Mytilus contain abundant antimicrobial peptides(AMPs)that play a key role in the innate immunity.However,heterologous production of these AMPs remains challenging due to their short sequences,multiple disulfide bonds,...Mytilus contain abundant antimicrobial peptides(AMPs)that play a key role in the innate immunity.However,heterologous production of these AMPs remains challenging due to their short sequences,multiple disulfide bonds,and high content of cationic amino acids,which hinder functional expression in prokaryotic systems such as Escherichia coli.To establish a eukaryotic recombinant expression system for the AMPs of mussel and obtain recombinant mussel AMPs for subsequent studies,we reported the successful recombinant expression of myticofensin B1,a novel defensin-like AMP identified previously in Mytiluscoruscus,using the eukaryotic host Pichia pastoris.The codon-optimized gene encoding the mature myticofensin-B1(composed of 65 amino acid residues,including 6 conserved cysteine residues)was cloned into a pPICZαA vector and expressed in P.pastoris GS115.Structural fidelity of the recombinant peptide was confirmed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),showing a molecular weight of 8849.9 Da,which was consistent with the theoretical prediction.Functional assays demonstrated a broad-spectrum antimicrobial activity of the recombinant myticofensin-B1,with stronger inhibition against Gram-negative bacteria.Scanning electron microscopy revealed different effects of the recombinant myticofensin-B1 against different bacteria.In addition,the recombinant myticofensin-B1 exhibited a very low hemolytic activity against sheep red blood cells and weak cytotoxicity against human A549 lung cancer cells.This study establishes P.pastoris as a powerful platform to produce functional mussel AMP and highlights the potential of the recombinant myticofensin-B1 as a therapeutic agent for aquaculture pathogens and infections.展开更多
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
[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
基金supported by the National Natural Science Foundation of China(32271580,42020104009)the Fundamental Research Funds for Zhejiang Provincial Universities and Research Institutes(JX6311101923)。
文摘Mytilus contain abundant antimicrobial peptides(AMPs)that play a key role in the innate immunity.However,heterologous production of these AMPs remains challenging due to their short sequences,multiple disulfide bonds,and high content of cationic amino acids,which hinder functional expression in prokaryotic systems such as Escherichia coli.To establish a eukaryotic recombinant expression system for the AMPs of mussel and obtain recombinant mussel AMPs for subsequent studies,we reported the successful recombinant expression of myticofensin B1,a novel defensin-like AMP identified previously in Mytiluscoruscus,using the eukaryotic host Pichia pastoris.The codon-optimized gene encoding the mature myticofensin-B1(composed of 65 amino acid residues,including 6 conserved cysteine residues)was cloned into a pPICZαA vector and expressed in P.pastoris GS115.Structural fidelity of the recombinant peptide was confirmed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),showing a molecular weight of 8849.9 Da,which was consistent with the theoretical prediction.Functional assays demonstrated a broad-spectrum antimicrobial activity of the recombinant myticofensin-B1,with stronger inhibition against Gram-negative bacteria.Scanning electron microscopy revealed different effects of the recombinant myticofensin-B1 against different bacteria.In addition,the recombinant myticofensin-B1 exhibited a very low hemolytic activity against sheep red blood cells and weak cytotoxicity against human A549 lung cancer cells.This study establishes P.pastoris as a powerful platform to produce functional mussel AMP and highlights the potential of the recombinant myticofensin-B1 as a therapeutic agent for aquaculture pathogens and infections.
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。
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.
