TMT定量蛋白质组学解析Rummeliibacillus suwonensis 3B-1 生长及己酸代谢机制

doi:10.13560/j.cnki.biotech.bull.1985.2023-0862

摘要/Abstract

摘要：

【目的】 从蛋白水平阐明水源拉梅尔芽孢杆菌（Rummeliibacillus suwonensis）的生长及己酸代谢机理，为水源拉梅尔芽孢杆菌的基因工程改造提供一定技术基础。【方法】 以R. suwonensis 3B-1为研究对象，应用串联质谱标签（tandem mass tags, TMT）蛋白组学技术对该菌在好氧与厌氧条件下的差异表达蛋白（differentially expressed proteins, DEPs）进行挖掘，并对鉴定到的DEPs进行亚细胞定位、GO功能富集、KEGG信号通路注释、蛋白相互作用等生物信息学分析。【结果】 从比较组中共鉴定获得810个DEPs，其中上调蛋白423个，下调蛋白387个，亚细胞定位到6个条目上，主要涉及细胞质蛋白，细胞膜蛋白和细胞壁等蛋白。GO功能富集分析结果显示，肽的生物合成、翻译和肽代谢过程等生物学过程；核糖体的结构组成和结构分子活性等分子功能；核糖体和核糖核蛋白复合物等细胞组分发生了显著变化。810个DEPs 注释到113条KEGG信号通路，主要涉及辅因子生物合成，双组分系统，磷酸戊糖代谢，糖酵解/糖异生，以及氧化磷酸化等信号通路。苯丙氨酸-tRNA连接酶β亚基和核黄素生物合成蛋白RibD在蛋白互作网络中关联度最高。【结论】 厌氧条件下，糖酵解途径中丙酮酸脱氢酶和丙酮酸激酶表达下调，氨基酸代谢和生物素蛋白连接酶等辅因子相关蛋白表达均呈现下调，表明该菌适合在好氧环境中生长。己酸合成方面，酰基辅酶A硫酯酶的表达量显著上调，同时，糖酵解/糖异生途径、三羧酸循环和磷酸戊糖途径为己酸合成提供了充足的前体物质和还原当量，共同促进了己酸合成。

关键词: TMT蛋白质组学, Rummeliibacillus suwonensis, 己酸, 生物信息学

Abstract:

【Objective】 This work aims to elucidate the growth and caproic acid metabolism mechanism of Rummeliibacillus suwonensis from protein level, and to further improve its metabolic capacity. It provides a technical basis for the genetic engineering of R. suwonensis. 【Method】 The differential expression proteins（DEPs）of strain R. suwonensis 3B-1 were extracted by tandem mass tags（TMT）proteomics in aerobic and anaerobic conditions. Its subcellular localization analysis, Gene Ontology（GO）functional analysis, KEGG signaling pathway annotation analysis, and protein-protein interaction were analyzed.【Result】 A total of 810 DEPs were identified, including 423 up-regulated proteins and 387 down-regulated proteins. A total of 725 DEPs were subcellular mapped to 6 items, mainly involving cytoplasmic proteins, cytoplasmic membrane proteins and cellwall proteins. GO enrichment of function analysis indicated that, biological processes such as peptide biosynthesis, translation, and peptide metabolism, molecular functions such as structural constituent and structural molecular activity of ribosomes, and cellular components such as ribosomes and ribonucleoprotein complexes have undergone significant changes. A total of 810 DEPs were annotated into 113 KEGG signaling pathways in KEGG database, which mainly were involved cofactor biosynthesis, two-component system, pentose phosphate pathway, glycolysis/gluconeogenesis and oxidative phosphorylation. The phenylalanine-tRNA ligase β subunit and the riboflavin biosynthesis protein RibD had the highest correlation in the protein interaction network. 【Conclusion】 Under anaerobic conditions, the expression of pyruvate dehydrogenase and pyruvate kinase in the glycolytic pathway was down-regulated. Additionally, proteins related to amino acid metabolism and biotin protein ligase cofactors were also downregulated. This indicates that the bacterium is better suited for growth in an aerobic conditions. Regarding the synthesis of caproic acid, the expression of acyl-CoA thioesterase was significantly up-regulated. In addition, the glycolysis/gluconeogenesis pathway, the tricarboxylic acid cycle and the pentose phosphate pathway provided sufficient precursors and reduction equivalents for caproic acid synthesis, which jointly promoted caproic acid synthesis.

