LIST OF TABLES
4. CLONING OF NOVEL COWPEA TRANSCRIPTION FACTORS AND THEIR FUNCTIONAL CHARACTERIZATION IN YEAST
4.3 RESULTS
4.3.6 LCMS-based profiling of differentially accumulated metabolites (DAMs)
4.3.6.4 Energy generating and secondary metabolic pathways
Pathways such as pyruvate metabolism, TCA cycle, glycolysis, etc., involved in energy conversion were also impacted in the transgenic strains (Table 2, and Supplementary Table S11). Pyruvaldehyde and lactoylglutathione, intermediates of pyruvate metabolism, were accumulated. Acetyldihydrolipoamide, oxidizing pyruvate into acetyl-CoA to enter the TCA cycle, increased. The oxalosuccinate and glycolate, products of the TCA and glyoxylate cycle, respectively, accumulated. Glucose, glucose 6-phosphate, and glyceraldehyde 3-phosphate, the key metabolites in glycolysis and pentose phosphate pathway, were exhausted in the transgenic strain, indicating the improved glycolytic flux. Nevertheless, the cells were rich in other sugars like sorbose, cellobiose, and lactulose, along with galactose pathway metabolites such as melibitol, epimelibiose, and 1-phospho-α-galacturonate, which were not detected in the wild type strain. The glucosamine and glucosamine 6-phosphate, precursors for cell wall sugars, seemed exhausted to synthesize UDP- N-acetylglucosamine and UDP-xylose. In addition to the sugars and cellular polysaccharides, lipids, fatty acids, and other metabolites regulating membrane homeostasis and stress tolerance such as carnitine, glycerophosphocholine, etc.
[399, 400], were also enhanced in the transgenic strain, as listed in Table 2. Flavonoids like kaempferol and catechin, responsible for the antioxidant activity and alcohol tolerance, respectively, were increased. N-(3-oxo-hexanoyl)-homoserine lactone involved in quorum sensing and stress tolerance was also enhanced. In contrast, metabolites having anti-growth effects such as 5-hydroxydopamine and miglitol via inhibiting respiration and carbohydrate metabolism, respectively, decreased. Sialic acids like N-Acetyl-9-O-lactoyl neuraminic acid and their precursors were accumulated in transgenic strain, which could increase cell-negative charge, hence explaining the reduced aggregation in the VuNAC- expressing cells [401].
Table 4.3. Annotation of metabolites detected by LC-MS
S.N. Compounds Description m/z RT log FC
(log phase)
log FC (stationary)
VuNAC 1
VuNAC 2
VuNAC 1
VuNAC 2 PYRIMIDINE METABOLISM
1 Orotidylic acid de novo pyrimidine precursors 390.2 5.9 -7.74 -7.74 - -
2 UMP salvage pyrimidine and RNA
precursors
307.2 10.7 16.23 15.60 0.00 7.69
3 CMP RNA precursors 345.0 0.7 0.00 7.64 - -
4 2',3'-Cyclic CMP secondary messengers 343.1 0.8 8.22 0.00 - -
5 CDP DNA and RNA precursors 445.0 0.7 0.55 8.12 8.14 0.00
6 dUDP thymidine precursors 388.2 6.5 -15.80 -15.80 7.04 6.95
7 dTMP DNA precursors (thymidine) 342.2 0.7 -1.99 -9.51 15.68 0.00
8 Cytidine salvage pyrimidine precursors 281.2 7.3 18.09 17.25 - -
9 2'-Deoxyuridine 270.2 14.4 - - 15.52 7.87
10 Thymidine 288.2 12.5 6.94 0.00 7.12 0.00
PURINE METABOLISM
11 AICAR do novo purine precursor 321.2 0.7 -7.16 -7.16 - -
