Comparative metabolomics analysis of the key metabolic nodes in propionic acid synthesis in Propionibacterium acidipropionici

Ningzi Guana, Jianghua Lia, b, Hyun-dong Shinc, Jing Wud, Guocheng Dua, b, Zhongping Shib*, Long Liua, b*, Jian Chena,b

aKey Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, JiangnanUniversity, Wuxi 214122, China

bKey Laboratory of Industrial Biotechnology, Ministry of Education, JiangnanUniversity, Wuxi 214122, China

cSchool of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta30332, USA

d National Key Laboratory of Food Science and Technology, JiangnanUniversity, Wuxi 214122, China

*Corresponding authors: Zhongping Shi, Tel.: +86-510-85918292, Fax: +86-510-85326276, E-mail: ; Long Liu, Tel.: +86-510-85918312, Fax: +86-510-85918309, E-mail: .

Table S1: Intracellular metabolites found in P. acidipropionici.

Classification / Metabolites / PubChem ID / Ion mode / Score / m/za / RTb (min) / MS2
Sugar / Deoxyribose / 46173726 / + / 2.63 / 135.07/- / 28.5/- / -
Sugar / CDP-glucose / 439244 / + / 3.75 / 566.08/- / 22.4/- / -
Sugar / Chitobiose / 439544 / + / 17.64 / 425.18/- / 29.9/- / -
Sugar / Erythrose 4-phosphate / 122357 / + / 16.49 / 201.02/- / 18.3/- / -
Sugar / Fructose 1,6-bisphosphate / 172313 / - / 9.71 / 338. 99/338.99 / 21.5/21.6 / -
Sugar / Fructose 6-phosphate / 439160 / - / 15.30 / 259.02/259.02 / 15.6/15.6 / -
Sugar / Glucosamine / 439213 / + / 49.16 / 180.09/180.09 / 4.9/4.9 / -
Sugar / Glucosamine 6-phosphate / 440997 / + / 7.23 / 260.05/- / 28.0/- / -
Sugar / Glucose / 5793 / - / 0.90 / 179.06/- / 22.0/- / -
Sugar / N-Acetyl glucosamine / 439174 / + / 15.81 / 222.09/222.09 / 5.6/5.7 / 204.09, 186.08
Sugar / Raffinose / 439242 / + / 0.25 / 505.18/- / 24.1/- / -
Sugar / Ribose / 5779 / + / 23.38 / 151.06/- / 11.1/- / -
Sugar / Ribose 5-phosphate / 439167 / + / 1.81 / 231.03/- / 3.7/- / -
Sugar / Sedoheptulose 1,7-bisphosphate / 164735 / + / 2.95 / 371.01/- / 11.0/- / -
Sugar alcohol / Glycerol / 753 / + / 43.23 / 93.06/- / 8.1/- / -
Sugar alcohol / Mannitol / 6251 / + / 23.75 / 183.09/- / 8.2/- / -
Sugar alcohol / Mannitol 1-phosphate / 130418 / + / 0.23 / 263.05/- / 18.8/- / -
Sugar alcohol / sn-Glycerol 3-phosphate / 439162 / + / 0.72 / 173.02/- / 3.1/- / -
Sugar alcohol / Xylitol / 6912 / + / 2.18 / 153.08/- / 8.0/- / -
Organic acid / 2-Oxoadipate / 71 / - / 2.04 / 161.04/- / 22.9/- / -
Organic acid / 2-Oxobutanoate / 58 / + / 4.38 / 103.04/- / 8.6/- / 57.03
Organic acid / 2-Oxoglutarate / 51 / + / 0.34 / 147.03/- / 7.5/- / -
Organic acid / 3-Phospho-glycerate / 439183 / + / 0.99 / 187.00/- / 7.9/- / -
Organic acid / 4-Hydroxybenzoate / 135 / + / 3.93 / 139.04/- / 11.8/- / -
