IrC nVl lN nt0 lhe cul1 should reslllt in the

Tm! !n\>l a sell mutated at a particular site. The \ \i.riiioii of a selectable marker into the cloned HN would avist in the recovery of cells incorporating 'he modified ucne(s). Thus, such systems still depend !m ilie development of methods to recover cells displaying the desirable feature.

The -ep.ii'iiion of desirable mutants from the other Miniw'i-x ôi .1 mutation treatment is similar to the ^.kaiini ol dc-irable organisms from nature. Where possible, the mutant isolation procedure should use the unpriced cluucteristic of the desired mutant as a selcciiw i.icti'i. Presumably, superior productivity is a result of a diversion of precursors into the product and -oi a modification of the control mechanisms limiting the level of production. Thus, a knowledge of the hio-vmhetic route and the mechanisms of control of the biosynthesis of the product should enable the prediction of a 'blueprint' of the desirable mutant. Such a 'bluepi int' might then enable the design of isolation techniques which would give the desired mutants a selective advantage over the other types present. Knowledge of biosynthetic routes and control mechanism-. are more detailed for primary metabolites and, ilvielore, the use of selective pressure in mutant isolation i- more common in the fields of amino acid, iiuclcoiide and enzyme production than in secondary niel.ibnlite production. However, considerable progress has also been made in the design of such procedures t.u lhe isolation of secondary metabolite over-pro-

dlliYlv

The selection of induced mutants synthesizing improved levels of primary metabolites sion. Feedback inhibition is the situation where the end product of a biochemical pathway inhibits the activity of an enzyme catalysing one of the reactions (normally the first reaction) of the pathway. Inhibition acts by the end product binding to the enzyme at an allosteric site which results in interference with the attachment of the enzyme to its substrate. Feedback repression is the situation where the end product (or a derivative of the end product) of a biochemical pathway prevents the synthesis of an enzyme (or enzymes) catalysing a reaction (or reactions) of the pathway. Repression occurs at the gene level by a derivative of the end product combining with the genome in such a way as to prevent the transcription of the gene into messenger RNA, thus resulting in the prevention of enzyme synthesis.

Feedback inhibition and repression frequently act in concert in the control of biosynthetic pathways, where inhibition may be visualized as a rapid control which switches off the biosynthesis of an end product and repression as a mechanism to then switch off the synthesis of temporarily redundant enzymes. The control of pathways giving rise to only one product (i.e. unbranched pathways) is normally achieved by the first enzyme in the sequence being susceptible to inhibition by the end product and the synthesis of all the enzymes being susceptible to repression by the end product, as shown in Fig. 3.5.

The control of biosynthetic pathways giving rise to a number of end products (branched pathways) is more complex than the control of simple, unbranched sequences. The end products of the same, branched biosynthetic pathway are rarely required by the microorganism to the same extent, so that if an end product exerts control over a part of the pathway common to two, or more, end products then the organism may suffer deprivation of the products not participating in the control. Thus, mechanisms have evolved which enable the level of end products of branched pathways to be controlled without depriving the cell of essential intermediates. The following descriptions of these mechanisms are based on the effect of the control, which may be arrived at by inhibition, repression or a combination of both systems.

Hel.ire considering the methods used for the selection oi mutants producing improved levels of primary mciaiMlites it is necessary to study the mechanisms of control of their biosynthesis such that the 'blueprints', 'elciu-d to above, may be drawn accurately. The levels I Pnmary metabolites in micro-organisms are regulated by feedback control systems. The major systems iii\\)lvi-i| are feedback inhibition and feedback repres

---^ Feedback inhibition

~~I r" Feedback repression

---^ Feedback inhibition

~~I r" Feedback repression

Flo. 3.5. The control of a biosynthetic pathway converting precursor A to end product E via the intermediates B, C and D.

Fig. 3.6. The control of a biosynthetic pathway by the concerted 50%_____'

effects of products D and F on the first enzyme of the pathway.

Concerted or multivalent feedback control. This control system involves the control of the pathway by more than one end product — the first enzyme of the pathway is inhibited or repressed only when all end products are in excess, as shown in Fig. 3.6.

Co-operative feedback control. The system is similar to concerted control except that weak control may be effected by each end product independently. Thus, the presence of all end products in excess results in a synergistic repression or inhibition. The system is illustrated in Fig. 3.7 and it may be seen that for efficient control to occur when one product is in excess there should be a further control operational immediately after the branch point to the excess product. Thus, the reduced flow of intermediates will be diverted to the product which is still required.

Cumulative feedback control. Each of the end products of the pathway inhibits the first enzyme by a certain percentage independently of the other end products. In Fig. 3.8 both D and F independently reduce the activity of the first enzyme by 50%, resulting in total inhibition when both products are in excess. As in the case of co-operative control, each end product must exert control immediately after the branch point so that the common intermediate, B, is diverted away from the pathway of the product in excess.

Sequential feedback control. Each end product of the pathway controls the enzyme immediately after the i------------1

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