12 FAICAR 317.2 6.7 -16.54 -16.54 15.15 0.00
13 IMP salvage purine precursor 408.2 13.7 16.75 15.19 7.83 0.00
14 XMP 363.2 14.9 7.35 -0.04 - -
15 Hypoxanthine 136.0 0.8 -8.60 0.23 -15.69 -15.69
16 GDP RNA precursor 465.2 5.1 0.17 8.99 - -
17 ATP RNA precursor, energy currency 489.2 0.5 7.47 17.89 -9.00 -9.00
18 dATP DNA precursor 508.2 6.2 -7.75 -0.62 -7.35 -7.35
19 dAMP secondary messengers 333.2 2.9 -1.42 -1.31 - -
VITAMIN B6 AND THIAMINE METABOLISM
20 Pyridoxine 5'-phosphate vitamin B6 269.1 9.6 7.76 14.90 - -
21 Pyridoxal 5'-phosphate (PLP) 246.2 5.4 -16.47 -16.47 - -
22 HMP-P thiamine precursor 218.1 1.2 -19.21 -19.21 16.84 0.00
23 Thiamine TPP co-enzyme precursor 264.1 0.5 -1.30 -0.72 - -
24 Thiamine diphosphate (TDP) thiamine derivative, cofactor 423.3 11.6 15.49 16.55 - -
25 Thiamine pyrophosphate (TPP) coenzyme 442.3 16.8 8.42 -0.09 - -
FOLIC ACID AND PTERIN METABOLISM 26 2,5-Diamino-6-(5'-
phosphoribosylamino)-4- pyrimidineone
biopterin and flavin precursor 336.2 16.4 8.71 0.00 -7.41 -0.08
27 2,5-Diamino-4-hydroxy-6-(5- phosphoribosylamino) pyrimidine
335.2 12.5 0.00 14.14 - -
28 7,8-Dihydroneopterin biopterin precursor 254.2 16.5 9.12 9.07 0.00 18.58
29 Tetrahydrofolic acid (THF) folate derivative 467.4 15.8 7.28 0.00 - -
30 5,10-Methenyltetra-hydrofolic acid
460.4 18.3 -7.90 -7.90 -8.47 -8.47
RIBOFLAVIN METABOLISM
31 6,7-Dimethyl-8-(1-D-ribityl) lumazine riboflavin intermediate 325.3 17.2 -9.04 -0.25 - -
32 FMN cofactor, riboflavin derivative 456.4 11.3 6.79 0.00 - -
33 FMNH2 462.3 13.0 - - 0.00 15.90 PANTOTHENATE AND COENZYME A METABOLISM
34 Pantothenic Acid coenzyme A precursor 218.1 1.2 -19.21 -19.21 -5.05 -2.24
35 4-Phosphopantothenoyl cysteine coenzyme A intermediate 406.1 3.4 9.22 18.78 0.00 14.70
36 Pantetheine 278.1 6.2 18.31 16.71 0.00 8.46
NAD/NADH METABOLISM
37 NaMN vitamin B3/NAD precursor 374.2 5.3 -8.65 -8.65 18.39 0.00
38 NADH cofactor 665.1 0.8 0.00 8.02 -17.38 -17.38
39 Nicotinate ribotide vitamin B3/NAD salvage precursor 273.3 10.6 0.51 0.33 -7.80 -7.80
40 Nicotinamide vitamin B3 103.1 0.5 9.06 0.00 - -
GLUTAMATE, ASPARTATE AND ALANINE METABOLISM 41 Glutamate TCA intermediate, nucleotides and amino
acid precursor
147.1 0.6 -8.42 -7.32 - -
42 L-Aspartic acid 132.1 17.0 -8.88 -0.23 - -
43 L-Aspartyl-4-phosphate aspartate derivative, amino acid precursor 273.1 0.5 7.53 0.00 - - 44 Homoserine lysine, threonine and cysteine
precursor
119.1 0.5 7.03 7.62 -8.76 -8.76
GLUTATHIONE METABOLISM
45 Pyroglutamic acid glutathione precursor 167.1 0.8 -19.27 -2.00 - -
46 Reduced Glutathione (GSH) anti-oxidant 307.1 0.7 20.68 20.27 -2.96 -0.17
47 Oxidized Glutathione (GSSH) 612.2 0.7 2.32 2.34 -2.06 -0.44
HISTIDINE, ARGININE AND LYSINE METABOLISM
48 Imidazole acetol-phosphate 201.1 1.5 -1.03 -1.18 - -
49 2-(3-Carboxy-
3(methylammonio)propyl)- L- histidine
histidine derivative 251.1 11.7 8.25 7.96 - -
50 Nitro-L- Arginine arginine derivative, NO inhibitor 236.1 1.9 8.30 0.00 - -
51 L-2-Aminoadipic acid lysine precursor 143.1 0.7 20.65 20.32 0.29 0.78
52 Lysine nitrogen nutrient, histone component 146.1 0.5 7.46 8.44 - -
