Biliverdin,a bile pigment hydrolyzed from heme by heme oxygenase(HO),serves multiple functions in the human body,including antioxidant,anti-inflammatory,and immune response inhibitory activities.Biliverdin has great p...Biliverdin,a bile pigment hydrolyzed from heme by heme oxygenase(HO),serves multiple functions in the human body,including antioxidant,anti-inflammatory,and immune response inhibitory activities.Biliverdin has great potential as a clinical drug;however,no economic and efficient production method is available currently.Therefore,the production of biliverdin by the biotransformation of exogenous heme using recombinant HO-expressing yeast cells was studied in this research.First,the heme oxygenase-1 gene(HO1)encoding the inducible plastidic isozyme from Arabidopsis thaliana,with the plastid transport peptide sequence removed,was recombined into Pichia pasto-ris GS115 cells.This resulted in the construction of a recombinant P.pastoris GS115-HO1 strain that expressed active HO1 in the cytoplasm.After that,the concentration of the inducer methanol,the induction culture time,the pH of the medium,and the concentration of sorbitol supplied in the medium were optimized,resulting in a significant improvement in the yield of HO1.Subsequently,the whole cells of GS115-HO1 were employed as catalysts to convert heme chloride(hemin)into biliverdin.The results showed that the yield of biliverdin was 132 mg/L when hemin was added to the culture of GS115-HO1 and incubated for 4 h at 30°C.The findings of this study have laid a good foundation for future applications of this method for the economical production of biliverdin.展开更多
Pectin lyase(PMGL)is an industrially important enzyme with widespread applications in the food,paper,and textile industries,owing to its capacity for direct degradation of highly esterified pectin.In this study,PMGL-B...Pectin lyase(PMGL)is an industrially important enzyme with widespread applications in the food,paper,and textile industries,owing to its capacity for direct degradation of highly esterified pectin.In this study,PMGL-Ba derived from Bacillus licheniformis underwent mining and heterologous expression in P.pastoris.Furthermore,diverse strategies,encompassing the optimization of expression cassette components,elevation of gene dosage,and co-expression of chaperone factors,were employed to augment PMGL-Ba production in P.pastoris.The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements.By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup,a strain yielding high PMGL-Ba production was achieved.In shake flask fermentation lasting 144 h,the total protein concentration reached 1.81 g/L,and the enzyme activity reached 1821.36 U/mL.For further scale up production,high-density fermentation transpired in a 5 L fermenter for 72 h.Remarkably,the total protein concentration increased to 12.49 g/L,and the enzyme activity reached an impressive 12668.12 U/mL.The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications.展开更多
[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was ...[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.展开更多
[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Metho...[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZa-A yeast expression vector to construct the secretory recombinant expression vector pPICZa-A-P97R1. The plasmid pPICZa-A-p97R1 linearized by Sac I was transformed into P. pastoris GSl15 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting anal.wsis. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secre- tory amount of 499μg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for anti- body detection and genetically engineered vaccine to Mhp.展开更多
基金financially supported by the Key Research and Development Program of Zhejiang Province(2021C03088-2).
文摘Biliverdin,a bile pigment hydrolyzed from heme by heme oxygenase(HO),serves multiple functions in the human body,including antioxidant,anti-inflammatory,and immune response inhibitory activities.Biliverdin has great potential as a clinical drug;however,no economic and efficient production method is available currently.Therefore,the production of biliverdin by the biotransformation of exogenous heme using recombinant HO-expressing yeast cells was studied in this research.First,the heme oxygenase-1 gene(HO1)encoding the inducible plastidic isozyme from Arabidopsis thaliana,with the plastid transport peptide sequence removed,was recombined into Pichia pasto-ris GS115 cells.This resulted in the construction of a recombinant P.pastoris GS115-HO1 strain that expressed active HO1 in the cytoplasm.After that,the concentration of the inducer methanol,the induction culture time,the pH of the medium,and the concentration of sorbitol supplied in the medium were optimized,resulting in a significant improvement in the yield of HO1.Subsequently,the whole cells of GS115-HO1 were employed as catalysts to convert heme chloride(hemin)into biliverdin.The results showed that the yield of biliverdin was 132 mg/L when hemin was added to the culture of GS115-HO1 and incubated for 4 h at 30°C.The findings of this study have laid a good foundation for future applications of this method for the economical production of biliverdin.
基金supported by the National Key R&D Program of China(NO.2021YFC2100400).
文摘Pectin lyase(PMGL)is an industrially important enzyme with widespread applications in the food,paper,and textile industries,owing to its capacity for direct degradation of highly esterified pectin.In this study,PMGL-Ba derived from Bacillus licheniformis underwent mining and heterologous expression in P.pastoris.Furthermore,diverse strategies,encompassing the optimization of expression cassette components,elevation of gene dosage,and co-expression of chaperone factors,were employed to augment PMGL-Ba production in P.pastoris.The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements.By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup,a strain yielding high PMGL-Ba production was achieved.In shake flask fermentation lasting 144 h,the total protein concentration reached 1.81 g/L,and the enzyme activity reached 1821.36 U/mL.For further scale up production,high-density fermentation transpired in a 5 L fermenter for 72 h.Remarkably,the total protein concentration increased to 12.49 g/L,and the enzyme activity reached an impressive 12668.12 U/mL.The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications.
文摘[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.
基金Supported by the National Natural Science Foundation of China(31100136)the Special Fund for Independent Innovation of Agricultural Science and Technology in Jiangsu Province[CX(12)5051]~~
文摘[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZa-A yeast expression vector to construct the secretory recombinant expression vector pPICZa-A-P97R1. The plasmid pPICZa-A-p97R1 linearized by Sac I was transformed into P. pastoris GSl15 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting anal.wsis. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secre- tory amount of 499μg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for anti- body detection and genetically engineered vaccine to Mhp.
