Table 1.

Genes used in combinatorial cloning and their metabolic functions. Mod column shows which gene was deleted “- “, or upregulated “+”, Modifications of genes with indicated ‘-’ mode appear in the text with ‘D’ prefix (Dtdh).

Gene name	Enzyme Name	Role	Expected effect	Mod	Reference
thrABC	Bifunctional aspartokinase/homoserine dehydrogenase 1, homoserine kinase, and threonine synthetase	Thr/Lys/Met biosynthesis	Amplification should activate threonine overproduction	+	(Kozlov Iu et al., 1980)
asd	Aspartate semialdehyde dehydrogenase	Thr/Met biosynthesis	Amplification should activate threonine overproduction	+	(Debabov 2003)
lysC	Lysine-sensitive aspartokinase 3	Thr/Lys/Met biosynthesis	Increased expression of lysine feedback resistant allele is expected to improve production	–	(Ogawa-Miyata et al., 2001)
metL	Bifunctional aspartokinase/homoserine dehydrogenase 2	Thr/Lys/Met biosynthesis	Deletion/Increased expression are expected to affect methionine and SAM production	–	(Neidhardt and Curtiss 1996)
rhtA	Threonine/homoserine exporter	Threonine export	Increased expression is expected to improve threonine production	+ or -	(Livshits et al., 2003)
aceBA	Malate synthase A and Isocitrate lyase	Carbon backbone	Increased expression of the glyoxylate shunt is expected to improve threonine production	+	(Liu et al., 2019)
ppc	Phosphoenolpyruvate carboxylase	Carbon backbone	Increased expression of ppc is expected to improve threonine production (more precursor available)	+	(Lee et al., 2007)
pyc	Pyruvate carboxylase	Carbon backbone	Increased expression facilitates the flow of carbon to oxaloacetate and aspartate in C. glutamicum, expected to cause the same effect in E.coli	+	(Peters-Wendisch et al., 2001)
ptsG	PTS system glucose-specific EIICB component	Carbon backbone	Deletion of the PTS system for glucose uptake is expected to make more precursor available	–	(Zhu et al., 2019)
dhaM	PEP-dependent dihydroxyacetone kinase, phosphoryl donor subunit	Carbon backbone	Deletion is expected to disrupt putative allosteric regulation of dihydroxyacetone kinase (encoded by dhaK) and negative regulation of dha operon	–	(Gutknecht et al., 2001) (Bachler et al., 2005)
zwf	Glucose-6-phosphate 1-dehydrogenase	Carbon backbone, Co-factor biosynthesis	Increased expression is expected to increase availability of threonine precursors (glucose-6-p and NADPH)	+	(Becker et al., 2007)
pntAB	NAD(P) transhydrogenase (membrane-bound)	Co-factor biosynthesis	Increased expression expected to compensate for NADPH depletion	+	(Liu et al., 2019)
aspC	Aspartate aminotranferase	Threonine biosynthesis/Competing pathways	Increased expression is expected to increase precursor pool	+	(Zhao et al., 2020)
lysA	Diaminopimelate decarboxylase	Competing pathways	Deletion is expected to make more precursors available	–	(Lee et al., 2007)
dapA	Dihydrodipicolinate synthase	Competing pathways	Deletion is expected to affect availability of all threonine precursors (aspartate, glutamate, NADPH)	–
tdh	L-threonine 3-dehydrogenase	Threonine catabolism	Deletion is expected to diminish threonine degradation	–	(Lee et al., 2007)