贝莱斯芽孢杆菌<i>PksN</i>生物信息学及表达分析

doi:10.7668/hbnxb.20195156

摘要/Abstract

摘要：

为探究贝莱斯芽孢杆菌DJ1菌株聚酮合酶PksN的理化性质和结构及聚酮化合物的生物合成过程中PksN的表达情况。利用生物信息学、分子对接技术分析了贝莱斯芽孢杆菌DJ1聚酮合酶PksN的同源序列、理化性质、蛋白结构、配体与受体结合位点、小分子化合物和蛋白质互作网络,并利用实时荧光定量PCR测定了贝莱斯芽孢杆菌DJ1发酵不同时间点PksN的表达。结果表明,贝莱斯芽孢杆菌PksN属于PksD家族,与Bacillus的PksN相似性为100%,含有55个开放阅读框,5个保守结构域,其中包括KR结构域。PksN共有2 051个氨基酸,分子量为226.05 ku,等电点为5.59,分子式为C₁₀₀₂₈H₁₅₇₁₉N₂₇₅₃O₃₀₅₀S₇₅,是一种不稳定蛋白,位于细胞质膜,PksN以α-螺旋为主要结构,并预测了聚酮合酶PksN的三级结构。分子对接结果表明,PksN能利用乙酰辅酶A、丙酰辅酶A、异戊酰辅酶A、丙二酰辅酶A、甲基丙二酰辅酶A和乙基丙二酰辅酶A作为底物合成聚酮化合物,互作蛋白中排名前十的蛋白均与聚酮类化合物合成有关,实时荧光定量PCR结果表明,贝莱斯芽孢杆菌DJ1发酵48 h PksN表达量最高,可确定后续提取聚酮化合物的最佳时间点。

关键词: 贝莱斯芽孢杆菌, 聚酮合酶, 生物信息, 结构预测, 分子对接, 基因表达

Abstract:

To explore the physicochemical properties,structure,and expression of the polyketide synthase PksN during polyketones biosynthesis in Bacillus velezensis DJ1 strain.It employed bioinformatics and molecular docking techniques to analyze the homologous sequences,physicochemical properties,protein structure,ligand-receptor binding sites,small molecule compounds,and protein-protein interaction networks of PksN.Additionally,Real-time Quantitative PCR was used to measure the expression of PksN at different time points during fermentation of Bacillus velezensis DJ1.The results showed that PksN belongs to the PksD family and shares 100% similarity with PksN gene of Bacillus.It contained 55 open reading frames(ORFs)and five conserved domains,including the KR domain.The protein consisted of 2 051 amino acids,with a molecular weight of 226.05 ku and an isoelectric point of 5.59.The molecular formula was C₁₀₀₂₈H₁₅₇₁₉N₂₇₅₃O₃₀₅₀S₇₅.PksN was an unstable protein located in the cytoplasmic membrane,and its secondary structure was predominantly α-helix.The tertiary structure of PksN was also predicted.Molecular docking results revealed that PksN can utilize acetyl-CoA,propionyl-CoA,isovaleryl-CoA,malonyl-CoA,methylmalonyl-CoA,and ethylmalonyl-CoA as substrates for polyketide biosynthesis.The top ten interacting proteins are all associated with polyketide biosynthesis.Real-time Quantitative PCR results indicated that the expression of PksN in Bacillus velezensis DJ1 peaked at 48 hours of fermentation,identifying the optimal time point for the subsequent extraction of polyketides.

Key words: Bacillus velezensis, Polyketide synthase, Bioinformatics, Structural prediction, Molecular docking, Gene expression

中图分类号:

Q936微生物生物化学

刘伟, 闫更轩, 胡基华, 田缘, 于冲, 毛雪, 张淑梅. 贝莱斯芽孢杆菌PksN生物信息学及表达分析[J]. 华北农学报, 2025, 40(S1): 292-299. doi: 10.7668/hbnxb.20195156.

