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Unlocking Nature's Toolbox: glutamate-inducible recombinant protein production from the Komagatella phaffii PEPCK promoter.
Rajak, Neetu; Dey, Trishna; Sharma, Yash; Bellad, Vedanth; Rangarajan, Pundi N.
Afiliación
  • Rajak N; Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India.
  • Dey T; Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India.
  • Sharma Y; Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India.
  • Bellad V; Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India.
  • Rangarajan PN; Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India. pnr@iisc.ac.in.
Microb Cell Fact ; 23(1): 66, 2024 Feb 24.
Article en En | MEDLINE | ID: mdl-38402195
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 (PPEPCK) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins.

RESULTS:

PPEPCK-RBD/GFP expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from PPEPCK. The traditional, methanol-inducible alcohol oxidase 1 promoter (PAOX1) 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 PPEPCK that is comparable or even higher than that from PAOX1.

CONCLUSIONS:

We have designed an induction medium for recombinant protein production from K. phaffii PPEPCK 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 PPEPCK equaled and even surpassed those 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.
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Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Metanol / Saccharomycetales Idioma: En Revista: Microb Cell Fact Asunto de la revista: BIOTECNOLOGIA / MICROBIOLOGIA Año: 2024 Tipo del documento: Article País de afiliación: India

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Metanol / Saccharomycetales Idioma: En Revista: Microb Cell Fact Asunto de la revista: BIOTECNOLOGIA / MICROBIOLOGIA Año: 2024 Tipo del documento: Article País de afiliación: India