Identification and Classification of Principal Nodes, Fructose, Phosphate, Phosphoenolpyruvate, Metabolic engineering

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Metabolic Engineering for Over production of Metabolites

	Identification and Classification of Principal Nodes - In the above example of lysine production, although, there may be as many as 30 branch points, but significant changes in flux distribution occur at only three nodes, when lysine production increases from 35% to 75% (Fig. 25.3B and 25.3C). These include glucose-6-phosphate (G6P), phosphoenolpyruvate (PEP), and pyruvate (Py). For yield above 60%, fructose-6 phosphate (F6P) becomes a principal node in place of G6P (for details consult Stephanopoulos and Vallino, 1991). Identification of these nodes in any metabolic pathway is a prerequisite for any exercise involving metabolic engineering.
Identification of principal nodes, where flux distribution needs to be altered also depends on whether a network or subnetwork architecture is dependent (where each node contributes to a consumed component of final product; e.g. OaA and Py in lysine network) or independent (where metabolites produced from principal nodes do not condense. The metabolic engineering in a dependent pathway is much more difficult than in a.1 independent pathway. The yield of desired product in an independent" network can be improved simply by altering flux partitioning at anyone of the principal nodes. In a dependent network, on the other hand, flux partitioning should be altered at both dependent nodes, so that flux partitioning at these nodes is altered in a co ordinate manner.
The principal nodes are classified into three main categories depicted in flexible nodes are those, where flux alterations can be made easily to meet the demands of metabolic engineering without any adverse effects. Such flexible nodes are often controlled by feedback inhibition in each branch. One of the examples of flexible nodes is aspartate semiadehyde (ASA), where flux can be redirected towards lysine synthesis by deregulating feedback inhibition of aspartokinase by lysine and threonine (ii) Weakly rigid nodes are those, where one branch is dominant with no feedback inhibition and the other is subordinate with feedback inhibition. In such a case even if subordinate branch is deregulated, to get a specific product P, a significant fraction of the flux still enters the dominant branch due to enzyme affinity, so that the yield of desired product is limited.
Therefore, not only deregulation of subordinate branch, but also attenuation of the activity of dominant branch and/or amplification of the activity of subordinate branch will be required. Such weakly rigid nodes are often found in catabolic pathways leading to CO₂ formation. One such example is isocitrate branch point between glyoxylate shunt and TCA cycle in E.coli cultures grown on acetate. When glucose is added to this culture, the flux supported by isocitrate lyase (ICL, which is the first enzyme of glyoxylate shunt) drops to zero (although glyoxylate shunt is fully active) and isocitrate dehydrogenase (ICDH) of TCA cycle exhibits complete dominance over ICL, because Michaelis constant (Km) of ICDH for isocitrate is 1/75 that of ICL.
	However, if the activity of ICDH is attenuated by 60% or more, a significant fraction of isocitrate can be diverted into glyoxylate shunt (ICDH activity can be attenuated by phosphorylation). Therefore, for preferential flux distribution towards glyoxylate shunt, feedback inhibition of ICL needs to be deregulated and ICDH activity needs to be attenuated. (iii) Strongly rigid nodes are those, in which feedback inhibition by a metabolite in its own branch is associated with transactivation of enzyme in an opposite branch. In B.subtilis, histidine activates anthranilate synthetase, an enzyme involved in tryptophan synthesis, so that B. subtilis can grow in presence of 5-methyltryptophan, an antimetabolite of tryptophan.

Such transactivation is also described as metabolic Interlock or compensatory control of activation. Remote effectors may also be involved in activation. Thus there are significant coupling interactions associated with these strongly rigid nodes, so that flux partitioning can not be easily modified. This may require alterations both in substrate affinity and the effectors involved.