title: Comparative performance of S-adenosyl-L-methionine biosynthesis and degradation in Pichia pastoris using different promoters and novel consumption inhibitors.
abstract: The yeast Pichia pastoris is a widely used host for recombinant protein expression, and has recently been engineered for whole-cell biocatalysis. The inducible P(AOX) and constitutive P(GAP) promoters are commonly employed. In this study, the S-adenosyl-L-methionine (SAM) biosynthesis and degradation efficiency of two P. pastoris strains were compared, and novel inhibitors that suppress SAM degradation were characterized. The strains exhibited clear physiological differences. P(GAP)-Pichia showed higher transcription and activity of SAM synthetase, and the rapid cell growth led to higher levels of spermidine synthesis from SAM. In contrast, P(AOX)-Pichia synthesized higher levels of glutathione from SAM, and this strain responded to hydrogen peroxide formation during methanol utilization. Aristeromycin proved an efficient inhibitor of SAM degradation in P(AOX)-Pichia; 0.02 mg/L led to a 36.36% reduction in the ratio of glutathionine:SAM, and SAM accumulation was enhanced by 7.74% to 11.83 g/L. Ethanol was an even more efficient inhibitor of SAM consumption in P(GAP)-Pichia; 8 g/L resulted in a 73.68% decrease in the ratio of SPD:SAM, and SAM production was elevated by 54.55% to 0.17 g/L/h.
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, and State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Ju
authors.initials: J
authors.lastname: Chu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. Electronic address: juchu@ecust.edu.cn.
authors.forename: Siliang
authors.initials: S
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Yingping
authors.initials: Y
authors.lastname: Zhuang
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics; D000431:Ethanol / Q000494:pharmacology; D005285:Fermentation; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic; D005978:Glutathione / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D008716:Methionine Adenosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013095:Spermidine / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C030034:aristeromycin; D000431:Ethanol; D012436:S-Adenosylmethionine; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008716:Methionine Adenosyltransferase; D005978:Glutathione; D000241:Adenosine; D013095:Spermidine; D000432:Methanol
keywords: Biosynthesis; Constitutive promoter; Consumption; Inducible promoter; Pichia pastoris; S-adenosyl-l-methionine