Unlocking Nature’s Toolbox: glutamate-inducible recombinant protein production from the Komagatella phaffii PEPCK promoter

Abstract

Abstract Background Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase (PEPCK) is induced by glutamate. Glutamate-inducible synthesis of these enzymes is regulated by Rtg1p, a cytosolic, basic helix-loop-helix protein. Here, we report food-grade monosodium glutamate (MSG)-inducible recombinant protein production from K. phaffii PEPCK promoter (P PEPCK ) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins. Results P PEPCK -RBD/GFP expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from P PEPCK . The traditional, methanol-inducible alcohol oxidase 1 promoter (P AOX1 ) was used as the benchmark. Initial studies carried out with MSG as the inducer resulted in low recombinant protein yield. A new strategy employing MSG/ethanol mixed feeding improved biomass generation as well as recombinant protein yield. 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 P PEPCK that is comparable or even higher than that from P AOX1 . Conclusions We have designed an induction medium for recombinant protein production from K. phaffii P PEPCK in shake flask cultivations. It consists of 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. Substitution of ammonium sulphate with 0.5% urea is optional. Carbon source was replenished every 24 h during 72 h induction period. Under these conditions, GFP and RBD yields from P PEPCK equaled and even surpassed those from P AOX1 . 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 P PEPCK in bioreactors.

Keywords

• Komagataella phaffii
• PEPCK promoter
• Glutamate inducible expression system
• Monosodium glutamate