LIU Wei, YAN Gengxuan, HU Jihua, TIAN Yuan, YU Chong, MAO Xue, ZHANG Shumei. Bioinformatics and Expression Analysis of PksN in Bacillus velezensis Polyketide Synthase[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(S1): 292-299. doi: 10.7668/hbnxb.20195156.

http://www.hbnxb.net/CN/Y2025/V40/IS1/292

图/表 9

表1 实时荧光定量PCR引物序列

Tab.1 Real-time PCR primer sequence

基因Gene	引物序列(5'-3') Primer sequence(5'-3')
16S	GCTTCGGCTACCACTTACAGAT
	TCAGGTCGGCTACGCATCG
PksN	ATCGCCGATTTTGATCCTGAGTTC
	ATGTCCTCGTATATCCCGCATCC

图1 PksN磷酸化位点预测分析及疏水性分析结果

Fig.1 PksN phosphorylation site prediction and hydrophobicity analysis results

图2 PksN二级结构

Fig.2 PksN secondary structure

图3 PksN 三维结构

Fig.3 PksN three-dimensional structure

表2 PksN与小分子之间的结合得分

Tab.2 Binding scores between PksN and small molecules

小分子Micromolecule	得分Score
乙酰辅酶A Acetyl-CoA	-12.118 781 1
丙酰辅酶A Propionyl-CoA	-11.694 555 3
异戊酰辅酶A Isovaleryl-CoA	-11.775 791 2
丙二酰辅酶A Malonyl-CoA	-12.488 548 3
甲基丙二酰辅酶A Methylmalonyl-CoA	-12.189 192 8
乙基丙二酰辅酶A Ethylmalonyl-CoA	-13.497 603 4
甲酸Formic acid	-3.180 979 0
乙酸Acetic acid	-3.472 922 3
丙酸Propionic acid	-3.856 440 3
丁酸Butyric acid	-4.132 806 3

图4 PksN与小分子之间的结合模式1 A.乙酰辅酶A;B.丙酰辅酶A;C.异戊酰辅酶A;D.丙二酰辅酶A; E.甲基丙二酰辅酶A;F.乙基丙二酰辅酶A。Polar.极性;Acidic.酸性;Basic.碱性;Greasy.疏水性;Proximity contour.临近轮廓;Sidechain acceptor.侧链受体;Sidechain donor.侧链供体;Backbone acceptor.主链受体;Backbone donor.主链供体;Ligand exposure.配体暴露;Receptor exposure.受体暴露;Solvent residue.溶剂残基;Metal complex.金属复合物;Solvent contact.溶剂接触;Metal/ion contact.金属/离子接触;Arene-arene.芳环-芳环相互作用;Arene-H.芳环-氢相互作用;Arene-cation.芳环-阳离子相互作用。图5同。

Fig.4 Binding patterns 1 between PksN and small molecules A.Acetyl CoA;B.Propionyl-CoA;C.Isovaleryl CoA;D.Malonyl-CoA; E.Methylmalonyl-CoA;F.Ethyl malonyl-CoA.Polar.Polar;Acidic.Acidic;Basic.Basic;Greasy.Hydrophobic;Proximity contour.Proximity contour;Sidechain acceptor.Sidechain acceptor;Sidechain.Denotes sidechain donor;Backbone acceptor.Backbone acceptor;Backbone.Backbone donor;Ligand exposure.Ligand exposure;Receptor exposure.Receptor exposure;Solvent residue.Solvent residue;Metal complex.Metal complex;Solvent contact.Solvent contact;Metal/ion contact.Metal/ion contact;Arene-arene.Arene-arene interaction;Arene-H.Arene-hydrogen interaction;Arene-cation.Arene-cation interaction.The same as Fig.5.

图5 PksN与小分子之间的结合模式2 A.甲酸;B.乙酸;C.丙酸;D.丁酸。

Fig.5 Binding patterns 2 between PksN and small molecules A.Formic acid;B.Acetic acid;C.Propionic acid;D.Butyric acid.

图6 PksN蛋白互作网络

Fig.6 PksN protein interaction network

图7 不同发酵时间PksN相对表达量

Fig.7 Relative expression of PksN at different fermentation times

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贝莱斯芽孢杆菌PksN生物信息学及表达分析

Bioinformatics and Expression Analysis of PksN in Bacillus velezensis Polyketide Synthase

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