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Pichia pastoris)<\/jats:italic> 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 <jats:italic>K. phaffii PEPCK<\/jats:italic> promoter (<jats:italic>P<\/jats:italic><jats:sub><jats:italic>PEPCK<\/jats:italic><\/jats:sub>) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p><jats:italic>P<\/jats:italic><jats:sub><jats:italic>PEPCK<\/jats:italic><\/jats:sub><jats:italic>-RBD\/GFP<\/jats:italic> expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from <jats:italic>P<\/jats:italic><jats:sub><jats:italic>PEPCK<\/jats:italic><\/jats:sub>. The traditional, methanol-inducible alcohol oxidase 1 promoter (<jats:italic>P<\/jats:italic><jats:sub><jats:italic>AOX1<\/jats:italic><\/jats:sub>) 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\u2013120 A<jats:sub>600<\/jats:sub> units\/ml was achieved after 72\u00a0h of induction in shake flask cultivations, resulting in recombinant protein yield from <jats:italic>P<\/jats:italic><jats:sub><jats:italic>PEPCK<\/jats:italic><\/jats:sub> that is comparable or even higher than that from<jats:italic> P<\/jats:italic><jats:sub><jats:italic>AOX1<\/jats:italic><\/jats:sub>.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>We have designed an induction medium for recombinant protein production from <jats:italic>K. phaffii P<\/jats:italic><jats:sub><jats:italic>PEPCK<\/jats:italic><\/jats:sub> in shake flask cultivations. It consists of 1.0% yeast extract, 2.0% peptone, 0.17% yeast nitrogen base with ammonium sulfate, 100\u00a0mM potassium phosphate (pH 6.0), 0.4\u00a0mg\/L biotin, 2.0% MSG, and 2% ethanol. Substitution of ammonium sulphate with 0.5% urea is optional. Carbon source was replenished every 24\u00a0h during 72\u00a0h induction period. Under these conditions, GFP and RBD yields from <jats:italic>P<\/jats:italic><jats:sub><jats:italic>PEPCK<\/jats:italic><\/jats:sub> equaled and even surpassed those from <jats:italic>P<\/jats:italic><jats:sub><jats:italic>AOX1<\/jats:italic><\/jats:sub>. 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 <jats:italic>K. phaffii P<\/jats:italic><jats:sub><jats:italic>PEPCK<\/jats:italic><\/jats:sub> in bioreactors.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12934-024-02340-1","type":"journal-article","created":{"date-parts":[[2024,2,24]],"date-time":"2024-02-24T09:02:09Z","timestamp":1708765329000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Unlocking Nature\u2019s Toolbox: glutamate-inducible recombinant protein production from the Komagatella phaffii PEPCK promoter"],"prefix":"10.1186","volume":"23","author":[{"given":"Neetu","family":"Rajak","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Trishna","family":"Dey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yash","family":"Sharma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vedanth","family":"Bellad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pundi N.","family":"Rangarajan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,2,24]]},"reference":[{"key":"2340_CR1","doi-asserted-by":"publisher","first-page":"20588","DOI":"10.1074\/jbc.M116.740191","volume":"291","author":"U Sahu","year":"2016","unstructured":"Sahu U, Rangarajan PN. Methanol expression regulator 1 (Mxr1p) is essential for the utilization of amino acids as the sole source of carbon by the methylotrophic yeast. Pichia pastoris J Biol Chem. 2016;291:20588.","journal-title":"Pichia pastoris J Biol Chem"},{"key":"2340_CR2","doi-asserted-by":"publisher","first-page":"16647","DOI":"10.1074\/jbc.RA118.004486","volume":"293","author":"T Dey","year":"2018","unstructured":"Dey T, Rao KK, Khatun J, Rangarajan PN. The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris. J Biol Chem. 2018;293:16647\u201360.","journal-title":"J Biol Chem"},{"key":"2340_CR3","doi-asserted-by":"publisher","first-page":"25","DOI":"10.1016\/j.bbrc.2021.10.015","volume":"581","author":"T Dey","year":"2021","unstructured":"Dey T, Rangarajan PN. Carbon starvation-induced synthesis of GDH2 and PEPCK is essential for the survival of Pichia pastoris. 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U.S. patent is pending (Application No.18\/266,357). The glutamate-inducible <i>K. phaffii<\/i> expression system has been licensed to Biogrammatics Inc., USA.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"66"}}