Table 1.

Graph-based, Stoichiometry-based, and retrosynthesis-based pathway design tools and their characteristics.

Category	Name	Database	Network representation	Network pruning	Search algorithm	Pathway ranking	Reference
Graph-based	ReTrace	KEGG	Bipartite graph	Atom mapping	Heuristic search	Atom conservation and pathway length	[23]
	PathComp	KEGG	Substrate graph	–	Depth-first search (DFS)	–	[26]
	MetaRoute	KEGG	Reaction graph	Weighted graph and atom mapping	Eppstein's k-shortest path	Atom conservation and metabolite connectivity	[51]
	Pathway Hunter Tool	KEGG	Substrate graph	–	Breadth-first search (BFS) with (Higher-order horn logic) HOHL	Structure similarity and pathway length	[63]
	FMM	KEGG	Substrate graph	Manual cofactor removal	BFS	Compare pathway across organisms	[64]
	RouteSearch	MetaCyc	Substrate graph	Atom mapping	Branch and Bound	Atom conservation and pathway length	[65]
	MRE	KEGG	Substrate graph	Weighted graph	Yen's loopless k-shortest path	Thermodynamics and genes from host organism	[66]
	CMPF	KEGG, RPAIR	Bipartite graph	Weighted graph	Bounded depth path enumeration	Metabolite connectivity, reaction occurrence frequency, and pathway switching	[67]
	NeAT	MetaCyc	Bipartite graph	Weighted graph	Takahashi–Matsuyama, Pairwise K-shortest paths, and kWalks	Metabolite connectivity	[68]
	LPAT/BPAT	KEGG	Bipartite graph	Atom mapping	BPAT-M Search	Atom conservation and pathway length	[69], [139]
	Rahnuma	KEGG	Hypergraph	Phylogeny or sub-network	DFS	–	[70]
	Metabolic Tinker	CHEBI, Rhea	Hypergraph	Weighted graph	Heuristic search	Pathway length, structure similarity, and thermodynamics	[71]
	FogLight	KEGG, MetaCyc	Hypergraph	And/Or graph	Brute-force search	Pathway length	[73]
	MRSD	KEGG	substrate graph	Weighted graph	Eppstein's k-shortest path	Reaction occurrence frequency	[78]
	DESHARKY	KEGG	–	Phylogeny	Monte Carlo	Metabolic burden	[88]
Stoichiometry-based	optStoic	KEGG, MetRxn	S matrix	Design overall stoichiometry	MILP	Pathway length or total metabolic flux	[24]
	PathTracer	BIGG, iJO1366	Substrate graph, S matrix	Atom mapping (MapMaker)	MILP	Pathway length or most active path	[50]
	CFP	BIGG	Substrate graph, S matrix	Atom mapping (carbon exchange network)	MILP	Pathway length	[75]
	METATOOL 5.0/k-shortest EFM	BIGG, iAF1260	S matrix	–	MILP	Pathway length	[76], [89]
	OptStrain	KEGG	S matrix	–	MILP	Number of heterologous reactions	[99]
Retrosynthesis-based	Simpheny	BIGG	Substrate graph	molecule sizes	Retrosynthetic enumeration	Pathway length, thermodynamics, product yield, number of known metabolites/enzymes, and existence of reaction operators	[5]
	GEM-Path	BIGG, iJO1366	Substrate graph	Third level EC number and substrate similarity	Retrosynthetic enumeration	Thermodynamics and product yield	[57]
	XTMS/RetroPath/RetroPath 2.0	MetaCyc, BioCyc	S matrix	Molecular signature with predetermined distance	Retrosynthetic enumeration and MILP	Thermodynamics, gene prediction, pathway length, number of putative steps, and product yield	[52], [82], [84]
	BNICE	KEGG, ATLAS	Substrate graph	Qualitative/Quantitative pruning	Retrosynthetic enumeration	Pruning criteria assessment (thermodynamics, pathway length, etc.)	[8], [53]
	UM-PPS	UM-BBD	Substrate graph	Rule priority	Retrosynthetic enumeration	–	[56]
	PathPred	KEGG, RPAIR	Substrate graph	Structure similarity	Retrosynthetic enumeration	Compound similarity and pathway score	[54]
	Route Designer	MOS, Beilste Crossfile	Substrate graph	Heuristics and user defined limits	Retrosynthetic enumeration	Weighted function (wastage, example counts, and balanced disconnections.)	[55]
	SimIndex/SimZyme	BRENDA	Substrate graph	Structure similarity	Byers–Waterman type pathway search	Pathway length	[83]
	Method by Cho et al.	KEGG	Substrate graph	–	Retrosynthetic enumeration	Combination of five priority factors	[22]