Key words: TMT proteomics, Rummeliibacillus suwonensis, caproic acid, bioinformatics

陈晓松, 刘超杰, 郑佳, 乔宗伟, 罗惠波, 邹伟. TMT定量蛋白质组学解析Rummeliibacillus suwonensis 3B-1 生长及己酸代谢机制[J]. 生物技术通报, 2024, 40(3): 135-145.

CHEN Xiao-song, LIU Chao-jie, ZHENG Jia, QIAO Zong-wei, LUO Hui-bo, ZOU Wei. Analyzing the Growth and Caproic Acid Metabolism Mechanism of Rummeliibacillus suwonensis 3B-1 by Tandem Mass Tag-based Quantitative Proteomics[J]. Biotechnology Bulletin, 2024, 40(3): 135-145.

图/表 9

图1 3B-1好氧和厌氧条件生长及代谢情况 A：好氧和厌氧条件下3B-1生长曲线图；B：好氧和厌氧条件下3B-1丁酸、乙酸产量图

Fig. 1 Growth and metabolism of 3B-1 under aerobic and anaerobic conditions A : 3B-1 growth curves under aerobic and anaerobic conditions. B : Butyric acid and caproic acid production of 3B-1 under aerobic and anaerobic conditions

表1 BCA蛋白定量结果

Table 1 BCA protein quantification results

样品名称 Sample	编号 Sample number	蛋白浓度 Protein concentration/(μg·μL^-1)	蛋白总量 Total protein/μg
RH1	1	1.475	885
RH2	2	3.197	1 918.2
RH3	3	1.647	988.2
RY1	4	1.268	760.8
RY2	5	1.954	1 172.4
RY3	6	1.608	964.8

图2 蛋白样品质量控制 A：蛋白样品SDS-PAGE图；B：肽段离子质量偏差分布图；C：肽段离子得分分布图

Fig. 2 Quality control of protein sample A: SDS-PAGE of protein samples. B: Distribution plot of peptide ion mass deviations. C: Distribution plot of peptide ion scores

图3 蛋白鉴定和差异表达结果 A：蛋白质的鉴定与定量结果图；B：蛋白质定量差异结果柱状图；C：比较组差异显著性火山图；D：比较组DEPs聚类分析图

Fig. 3 Results of protein identification and differential expression A: Result graph of protein identification and quantification. B: Bar graph of protein quantification differences. C: Volcano plot of significant differences in the comparative group. D: Analysis graph of clustering of DEPs in the comparative group

图4 比较组DEPs的GO注释图 A：比较组DEPs的GO注释图；B：比较组DEPs的生物过程分类下GO功能富集气泡图；C：比较组DEPs的分子功能分类下GO功能富集气泡图D：比较组DEPs的细胞组分分类下GO功能富集气泡图

Fig. 4 GO annotation map of DEPs in comparative group A : GO annotation map of DEPs in the comparative group. B : GO functional enrichment bubble diagram under the biological process classification of DEPs in the comparative group. C: GO functional enrichment bubble diagram under the molecular function classification of DEPs in the comparative group. D : GO functional enrichment bubble diagram under the cell component classification of DEPs in the comparative group

图5 KEGG富集代谢通路

Fig. 5 KEGG enriched metabolic pathways

图6 比较组DEPs互作网络分析结果 A:比较组DEPs网络互作图; B: DEPs互作网络中关联度最高的20个蛋白表达情况

Fig. 6 Results of the interaction network analysis of DEP in the comparative groups A: Network interaction graph of DEPs in the comparative groups. B: Expression patterns of the top 20 proteins with the highest correlation in the DEPs interaction network

