ability to produce glycosylation patterns adequate for drug safety and efficacy. Despite the advantages of being unicellular eukaryotes with efficient protein-folding and posttranslational-modification capabilities, yeasts are used for the production of only a small number of therapeutic proteins, because their high-mannose glycosylation patterns are not optimal for drug safety and efficacy.Glycoengineered Pichia pastoris has recently emerged as a promising production host for the manufacture of therapeutic ...

Protein O-mannosyltransferases (PMTs) catalyze the initial reaction of protein O-mannosylation by transferring the first mannose unit onto serine and threonine residues of a nascent polypeptide being synthesized in the endoplasmic reticulum (ER). The PMTs are well conserved in eukaryotic organisms, and in vivo defects of these enzymes result in cell death in yeast and congenital diseases in humans. A group of rhodanine-3-acetic acid derivatives (PMTi) specifically inhibits PMT activity both in vitro and in vivo. As such, these chemical compounds have been effectively used to minimize the extent of O-mannosylation on heterologously produced proteins from different yeast expression hosts. However, very little is known about how these PMT-inhibitors interact with the PMT enzyme, or what structural features of the PMTs are required for inhibitor-protein interactions. To better understand the inhibitor-enzyme interactions, and to gain potential insights for developing more effective PMT-inhibitors, we isolated PMTi-resistant mutants in Pichia pastoris. In this study, we report the identification and characterization of a point mutation within the PpPMT2 gene. We demonstrate that this F664S point mutation resulted in a near complete loss of PMTi sensitivity, both in terms of growth-inhibition and reduction in O-mannosylglycan site occupancy. Our results provide genetic evidence demonstrating that the F664 residue plays a critical role in mediating the inhibitory effects of these PMTi compounds. Our data also indicate that the main target of these PMT-inhibitors in P. pastoris is Pmt2p, and that the F664 residue most likely interacts directly with the PMTi-compounds.