Organic acid / 6-Phospho-gluconate / 91493 / + / 1.47 / 277.03/- / 18.7/- / -
Organic acid / Acetate / 176 / + / 21.47 / 60.03/- / 4.8/- / -
Organic acid / Acrylate / 6581 / + / 7.23 / 73.03/- / 24.7/- / -
Organic acid / Anthranilate / 227 / + / 37.85 / 138.06/- / 7.8/- / -
Organic acid / Barbiturate / 6211 / + / 2.66 / 129.03/- / 4.4/- / -
Organic acid / Benzoate / 243 / + / 30.27 / 123.04/- / 11.2/- / -
Organic acid / Chorismate / 12039 / + / 12.56 / 227.06/- / 12.2/- / -
Organic acid / Citrate / 311 / - / 16.99 / 191.02/- / 4.0/- / 174.93
Organic acid / Fumarate / 444972 / - / 4.40 / 115.01/- / 5.9/- / 71.02
Organic acid / Glucarate / 33037 / - / 2.77 / 209.03/- / 19.6/- / -
Organic acid / Gluconate / 10690 / + / 22.18 / 197.07/- / 16.4/- / -
Organic acid / Glucuronate / 94715 / + / 9.27 / 195.05/- / 4.0/- / -
Organic acid / Glycolate / 757 / + / 2.60 / 77.02/- / 7.9/- / -
Organic acid / Glyoxylate / 760 / + / 11.81 / 75.01/- / 4.3/- / -
Organic acid / Lactate / 107689 / - / 26.62 / 89.02/- / 6.1/- / -
Organic acid / Lipoate / 864 / + / 1.43 / 207.05/- / 16.9 / -
Organic acid / Malate / 222656 / - / 3.49 / 133.02/133.02 / 5.8/5.8 / 115.01
Organic acid / Mevalonate / 439230 / + / 12.02 / 149.08/- / 8.6/- / -
Organic acid / Phenylpyruvate / 997 / + / 1.34 / 165.06/- / 8.1/- / -
Organic acid / Phosphoenolpyruvate / 1005 / + / 5.46 / 168.99/- / 11.9/- / -
Organic acid / Pyruvate / 1060 / + / 3.75 / 89.02/- / 7.9/- / -
Organic acid / Succinate / 1110 / - / 0.59 / 117.02/117.02 / 5.9/5.9 / -
Amino acid / 1-Aminocyclopropane-1-carboxylate / 535 / + / 3.38 / 102.06/- / 8.6/- / -
Amino acid / 2,6-Diaminoheptanedioate / 439283 / + / 10.72 / 191.10/- / 7.9/- / -
Amino acid / 2-Aminomuconate / 5459864 / + / 2.10 / 158.05/- / 10.9/- / -
Amino acid / 5-Hydroxy-tryptophan / 439280 / + / 24.25 / 221.09/- / 4.9/- / 175.08
Amino acid / 7,8-Diaminononanoate / 652 / + / 8.12 / 189.16/- / 14.2/- / -
Amino acid / Alanine / 5950 / + / 17.23 / 90.06/90.06 / 6.8/6.8 / -
Amino acid / Arginine / 6322 / + / 25.20 / 175.12/175.12 / 4.1/4.0 / 130.10, 158.10
Amino acid / Arogenate / 439319 / + / 17.22 / 228.09/- / 17.7/- / 183.07
Amino acid / Aspartate / 5960 / + / 17.97 / 134.05/134.05 / 5.5/5.5 / 88.04, 116.03
Amino acid / beta-Alanyl arginine / 440637 / + / 2.09 / 246.16/- / 21.4/- / -
Amino acid / Citrulline / 9750 / + / 10.15 / 176.10/176.10 / 15.5/15.6 / -
Amino acid / Cystathionine / 834 / + / 2.31 / 223.08/- / 4.4/- / 178.08
Amino acid / Cysteine / 5862 / + / 5.51 / 122.03/- / 10.6/- / 76.02
Amino acid / GABA / 119 / + / 23.27 / 104.07/104.07 / 4.2/4.2 / -
Amino acid / Glutamate / 33032 / + / 9.28 / 148.06/- / 5.6/- / -
Amino acid / Glycine / 750 / + / 31.44 / 76.04/- / 6.1/-/- / -