表2 比较组蛋白互作网络中关联度最高的20个蛋白质

Table 2 Top 20 proteins with the highest correlation in the interaction network of DEPs

蛋白编号Protein	基因名称Gene name	蛋白描述Annotation
A0A3N6AUL8;A0A511BXG9	pheT	Phenylalanine--tRNA ligase beta subunit
A0A143HEW7	—	Riboflavin biosynthesis protein RibD
A0A3N5ZVL3	rpoA	DNA-directed RNA polymerase subunit alpha
A0A143HAI0;A0A3N5ZWK1	lepA	Elongation factor 4
A0A143HG81	tuf	Elongation factor Tu
A0A143HAK3;A0A3N5ZWQ3	rpsB	30S ribosomal protein S2
A0A3N6AF85	rplB	50S ribosomal protein L2
A0A3N6AU75;A0A511BUT3	infB	Translation initiation factor IF-2
A0A3N5ZVV7	rplE	50S ribosomal protein L5
A0A143HGW1;A0A3N5ZVN4	fusA	Elongation factor G
A0A143HGU0;A0A3N6AT98	rpsE	30S ribosomal protein S5
A0A3N5ZVM8	rpsC	30S ribosomal protein S3
A0A143HFJ7;A0A3N5ZWH0	rpsG	30S ribosomal protein S7
A0A143HFH8	rplD	50S ribosomal protein L4
A0A3N5ZWT6	cinA	Putative competence-damage inducible
A0A143HHE1;A0A3N6AER5	atpA	ATP synthase subunit alpha
A0A511C0H3	rplR	50S ribosomal protein L18
A0A143HFH6;A0A3N5ZWE8	rpsK	30S ribosomal protein S11
A0A143H9I3;A0A3N6AU15	ftsY	Signal recognition particle receptor FtsY
A0A3N5ZTP5	birA	Bifunctional ligase/repressor BirA

图7 DEPs在中心代谢和己酸代谢途径中的分布 “+”表示该酶表达量上调，“-”表示该酶表达量下调。（1）：己糖激酶；（2）：ATP依赖的6-磷酸果糖激酶；（3）：磷酸甘油酸激酶；（4）：果糖二磷酸醛缩酶；（5）：2 -磷酸甘油酸脱水酶；（6）：丙酮酸激酶；（7）：柠檬酸合酶；（8）：异柠檬酸脱氢酶；（9）：α-酮戊二酸脱氢酶复合体；（10）：琥珀酸脱氢酶；（11）：延胡索酸酶；（12）：苹果酸脱氢酶；（13）：乙酰辅酶A乙酰转移酶；（14）：3-羟基丁酰辅酶A脱氢酶；（15）：丁酰辅酶A脱氢酶；（16）：3-羟基丁酰辅酶A脱水酶；（17）：酰基辅酶A硫酯酶

Fig. 7 Distribution of DEPs in the central metabolic and caproic acid metabolic pathways “ +” indicates that the expression of the enzyme is up-regulated, “-” indicates that the expression of the enzyme is down-regulated. （1）: Hexokinase.（2）: ATP-dependent 6-phosphofructokinase.（3）: Phosphoglycerate kinase.（4）: Fructose diphosphate aldolase.（5）: 2-phosphoglycerate dehydratase.（6）: Pyruvate kinase.（7）: Citrate synthase.（8）: Isocitrate dehydrogenase.（9）: α-ketoglutarate dehydrogenase complex.（10）: Succinate dehydrogenase.（11）: Fumarase.（12）: Malate dehydrogenase.（13）: Acetyl-CoA acetyltransferase.（14）: 3-hydroxybutyryl-CoA dehydrogenase.（15）: Butyryl-CoA dehydrogenase.（16）: 3-hydroxybutyryl-CoA dehydratase.（17）: Acyl-CoA thioesterase

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