Analyzing the Growth and Caproic Acid Metabolism Mechanism of Rummeliibacillus suwonensis 3B-1 by Tandem Mass Tag-based Quantitative Proteomics

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

Abstract

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

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.

Figures/Tables 9

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

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

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

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

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

Fig. 5 KEGG enriched metabolic pathways

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

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

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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