Amino acid / Histidine / 773 / + / 15.80 / 156.08/- / 29.1/- / 110.07
Amino acid / Homocysteine / 778 / + / 4.15 / 136.04/- / 13.0/- / -
Amino acid / Leucine / 6106 / + / 50.00 / 132.10/- / 7.8/- / -
Amino acid / Methionine / 6137 / + / 3.51 / 150.06/- / 24.9/- / 105.04
Amino acid / N-Acetyl glutamate / 439174 / + / 7.18 / 190.07/- / 15.9/- / -
Amino acid / O-Phospho homoserine / 151187 / + / 7.23 / 200.03/- / 4.8/- / -
Amino acid / O-Phospho serine / 68841 / + / 4.01 / 186.02/- / 20.6/- / -
Amino acid / Phenylalanine / 6140 / + / 22.98 / 166.09/- / 13.9/- / 120.08
Amino acid / Proline / 145742 / + / 12.92 / 116.07/116.07 / 37.5/- / -
Amino acid / S-Adenosyl-homocysteine / 439155 / + / 1.32 / 385.13/- / 25.2/- / -
Amino acid / S-Adenosyl-methionine / 34755 / + / 2.41 / 399.15/- / 21.6/- / -
Amino acid / Serine / 5951 / + / 71.71 / 106.05/106.05 / 4.4/4.4 / 88.04
Amino acid / Threonine / 6288 / + / 24.28 / 120.08/- / 11.8/- / -
Amino acid / Tryptophan / 6305 / + / 10.32 / 205.10/- / 12.8/- / 188.07
Amino acid / Tyrosine / 6057 / + / 14.40 / 182.08/- / 8.1/- / -
Amino acid / Valine / 2687 / + / 10.39 / 118.09/- / 4.1/- / -
Peptide / Glutathione / 124886 / + / 12.78 / 308.09/- / 19.2/- / 234.03
Nucleotide / 5'-Methylthioadenosine / 439176 / + / 10.22 / 298.10/- / 18.3/- / -
Nucleotide / 5,6-Dihydrouracil / 649 / + / 8.23 / 115.05/- / 14.2/- / -
Nucleotide / Adenine / 190 / + / 5.35 / 136.06/- / 11.3/- / -
Nucleotide / Adenosine / 60961 / + / 24.57 / 268.10/- / 13.2/- / -
Nucleotide / ADP / 6022 / - / 8.10 / 426.02/- / 0.3/- / -
Nucleotide / AMP / 6083 / + / 21.95 / 348.07/- / 14.8/- / -
Nucleotide / ATP / 5957 / + / 21.14 / 508.00/- / 20.2/- / -
Nucleotide / CDP / 6132 / + / 1.73 / 404.03/- / 34.0/- / -
Nucleotide / CMP / 6131 / + / 0.37 / 324.06/- / 9.7/- / -
Nucleotide / CTP / 6176 / + / 1.53 / 483.99/- / 26.2/- / -
Nucleotide / Cytidine / 6175 / + / 3.72 / 244.09/- / 25.8/- / -
Nucleotide / Cytosine / 597 / + / 10.47 / 112.05/- / 10.0/- / -
Nucleotide / dADP / 188966 / - / 2.18 / 410.03/- / 1.1/- / -
Nucleotide / dATP / 15993 / - / 6.21 / 489.99/- / 19.5/- / -
Nucleotide / dCDP / 150855 / - / 9.11 / 386.02/- / 17.5/- / -
Nucleotide / Deoxyadenosine / 13730 / + / 51.56 / 252.11/- / 9.6/- / 134.99
Nucleotide / Deoxycytidine / 13711 / + / 6.87 / 228.10/- / 17.7/- / -
Nucleotide / dIDP / 439488 / + / 4.17 / 413.03/- / 23.2/- / -
Nucleotide / Dihydroorotate / 439216 / + / 19.18 / 159.04/- / 4.8/- / -
Nucleotide / dTMP / 9700 / + / 50.00 / 323.06/- / 21.3/- / -
Nucleotide / dUMP / 65063 / + / 0.46 / 309.05/- / 25.7/- / -
Nucleotide / FAD / 643975 / + / 0.26 / 786.17/- / 16.2/- / -
Nucleotide / FMN / 643976 / + / 12.33 / 457.11/- / 20.3/- / -
