为探究产酯酶库德里阿兹威毕赤酵母(Pichia kudriavzevii)混菌发酵的代谢机制,本研究使用串联质谱标签(tandem mass tags,TMT)定量蛋白质组学技术,对P.kudriavzevii JM5-4单菌纯培养以及与丁酸梭菌(Clostridium butyricum)GD1-1共培养...为探究产酯酶库德里阿兹威毕赤酵母(Pichia kudriavzevii)混菌发酵的代谢机制,本研究使用串联质谱标签(tandem mass tags,TMT)定量蛋白质组学技术,对P.kudriavzevii JM5-4单菌纯培养以及与丁酸梭菌(Clostridium butyricum)GD1-1共培养条件下的蛋白质组学进行分析。利用气相色谱-质谱联用仪检测单菌与混菌发酵时的代谢产物。结果显示,混菌发酵时发酵液中乙醇、乙酸、乙酸乙酯的产量分别为2.396、0.425 g/L和0.544 g/L,分别是单菌发酵的1.5、4.0倍和2.0倍。利用TMT定量蛋白质组学技术鉴定到3164个可定量蛋白质。共筛选到355个差异表达蛋白(differentially expressed proteins,DEPs),包括上调蛋白159个、下调蛋白质196个。基因本体论和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库功能富集分析显示,三羧酸循环、药物代谢、小分子代谢、细胞质大核糖体亚基、细胞质核糖体、烟酰胺腺嘌呤二核苷酸结合、氧化还原酶活性等定位蛋白质发生了显著性变化。利用CELLO软件对DEPs进行亚细胞定位,将257个蛋白质定位到8个不同的细胞器,主要分布在细胞质(129个)、线粒体(91个)、细胞核(19个)等。355个DEPs通过KEGG注释显示其主要涉及氧化磷酸化、辅因子的生物合成、三羧酸循环、糖酵解/糖异生等代谢亚系统。在混菌发酵时,糖酵解/糖异生(丙酮酸脱氢酶、醛脱氢酶等)、三羧酸代谢(柠檬酸合酶、乌头酸盐水合酶、富马酸水合酶、苹果酸脱氢酶等)、乙醛酸和二羧酸代谢(羟基丙酮酸还原酶、乙醛酸还原酶等)的DEPs全部上调,表明共培养条件下明显促进了JM5-4的生长与代谢。此外,涉及酯酶(S-甲酰谷胱甘肽水解酶、核糖核酸酶、海藻糖磷酸酶)的DEPs全部上调,表明混菌发酵对JM5-4产酯酶能力具有一定的积极作用。本研究可为探究白酒酿造过程中的混菌发酵机制、菌株代谢特性以及提高白酒风味品质提供一定的理论指导。展开更多
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
文摘为探究产酯酶库德里阿兹威毕赤酵母(Pichia kudriavzevii)混菌发酵的代谢机制,本研究使用串联质谱标签(tandem mass tags,TMT)定量蛋白质组学技术,对P.kudriavzevii JM5-4单菌纯培养以及与丁酸梭菌(Clostridium butyricum)GD1-1共培养条件下的蛋白质组学进行分析。利用气相色谱-质谱联用仪检测单菌与混菌发酵时的代谢产物。结果显示,混菌发酵时发酵液中乙醇、乙酸、乙酸乙酯的产量分别为2.396、0.425 g/L和0.544 g/L,分别是单菌发酵的1.5、4.0倍和2.0倍。利用TMT定量蛋白质组学技术鉴定到3164个可定量蛋白质。共筛选到355个差异表达蛋白(differentially expressed proteins,DEPs),包括上调蛋白159个、下调蛋白质196个。基因本体论和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库功能富集分析显示,三羧酸循环、药物代谢、小分子代谢、细胞质大核糖体亚基、细胞质核糖体、烟酰胺腺嘌呤二核苷酸结合、氧化还原酶活性等定位蛋白质发生了显著性变化。利用CELLO软件对DEPs进行亚细胞定位,将257个蛋白质定位到8个不同的细胞器,主要分布在细胞质(129个)、线粒体(91个)、细胞核(19个)等。355个DEPs通过KEGG注释显示其主要涉及氧化磷酸化、辅因子的生物合成、三羧酸循环、糖酵解/糖异生等代谢亚系统。在混菌发酵时,糖酵解/糖异生(丙酮酸脱氢酶、醛脱氢酶等)、三羧酸代谢(柠檬酸合酶、乌头酸盐水合酶、富马酸水合酶、苹果酸脱氢酶等)、乙醛酸和二羧酸代谢(羟基丙酮酸还原酶、乙醛酸还原酶等)的DEPs全部上调,表明共培养条件下明显促进了JM5-4的生长与代谢。此外,涉及酯酶(S-甲酰谷胱甘肽水解酶、核糖核酸酶、海藻糖磷酸酶)的DEPs全部上调,表明混菌发酵对JM5-4产酯酶能力具有一定的积极作用。本研究可为探究白酒酿造过程中的混菌发酵机制、菌株代谢特性以及提高白酒风味品质提供一定的理论指导。
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.
