Table 9 False gene essentiality predictions resulting from isozymes or pathways not operational under the experimental conditions of Baba et al.

ydiB encodes an isozyme for the AroE shikimate dehydrogenase in EcoCyc. Johansson and Liden [81] suggest that the NAD ⁺/NADP ⁺ specificity of YdiB and high intracellular NAD ⁺ concentrations lead it to operate in the “reversed” shikimate dehydrogenase direction, as opposed to the biosynthetic direction toward chorismate.

cynT, a carbonic anhydrase in the cyn cyanate degradation operon, encodes an isozyme for the Can carbonic anhydrase reaction in EcoCyc. cynT is conditionally expressed in the presence of cyanate, which is absent in [32] minimal media conditions.

folM, a 7,8-dihydromonapterin reductase in the tetrahydromonapterin biosynthesis pathway with a weak 7,8-dihydrofolate reductase activity, encodes an isozyme for the FolA dihydrofolate reductase reaction in EcoCyc. folM is insufficiently expressed in vivo to supply E. coli growth requirements for tetrahydrofolate.

10-formyl-tetrahydrafolate formation by folD can be shortcircuited in EcoCyc–18.0–GEM by purN’s reversible phosphoribosylglycinamide formyltransferase activity. PurN’s k _{c a t} is substantially higher in the forward direction than in the reverse direction in vitro, but the in vivo reversibility of the enzyme is uncertain.

The threonine dehydrogenase Tdh and 2-amino-3-ketobutyrate CoA ligase Kbl provide an

alternate, threonine-based route to glycine synthesis from serine by the GlyA serine hydroxymethyltransferase. tdh and kbl are under the control of the Lrp leucine repressor system, and are conditionally expressed in the presence of leucine, which is absent in [32] minimal media conditions. Patrick et al.[77] determined that overexpression of Tdh, the LtaE low-specificity threonine aldolase, the YneH glutaminase, or Rsd anti-sigma factor led to rescue of glyA deletion mutants, and that YneH was also capable of rescuing serA null mutants.

guaB is essential for growth on glucose in [32], and [88] indicates that guaB mutants require guanine for growth. In EcoCyc, nucleotide salvage pathways allow the degradation of IMP to inosine, subsequent conversion of inosine to xanthine via XapA/DeoD and XdhA, and finally conversion of xanthine to XMP via Gpt.

The threonine dehydratase TdcB acts as an isozyme for the IlvA threonine deaminase in EcoCyc.tdcB is expressed only under anaerobic conditions. Patrick et al.[77] identified TdcB or EmrD multidrug efflux transporter overexpression as capable of rescuing ilvA deletion mutants.

The lipoyl-carrier protein N ⁶-octanoyl-L-lysine intermediate in lipoate synthesis can be produced by both LipB and LplA in EcoCyc. LplA is primarily involved in the assimilation of extracellularly sourced lipoate. See [90] for further details.

MalY’s β-cystathionine lyase activity is listed as an isozyme for MetC in EcoCyc. malY is involved in complex regulatory interactions, and its expression is repressed by malI in WT strains. Without appropriate signaling, malY is not expressed. Patrick et al.[77] rescued metC deletion mutants via MalY, Alr alanine racemase, or FimE phase-variation switch regulator overexpression.

E. coli’s three aspartokinases, ThrA, MetL, and LysC, are all isozymes for the aspartokinase reaction in

EcoCyc. The end-product inhibition of each of these enzymes should prevent aspartokinase gene KOs from being rescued by their isozymes, since adequate amino acid pools in the pathways of the remaining isozymes will inhibit their activity. Kim and Copley [36] suggest that metL is not expressed on glucose.

The NrdDE ribonucleoside diphosphate reductase acts as an isozyme for the NrdAB ribonucleoside

diphosphate reductase in EcoCyc. nrdDE is expressed only under anaerobic conditions.

EcoCyc–18.0–GEM can overcome deletion of serC and pdxB by using the thrB/ltaE route from

glycolaldehyde (produced by FolB and formed spontaneously from 3-hydroxypyruvate supplied from YeaB) for production of 4-phospho-hydroxy-threonine and subsequently pyridoxal-5’-phosphate [96, 97]. Replacement of serC and pdxB by these pathways requires overexpression of thrB or yeaB/nudL, and growth after overexpression is reported only for solid media [97], whereas the assays of [32] were conducted in liquid media. Patrick et al.[77] rescued pdxB deletion mutants with Tdh threonine dehydrogenase or PurF amidophosphoribosyl transferase overexpression, and serC deletion mutants with YneH glutaminase overexpression.

The PRPP biosynthesis II pathway can substitute for prs deletion in EcoCyc–18.0–GEM. This pathway is based on connection of ribose 5-phosphate through the DeoB phosphomutase to the PhnN ribose 1,5 bisphosphokinase activity via a putative ribose 1-phosphokinase activity [98, 99]. PhnN is part of the phn operon, [99, 101] whose expression is repressed under the 2 mM phosphate glucose minimal media conditions used by [100].

EcoCyc–18.0–GEM can overcome deletion of serB by synthesizing serine from threonine via Tdh and reversible action of GlyA. Ravnikar and Somerville [102] isolated pseudorevertants containing elevated levels of Tdh from ser deletions following growth on media supplemented with threonine, leucine, arginine, lysine, and methionine followed by growth on minimal media. Patrick et al.[77] additionally identified overexpression of Gph, HisB, and YtjC phosphatases as capable of rescuing serB deletion mutants.