PDF(Pubmed)
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.
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.
摘要：
背景：Komagataellaphaffii（又称巴斯德毕赤酵母）具有谷氨酸利用途径，其中谷氨酸脱氢酶2和磷酸烯醇丙酮酸羧激酶（PEPCK）的合成由谷氨酸诱导。这些酶的谷氨酸诱导合成受Rtg1p调节，细胞溶质，碱性螺旋-环-螺旋蛋白。这里,我们报道了使用绿色荧光蛋白（GFP）和SARS-CoV-2病毒（RBD）的受体结合域作为模型蛋白，从K.phafiiPEPCK启动子（PPEPCK）产生食品级味精（MSG）诱导的重组蛋白。
结果:将PPEPCK-RBD/GFP表达盒整合在基因组的两个不同位点,以提高来自PPEPCK的重组蛋白产量。传统的,甲醇诱导型醇氧化酶1启动子(PAOX1)用作基准。用MSG作为诱导物进行的初步研究导致低重组蛋白产量。采用MSG/乙醇混合补料的新策略改善了生物质生成以及重组蛋白产量。在摇瓶培养中诱导72小时后，细胞密度达到100-120A600单位/ml。导致来自PPEPCK的重组蛋白产量与来自PAOX1的重组蛋白产量相当或甚至更高。
结论：我们设计了一种诱导培养基，用于在摇瓶培养中从K.phafiiPPEPCK生产重组蛋白。它由1.0%酵母提取物组成，2.0%蛋白胨,含硫酸铵的0.17%酵母氮碱，100mM磷酸钾（pH6.0），0.4mg/L生物素，2.0%味精，和2%的乙醇。用0.5%尿素取代硫酸铵是任选的。在72h诱导期间每24h补充碳源。在这些条件下,PPEPCK的GFP和RBD产量等于甚至超过PAOX1的产量。与传统的甲醇诱导表达系统相比,谷氨酸诱导表达系统的诱导剂是无毒的，它们的代谢不产生甲醛和过氧化氢等有毒代谢产物。这项研究为MSG诱导型奠定了基础，在生物反应器中从K.phafiiPPEPCK生产工业规模的重组蛋白。
结果:将PPEPCK-RBD/GFP表达盒整合在基因组的两个不同位点,以提高来自PPEPCK的重组蛋白产量。传统的,甲醇诱导型醇氧化酶1启动子(PAOX1)用作基准。用MSG作为诱导物进行的初步研究导致低重组蛋白产量。采用MSG/乙醇混合补料的新策略改善了生物质生成以及重组蛋白产量。在摇瓶培养中诱导72小时后，细胞密度达到100-120A600单位/ml。导致来自PPEPCK的重组蛋白产量与来自PAOX1的重组蛋白产量相当或甚至更高。
结论：我们设计了一种诱导培养基，用于在摇瓶培养中从K.phafiiPPEPCK生产重组蛋白。它由1.0%酵母提取物组成，2.0%蛋白胨,含硫酸铵的0.17%酵母氮碱，100mM磷酸钾（pH6.0），0.4mg/L生物素，2.0%味精，和2%的乙醇。用0.5%尿素取代硫酸铵是任选的。在72h诱导期间每24h补充碳源。在这些条件下,PPEPCK的GFP和RBD产量等于甚至超过PAOX1的产量。与传统的甲醇诱导表达系统相比,谷氨酸诱导表达系统的诱导剂是无毒的，它们的代谢不产生甲醛和过氧化氢等有毒代谢产物。这项研究为MSG诱导型奠定了基础，在生物反应器中从K.phafiiPPEPCK生产工业规模的重组蛋白。