Nucleotide / GMP / 6804 / - / 2.56 / 362.05/- / 12.2/- / -
Nucleotide / Guanine / 764 / + / 45.89 / 152.06/- / 8.6/- / 135.04
Nucleotide / Guanosine / 6802 / + / 22.01 / 284.10/- / 13.0/- / -
Nucleotide / Hypoxanthine / 790 / + / 2.02 / 137.05/- / 8.5/- / -
Nucleotide / IDP / 6831 / + / 4.97 / 429.02/- / 30.0/- / -
Nucleotide / IMP / 8582 / + / 6.55 / 349.05/- / 15.9/- / -
Nucleotide / Inosine / 6021 / + / 3.62 / 269.09/- / 13.3/- / -
Nucleotide / Nicotinamide / 936 / + / 3.02 / 123.06/- / 11.0/- / -
Nucleotide / Nicotinamide ribonucleotide / 14180 / + / 8.64 / 335.06/- / 12.3/- / -
Nucleotide / Orotate / 967 / + / 6.84 / 157.03/- / 4.7/- / -
Nucleotide / Thiamin diphosphate / 1132 / - / 1.84 / 424.04/- / 29.2/- / -
Nucleotide / Thymidine / 5789 / - / 29.46 / 241.08/- / 26.0/- / -
Nucleotide / Thymine / 1135 / + / 12.20 / 127.05/- / 4.7/- / -
Nucleotide / UDP / 6031 / + / 10.18 / 405.01/- / 19.6/- / -
Nucleotide / UMP / 6030 / - / 10.89 / 323.03/- / 11.7/- / -
Nucleotide / XMP / 73323 / - / 14.95 / 363.03/- / 12.7/- / -
CoA-ester / Propionyl-CoA / 92753 / + / 47.21 / 824.15/- / 21.2/- / 807.13
Amide / Acrylamide / 6579 / + / 4.96 / 72.05/- / 5.1/- / -
Amine / Carnitine / 10917 / + / 4.37 / 162.11/- / 15.9/- / -
Amine / Agmatine / 199 / + / 1.61 / 131.13/- / 4.2/- / -
Amine / Choline / 305 / + / 0.91 / 105.11/- / 4.2/- / -
Hydrocarbon / 3-Phospho-glyceroyl phosphate / 439191 / + / 1.16 / 266.97/- / 37.7/- / -
Hydrocarbon / 2-Aminophenol / 5801 / + / 2.10 / 110.06/- / 9.5/- / -
Hydrocarbon / Catechol / 289 / + / 24.33 / 111.04/- / 7.9/- / -
Hydrocarbon / Geranylgeranyl diphosphate / 447277 / + / 7.15 / 451.20/- / 3.9/- / -
Hydrocarbon / Isopentenyl diphosphate / 1195 / + / 20.89 / 247.01/- / 17.6/- / -
Aldehyde / 4-Aminobutyraldehyde / 118 / + / 24.35 / 88.08/- / 7.4/- / -
Aldehyde / Phenylacetaldehyde / 998 / + / 14.86 / 121.07/- / 3.6/- / -
Azole / 5-Aminoimidazole / 484 / + / 11.58 / 84.06/- / 6.7/- / -
Azole / Biotin / 171548 / + / 0.92 / 245.10/- / 21.3/- / -
Ester / 5-Phosphoribosylamine / 439905 / + / 4.36 / 230.04/- / 4.8/- / 196.00
Ester / Adenylylselenate / 440758 / + / 28.21 / 475.97/- / 18.9/- / -
Pteridine / Riboflavin / 493570 / + / 8.01 / 377.15/- / 5.4/- / -
Pteridine / Tetrahydrofolate / 91443 / + / 20.99 / 446.18/- / 30.0/- / -
Nitrile / Acrylonitrile / 7855 / + / 0.11 / 54.03/- / 4.8/- / -
Ketone / Aminoacetone / 215 / + / 19.03 / 74.06/- / 23.2/- / -
Ketone / Glycerone / 670 / - / 26.62 / 89.02/- / 5.4/- / -
Ketone / Glycerone phosphate / 668 / + / 7.96 / 171.01/- / 4.3/- / -
Organophosphate / Acetyl phosphate / 186 / - / 25.53 / 138.98/- / 4.6/- / -
Sulfhydryl Compound / Pantetheine 4'-phosphate / 115254 / - / 7.20 / 357.09/- / 5.1/- / -