53 Hydroxylysine lysine analog, collagen biosynthesis 222.1 10.9 - - 7.20 14.44 54 Trimethyllysine histone component, carnitine biosynthesis 188.2 0.5 - - 8.47 17.21
LINEAR AND BRANCHED AMINO ACID METABOLISM
55 Glycine thiamine and amino acid precursor 103.1 0.5 -0.59 0.05 - -
56 DL-O-Phosphoserine serine intermediate, cysteine precursor 207.1 0.7 0.79 0.54 - -
57 L-Threonine isoleucine precursor 157.1 1.4 16.94 0.00 - -
58 Isoleucine promotes fermentation duration 113.1 2.7 18.61 9.24 - -
59 2-Isopropylmalic acid eucine precursor 204.1 0.5 -7.38 -7.38 -8.39 -8.39
60 3-Hydroxyisobutyric acid valine metabolite 86.0 0.6 -8.16 -8.20 0.00 16.50 SULFUR AMINO ACID METABOLISM
61 3-mercaptopyruvate cysteine metabolite 124.0 28.9 7.87 0.00 8.40 0.00
62 Methionine amino acid, prolongs log phase 148.1 4.7 19.42 9.28 -8.50 8.91 63 Methionine sulfoxide anti-oxidant, effects lifespan 165.1 0.7 - - 0.41 0.75 64 Adenosine phosphosulfate sulfur reduction pathway 427.0 0.6 - - -1.28 -9.41
AROMATIC AMINO ACID METABOLISM
65 Levodopa dopamine precursor 243.1 0.5 - - -7.09 -7.09
66 Metyrosine tyrosine derivative 177.1 3.1 17.45 0.00 - -
67 N-Formyl-L-tyrosine 231.2 9.0 0.00 14.01 - -
68 N'-Formylkynurenine tryptophan catabolite 217.2 7.6 9.06 -0.16 -7.05 -7.05
69 L-3-Hydroxykynurenine 223.2 9.7 0.08 -0.51 2.03 1.57
70 Quinolinic acid folate and pterin precursor 187.1 0.5 16.21 8.35 - -
PYRUVATE METABOLISM
71 Pyruvaldehyde pyruvate intermediate 71.1 2.7 0.00 8.37 - -
72 S-Lactoylglutathione 401.4 14.4 7.01 0.00 - -
73 S-Acetyldihydrolipoamide converts pyruvate into acetyl-CoA 248.1 0.6 16.24 0.00 7.98 7.56 TCA AND GLYOXYLATE CYCLE
74 Isocitric acid TCA cycle intermediates 174.1 0.4 -20.16 -20.16 - -
75 Oxalosuccinic acid 189.1 1.0 16.76 8.22 -16.83 -16.83
76 Succinic acid 117.1 0.6 -0.28 10.44 -0.39 -0.65
77 Fumaric acid 115.1 0.6 -9.52 0.13 - -
78 Glycolic acid glyoxalate derivative 118.1 1.9 9.34 0.00 - -
GLYCOLYSIS/ GLUCONEOGENESIS AND PENTOSE PHOSPHATE PATHWAY
79 Beta-D-Glucose hexose sugar 202.1 1.4 -9.11 -1.94 - -
80 D-Glyceraldehyde 3-phosphate glycolsysis end product 151.0 5.1 -8.63 -8.63 - -
81 D-Ribose 5-phosphate pentose sugar, purine precursor 276.1 6.6 -7.16 -7.16 - -
82 L-Sorbose hexose sugar 222.2 12.0 7.50 7.26 - -
83 D-Sedoheptulose 7- phosphate
heptose sugar derivative 328.0 12.0 0.00 7.93 - -
84 Cellobiose disaccharides 342.3 11.2 8.10 -0.16 - -
85 Lactulose disaccharides, synthetic sugar 342.1 0.5 9.92 11.01 - -
GALACTOSE METABOLISM
86 Melibitol disaccharide, galactose metabolism 361.2 1.1 9.07 0.00 - -
87 Epimelibiose 388.1 0.5 9.82 11.26 -9.69 -9.69
88 1-Phospho-alpha-D- galacturonate
galactose derivative 216.1 0.6 9.41 18.29 -15.84 2.85 AMINO AND NUCLEOTIDE SUGAR METABOLISM
89 Glucosamine 6-phosphate lipid and nucleoside sugar precursor 259.2 1.3 -10.10 -1.53 - -
90 UDP-GlcNAc cell wall precursor 607.1 0.6 9.44 9.37 - -
91 UDP-D-Xylose 596.1 0.7 - - 7.67 0.00
92 UDP-N-acetyl-D- galactosamine
653.1 0.7 - - 0.00 16.57
LIPID AND FATTY ACID METABOLISM
