Control limits for accumulation of plant metabolites: brute force is no substitute for understanding.

Which factors limit metabolite accumulation in plant cells? Are theories on flux control effective at explaining the results? Many biotechnologists cling to the idea that every pathway has a rate limiting enzyme and target such enzymes first in order to modulate fluxes. This often translates into large effects on metabolite concentration, but disappointing small increases in flux. Rate limiting enzymes do exist, but are rare and quite opposite to what predicted by biochemistry. In many cases however, flux control is shared among many enzymes. Flux control and concentration control can (and must) be distinguished and quantified for effective manipulation. Flux control for several 'building blocks' of metabolism is placed on the demand side, and therefore increasing demand can be very successful. Tampering with supply, particularly desensitizing supply enzymes, is usually not very effective, if not dangerous, because supply regulatory mechanisms function to control metabolite homeostasis. Some important, but usually unnoticed, metabolic constraints shape the responses of metabolic systems to manipulation: mass conservation, cellular resource allocation and, most prominently, energy supply, particularly in heterotrophic tissues. The theoretical basis for this view shall be explored with recent examples gathered from the manipulation of several metabolites (vitamins, carotenoids, amino acids, sugars, fatty acids, polyhydroxyalkanoates, fructans and sugar alcohols). Some guiding principles are suggested for an even more successful engineering of plant metabolism.