Table 3. Composition of the in silico minimal media (IMMs) that allow growth of iPfa and essentiality.

Constitutive metabolites of the IMM		Essential1
Source of amino acids	L-Isoleucine; Oxyhemoglobin	E; NE
Source of inorganic sulfur	Sulfate; Sulfur donor	E; E
Source of inorganic iron	Heme	NE2
Cofactors and others not synthesized de novo	Biotin; Methylcobalamin; Thiamine; Cholesterol	E; E; E; E
Precursors for lipoylation	Lipoate; Apoprotein	E; E
Precursors of lipid components	Choline; N-Acylsphingosine	E; NE2
Precursor of FMN/FAD	Riboflavin	E
Precursors of isoprenoids	4-Hydroxybenzoate; HCO3-	E; E
Non-constitutive metabolites of the IMM3
Source of inorganic phosphate	Orthophosphate or Diphosphate	NE2
Precursors of CoA	Pantothenate or Pantetheine or N-((R)-Pantothenoyl)-L-cysteine	E
Source of pyridine ring (* and source of carbon)	Nicotinate D-ribonucleoside* or N-Ribosylnicotinamide* or Nicotinate or Nicotinamide	E
Source of DNA nucleotides (* and source of carbon)	S-adenosyl-L-methionine* or S-adenosyl-L-homocysteine* or S-adenosylmethioninamine* or Se-adenosyl-L-selenohomocysteine* or Adenosine* or Inosine* or Guanosine* or Xanthosine* or Adenine or Guanine or Hypoxanthine or Xanthine	NE2
Source of folate and derivatives	Tetrahydrofolate (THF) or 5,10-MethyleneTHF or 10-FormylTHF or 5-MethylTHF or 5,10-MethenylTHF or Dihydrofolate or Folate or Dihydropteroate or 4-Amino-4-deoxychorismate or Chorismate or 4-Aminobenzoate	E
Source of pyrimidine ring (precursors of UMP)	Orotate or (S)-Dihydroorotate	E
Source of C2/C4 for acetyl-CoA (precursors of nucleotide sugars)	Acetate or L-2-Amino-3-oxobutanoic acid	E

¹Phenotype observed in the simulation after one-by-one depletion of the substrate or group of substrates in the in silico rich medium of 236 substrates: E, essential; NE, non-essential.

²See the next result section for explanation.

³The 10,032 alternative IMMs are generated through the combination of the constitutive metabolites and one non-constitutive metabolite from each group reported in Table 3. Note that the following constraints in the combinations should be considered: (a) the IMM should provide a ribose-containing molecule, which serves as source of carbon (marked with * in Table 3) and (b) the presence of orthophosphate and S-adenosylmethioninamine in the same IMM is not allowed (S1 Methods for more details).