The recent advancement on metabolic systems engineering was reviewed based on our recent research results in terms of (1)metabolic signal flow diagram, (2)metabolic flux analysis (MFA) with or without NMR, (3)optimization of metabolic flux distribution (MFD), (4)modification of MDF by gene manipulation and by controlling culture environment, (5)identification of unknown pathways by NMR, and (6)metabolic control analysis (MCA). The main characteristics of metabolic engineering is to treat metabolism as a network or entirety instead of individual reactions. The applications were made for poly-3-hydroxybutyrate (PHB) production using Ralstonia eutropha and recombinant Escherichia coli, lactate production by recombinant Saccharomyces careuisiae, pyruvate production by vitamin auxotrophic yeast Toluropsis glabrata, and lysine production using Corynebacterium glutamicum. The characteristics of each approach were reviewed with their applications.