aThe numerical values representm/z ratios (detected/standard).

b RTrepresents the Retention times of metabolites (detected/standard).

c –represents that standard is missing.

Table S2:Culture optimization to determine the optimal combination of addition

Combination / PA production (g/L)
Control / 9.61±0.15
Malate+ Fumarate / 7.71±0.28
Malate+ Lactate / 14.38±0.06
Malate+ Succinate / 10.59±0.32
Malate+ Fumarate+ Lactate / 15.99±0.18
Malate+ Fumarate+ Succinate / 11.06±0.21
Malate+ Lactate+ Succinate / 15.73±0.27
Malate + Fumarate + Lactate + Succinate / 13.81±0.28
Fumarate+ Lactate / 15.47±0.07
Fumarate+ Succinate / 10.44±0.32
Fumarate+ Lactate+ Succinate / 16.99±0.0.26
Lactate+ Succinate / 13.55±0.38

Table S3:Culture optimization to determine the optimal concentrations of additions.

Concentration of lactate, fumarate and succinate(mM) / PA production (g/L)
Control / 9.61±0.15
70,20,10 / 14.76±0.38
70,20,20 / 15.36±0.29
70,20,30 / 14.22±0.27
70,30,10 / 15.53±0.27
70,30,20 / 15.42±0.30
70,30,30 / 13.62±0.29
70,40,10 / 14.07±0.34
70,40,20 / 14.51±0.23
70,40,30 / 8.28±0.33
90,20,10 / 16.16±0.37
90,20,20 / 17.16±0.30
90,20,30 / 15.87±0.35
90,30,10 / 16.40±0.45
90,30,20 / 15.77±0.26
90,30,30 / 14.38±0.29
90,40,10 / 16.18±0.34
90,40,20 / 14.30±0.28
90,40,30 / 8.99±0.12
105,20,10 / 16.23±0.30
105,20,20 / 16.43±0.33
105,20,30 / 17.58±0.39
105,30,10 / 17.04±0.34
105,30,20 / 15.03±0.38
105,30,30 / 10.45±0.18
105,40,10 / 14.72±0.26
105,40,20 / 10.07±0.20
105,40,30 / 8.23±0.32

Fig. S1: The relative content of discrepant intracellular metabolites in P. acidipropionici CGMCC 1.2230 and P. acidipropionici WSH1105 at middle exponential phase (A), late exponential phase (B) and the end of fermentation (C). The value represents the ratio of P. acidipropionici WSH1105 to P. acidipropionici CGMCC 1.2230.