Molecular dissection of Komagataella phaffii Rtg1–RtgX function and development of a glutamate-responsive protein expression platform

Abstract

In Saccharomyces cerevisiae, the basic helix-loop-helix leucine zipper (bHLH-LZ) proteins Rtg1 and Rtg3 form a heterodimeric transcription factor complex that mediates the retrograde response. In Candida albicans, these proteins regulate galactose and sphingolipid metabolism and contribute to virulence. In the methylotrophic yeast Komagataella phaffii (previously Pichia pastoris), Rtg1 (KpRtg1) regulates the expression of GDH2 (glutamate dehydrogenase 2) and PEPCK (phosphoenolpyruvate carboxykinase) post-transcriptionally during glutamate metabolism, and transcription of AOX1 (alcohol oxidase 1) during methanol metabolism. We identified a putative RTG3 homolog (NCBI accession AOA70166.1) that lacks the conserved leucine zipper motif and fails to interact with KpRtg1 in vitro when expressed as recombinant protein in Escherichia coli. We therefore designate this protein as KpRtgX. Here, we demonstrate that KpRtgX localizes to both cytosol and nucleus and functions as a dual regulator of gene expression. In the cytosol, KpRtgX post-transcriptionally regulates GDH2 and PEPCK, while in the nucleus it transcriptionally activates AOX1. Co-immunoprecipitation of epitope-tagged proteins expressed in K. phaffii reveals that KpRtgX physically associates with KpRtg1 via conserved arginine residues within its bHLH domain. Notably, KpRtgX protein is undetectable in ∆KpRtg1, indicating that its stability or expression is KpRtg1-dependent. Our study uncovers a unique variation of yeast Rtg1–Rtg3 signaling, wherein KpRtgX, a non-canonical Rtg3 homolog lacking the conserved leucine zipper motif interacts with KpRtg1 via conserved arginine residues in bHLH domain and functions as both transcriptional and post-transcriptional regulator of methanol and glutamate metabolism respectively. Synthesis of GDH2 and PEPCK is induced maximally by glutamate and this property was exploited to develop a novel glutamate-inducible recombinant protein expression system based on the K. phaffii PEPCK promoter. Using food-grade monosodium glutamate (MSG) and ethanol as the inducers, we demonstrate production of two recombinant proteins (green fluorescent protein, GFP and receptor binding domain of SARS-CoV-2 virus, RBD) from K. phaffii PEPCK promoter (PPEPCK). Initial studies carried out with MSG alone as the inducer resulted in low recombinant protein yield necessitating the inclusion of ethanol to improve biomass and recombinant protein yield. Media optimization led to the development of two culture media referred to as INDI-1 (1.0% yeast extract, 2.0% peptone, 0.17% yeast nitrogen base with ammonium sulfate, 100 mM potassium phosphate, pH 6.0, 0.4 mg/L biotin, 2.0% MSG, and 2% ethanol) and INDI-2 (1.0% yeast extract, 2.0% peptone, 0.17% yeast nitrogen base without ammonium sulphate, 0.5% urea, 100 mM potassium phosphate, pH 6.0, 0.4 mg/L biotin, 2.0% MSG, and 2% ethanol.) Cell density of 100–120 A600 units/ml was achieved after 72 h of induction in shake flask cultivations, resulting in recombinant protein yield from PPEPCK that is comparable or even higher than that from PAOX1. Compared to the traditional methanol-inducible expression system, the inducers of glutamate-inducible expression system are non-toxic and their metabolism does not generate toxic metabolites such as formaldehyde and hydrogen peroxide. This study sets the stage for MSG-inducible, industrial scale recombinant protein production from K. phaffii PPEPCK in bioreactors in collaboration with biotech companies.