STRAIN IMPROVEMENT IN VITRO AND

FEEDBACK REGULATION

The most dramatic examples of strain improvement come from the

applications of recombinant DNA technology which has resulted in organisms
producing compounds which they were not able to produce previously.

THE ISOLATION OF MUTANTS WHICH DO NOT PRODUCE FEEDBACK

INHIBITORS OR REPRESSORS( AUXOTROPHS )-
Mutants which do not produce certain feedback inhibitors or repressors may
be useful for the production of intermediates of unbranched pathways and
intermediates and end products of branched pathway. The mutants
do not produce some of the inhibitors or repressors ,the control of pathway is
lifted. Dulaney and Dulaney (1967) compared the spread in productivity of
chlortetracycline of natural variants of Streptomyces viridifaciens with the
spread in productivity of the survivors of an ultraviolet treatment.Although
there are more inferior producers amongst the survivors of the ultraviolet
treatment there are also strains producing more than twice the parental
level,far greater than the best of the natural variants.

-Isolation of analogue resistant mutants

An analogue is a compound which is very similar in structure to another
cimpound Mutants may be isolated which are resistant to the inhibitory
effects of the analogue and, if the site of toxicity of the analogue is the
mimicing of the control properties of the natural product,such mutants may
overproduce the compound to which the analogue is analogous provided that:
(i) The toxicity of the analogue is due to its mimicing the control properties of
the natural product.
(ii) The site of resistance of the resistant mutant is the site of control by the
end product
Second technique used for the isolation of mutants altered in the recognition
of control factor is the isolation of revertant mutant
ISOLATION OF REVERTANT MUTANTS-
.Auxotrophic mutants may revert to the phenotype of the mutant parent
A revertant of the mutant produces large concentrations of end product The
explanation of the behaviour of the revertant is that,with two mutations
having occurred at loci concerned with the production of enzyme the enzyme
of the revertant is
different from the enzyme of the original prototrophic strain and is not
susceptible to the control by p. Revertants may occur spontaneously or
mutagenic agents may be used to increase the frequency of occurrence

The use of recombination systems for the improvement of industrial micro-

organisms

Hopwood (1979) defined recombination ,in its broadest sense,as "any process
which helps to generate new combinations of genes that were originally
present in different individuals".The use of recombination mechanisms for the
improvement of industrial strains has increased significantly due to the
developments in recombinant DNA technology and the necessity to develop
new methods of strain improvement as the returns generated from mutation
and selection programmes decreased.
APPLICATION OF PARASEXUAL CYCLE-
Many industrially important fungi do not possess a sexual stage and therefore
it would appear difficult to achieve recombination in these organisms.
However, Pontecorvo et al. (1953) demonstrated that nuclear fusion and gene
segregation could take place outside,or in the absence of, the sexual
organs.The process was termed the parasexual cycle and has been
demonstrated in the imperfect fungi, A. niger and P. chrysogenum, as well as
the sexual fungus A. nidulans.
The major components of the parasexual cycle are the establishment of
heterokaryon,vegetative nuclear fusion and mitotic crossing over or
haploidization resulting in the formation of a recombinant.
THE APPLICATION OF PROTOPLAST
FUSION TECHNIQUES
.Cell fusion,followed by nuclear fusion, may occur between protoplasts of
strains which would otherwise not fuse and the resulting fused protoplast may
regenerate a cell wall and
grow as normal cell.Thus,protoplasts may be used to overcome some
recombination barrier.

THE APPLICATION OF RECOMBINANT

DNA TECHNIQUES-
The basic requirements for the in vitro transfer and expression of foreign
DNA in a host micro-organism as follows:
(j) A 'vector'DNA molecule(plasmid or phage) capable of entering the hostcell
and replicating within it. Ideally the vector should be small,
easily prepared and must contain at least one site where integration of foreign
DNA will not destroy an essential function.
(ij) A method of splicing foreign genetic information into the vector.
(iii) A method of introducing the vector foreign DNA recombinants into the
host cell and selecting for their presence.
(iv) A method of assaying for the 'foreign 'gene product of choice from the
population of recombinant created .

FEEDBACK REGULATION-

The major systems involved are feedback inhibition and feedback repression.
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.

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.

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.

Cumulative feedback control-

Each of the end products of the pathway inhibits the first enzyme by a certain
percentage independently of the other end product.
Sequential feedback control- Each end product of the pathway controls the
branch point to the product. The intermediates which then build up as a result
of this control earlier enzymes in the pathway .
Isoenzyme control-
Isoenzymes are enzymes which catalyse the same reaction but differ
in their control characteristics.

An understanding of the control of production of a metabolite may enable the

construction of 'blueprint 'of the most useful industrial mutant,i.e. one where
the production of the metabolite is not restricted by the organism's control
system. Such postulated mutants may be modified in three ways:
1.The organism may be modified such that the end products which control the
key enzymes of the pathway are lost from the cell due to some abnormality in
the permeability of the cell membrane.
2. The organism may be modified such that it does not produce the end
products which control the key enzymes of the pathway.
3. The organism may be modified such that it does not recognize the presence
of inhibiting or repressing levels of the normal control metabolites.