93 Phosphocholine represses phospholipid biosynthesis 183.1 13.0 -1.66 -0.25 -0.73 0.08 94 Glycerophosphocholine membrane lipid homeostasis, salt tolerance 257.1 0.6 2.84 2.31 -0.26 1.89 95 GPCho(O-16:0/2:0) membrane phospholipid, activator 523.4 15.9 10.71 0.78 8.78 9.64 97 GPEtn(18:0/0:0) glycerophosphorylethanolamine 541.3 14.4 8.11 8.08 -7.73 -7.73
98 GPEtn(16:0/0:0) 513.3 13.2 8.88 8.13 -10.30 -0.73
99 GPSer(18:1(9Z)/0:0) glycerophosphoserine 497.3 14.4 -7.88 0.60 -8.58 -8.42
100 Hexadecanedioic acid membrane lipid precursor 285.2 13.8 8.40 8.74 - -
101 7-palmitoleic acid unsaturated FA 236.2 16.5 8.26 0.00 16.65 8.35
102 2-methyl-tridecanedioic acid methyl FA, C13 257.2 12.1 10.07 10.15 0.00 15.35 103 3-Hydroxydodecanedioic acid hydroxy FA, C12 268.1 13.6 8.56 -0.07 8.13 8.09
104 Carnitine carnitine, FA oxidation 143.1 1.0 7.81 8.62 -16.49 -16.49
OTHER METABOLITES 104 N-Acetyl-D-mannosamine 6-
phosphate
sialic acid precursor 347.1 0.5 8.42 7.71 - -
105 N-Acetyl-9-O-lactoyl neuraminic acid
reduces cell-adhesion 363.1 5.2 8.43 0.00 1.17 0.45
106 Peonidin anthocyanin 305.2 9.3 -6.88 -6.88 16.49 8.37
107 Alloxanthin carotenoid, anti-oxidant and vitamin precursor
584.3 17.0 7.94 7.55 - -
108 Kaempferol flavonols, anti-inflammatory and antioxidant
324.2 13.4 9.55 9.68 - -
109 Catechin flavanols, alcohol and toxin resistance 289.3 10.8 -0.07 -0.51 1.66 1.30
110 Piceatannol stilbenes 272.2 14.2 16.05 15.83 - -
111 Epinephrine-like promotes glycogen break 165.1 1.2 22.23 22.02 0.90 0.42
112 Indole-3-carboxylic acid-like promotes sporulation 143.0 3.9 0.65 -8.80 18.18 8.20 113 Kinetin riboside-like promotes cell proliferation 347.1 5.9 8.06 7.97 - - 114 5-Hydroxydopamine drug inhibiting respiratory growth 151.1 0.7 -8.99 -8.89 - - 115 Miglitol drug inhibiting complex carbohydrate
catabolism
189.1 1.2 - - -10.17 -0.63
116 Sulfasalazine drug inhibiting biopterin biosynthesis 440.1 0.5 - - -3.05 -1.74 117 Sulfamerazine drug inhibiting the folic acid synthesis 246.1 0.5 - - -9.98 -9.98 118 Flumequine drug, anti-lipase, growth inhibition 307.1 0.7 - - -8.14 -8.14 119 N-(3-oxo-hexanoyl)-homoserine
lactone
quorum sensing, oxidative stress tolerance 212.1 0.7 8.35 16.27 - -
120 Metergoline Alkaloid inducing cell death 423.2 1.9 -8.62 -9.48 - -
Fig. 4.10 Pathway Enrichment Analysis. (A) Major altered pathways and their networks resulted from the differential accumulation of metabolites in the VuNAC1/2 expressing strains in different growth phases.
Fig. 4.10. Pathways impact analysis. (B) The bubble plot indicated alterations in the primary metabolic pathways in the order of the impact resulting from the differential accumulation of the metabolites due to transcriptional tuning of the genes/enzymes involved in the biosynthesis or metabolic conversions. The bubble size was proportional to the impact, and the color denoted statistical significance from highest (red) to lowest (white).
Fig. 4.11 Pictorial illustration of the remodeled pathways in VuNAC1-expressing strain in log phase.
(A) Nucleotide biosynthesis, (B) Vitamin biosynthesis, and (C) Amino acid biosynthesis. The pink spheres indicated accumulated metabolites, the blue spheres indicated exhausted metabolites, and the grey color indicated either no change or unavailability of information.