Industrial Biotechnology

Lesson 4
ISOLATION, SCREENING AND STRAIN
IMPROVEMENT

Isolation and Screening of

Industrial
Strain

Isolation of from the environment is by:

Collecting samples of free living
microorganism from anthropogenic or
natural habitats.
These isolates are then screened for
desirable traits.
Or by sampling from specific sites:
Mos with desired characteristics are
found among the natural microflora
After sampling of the organism the next
step is of enrichment.

Enrichment
Enrichment in batch or continuous
system on a defined growth media
and cultivation conditions are
performed to encourage the growth
of the organism with desired trait.
This will increase the quantity of the
desired organism prior to isolation
and screening.

Screening
Subsequent isolation as pure cultures on solid
growth media involves choosing or developing
the appropriate selective media and growth
conditions.
Next step to enrichment and isolation is
Screening.
The pure cultures must be screened for the
desired property; production of a specific
enzyme, inhibitory compound, etc.
Selected isolates must also be screened for
other important features, such as stability and,
where necessary, non-toxicity.

Screening
These isolation and screening
procedures are more easily, applied to
the search for a single microorganism.
The industrial microorganism should
ideally exhibit:
1. genetic stability
2. efficient production of the target
product, whose, route of biosynthesis,
should preferably be well characterized.

Screening
3. limited or no need for vitamins and
additional growth factors.
4. utilization of a wide range of low-cost
and readily available carbon sources
5. amenability to genetic manipulation;
6. safety, non-pathogenic and should not
produce toxic agents, unless there is the
target product;
7. ready harvesting from the fermentation; .
8. production of limited byproducts to ease
subsequent purification problems.

Culture Preservation
Streptomyces aureofaciens NRRL 2209
was the first microorganism deposited
in a culture collection in support of a
microbially based patent application.
Preservation of microbial cultures was
critical for all individuals and firms
engaged in the search for patentable
products from and patentable
processes by microorganisms.

Culture Preservation
Preservation of cultures by freezing,
drying, or a combination of the two
processes is highly influenced by
resistance of the culture to the damage
caused by rapid freezing, the dehydrating
effects of slow freezing, or damage caused
during recovery.
To minimize damage, agents have been
used that protect against ice formation by
causing the formation of glasses upon
cooling.

Culture Preservation
Methods to protect against the negative
effects of dehydration include adaptation
to lower effective water activity by preincubation in high osmotic pressure
solutions.
Damage caused by thawing after freezing
can be minimized by rapid melting and
by the composition of the medium used
for growth after preservation.

Culture Preservation
There are various preservation
methods .
To date, preservation in liquid
nitrogen is still the most successful
long-term method.

Serial Transfer
Based upon its ease of use, serial
transfer is often the first preservation
technique used by microbiologists.
The disadvantages of relying upon this
method for culture maintenance
include contamination, loss of genetic
and phenotypic characteristics, high
labor costs, and loss of productivity.

Preservation in Distilled
Water
This method (Castellani method, 50
years ago) was extensively tested on
594 fungal strains:
62% of the strains growing and
maintaining their original morphology.
In another study, 76% of yeasts,
filamentous fungi, and actinomycetes
survived storage in distilled water for
10 years.

Preservation in Distilled
Water
The pathogen Sporothrix schencki
concluded that even though longterm survival was good when this
procedure was used, there was a
noted loss in virulence.
Castellani technique should be
considered as one of the options for
practical storage of fungal isolates.

Preservation under Oil

One of the earlier preservation methods was
the use of mineral oil to prolong the utility of
stock cultures.
Mineral oil has been found to prevent
evaporation from the culture and
Decrease the metabolic rate of the culture
by limiting the supply of oxygen.
This method is more suitable than
lyophilization for the preservation of nonsporulating strains.

Lyophilization
One of the best methods for long-term
culture preservation of many microorganisms
is freeze-drying (lyophilization).
The commonly used cryoprotective agents
are skim milk (15% [wt/vol] for cultures
grown on agar slants and 20% for pelleted
broth cultures) or sucrose (12% [wt/vol] final
concentration).
It should be noted that some plasmid-containing bacteria are successfully
preserved by this method.

 Storage over Silica Gel

Neurospora has successfully been preserved
over silica gel.
Preservation on Paper
Drying the spores on some inert substrates
can preserve spore-forming fungi,
actinomycetes, and unicellular bacteria.
Fruiting bodies of the myxobacteria,
containing myxospores, may be preserved on
pieces of sterile filter paper and stored at
room temperature or at 6C for 5 to 15 years.
Preservation on Beads
The method involving preservation on beads
(glass, porcelain) , developed by Lederberg,
is successful for many bacteria.

Liquid Drying
To avoid the damage that freezing can cause, a
liquiddrying preservation process is applied.
It has effectively preserved organisms such as
anaerobes that are damaged by or fail to
survive freezing.
This procedure was preferred over lyophilization
for the maintenance of the biodegradation
capacity of six gram--negative bacteria capable
of degrading toluene.
Maliks liquid-drying method was also found to
be markedly superior to lyophilization for the
preservation of unicellular algae.

Cryopreservation
Microorganisms may be preserved at
- 5 to - 20C for 1, to 2 years by
freezing broth cultures or cell
suspensions in suitable vials.
Deep freezing of microorganisms
requires a cryoprotectant such as
glycerol or dimethyl sulfoxide (DMSO)
when stored at -70C or in the liquid
nitrogen at -156 to -196C.

Cryopreservation
Broth cultures taken in the mid-logarithmic to late logarithmic
growth phase are mixed with an
equal volume of 10 to 20% (vol/vol)
glycerol or 5 to 10% (vol/vol) DMSO.
Alternatively, a 10% glycerol-sterile
broth suspension of growth from agar
slants may be prepared.

Preservation in Liquid
Nitrogen
Storage in liquid nitrogen is clearly
the preferred method for
preservation of culture viability.

Protocol for Cryopreservation with

Cryoprotectants by a Two-stage
Freezing Process, and Revival of
Culture
After centrifugation the supernatant is
removed and the pellet, consisting of microbial
cells, is dissolved in an ice-cold solution
containing polyvinyl ethanol (10% [wt/vol]) and
glycerol (10% [wt/vol]) in a 1:1 ratio.
Due to the presence of polyvinyl ethanol, a
viscous thick cell suspension is obtained,
which is kept for about 30 minutes in an ice
bath for equilibration.

Protocol for Cryopreservation with

Cryoprotectants by a Two-stage
Freezing Process, and Revival of
Culture

During equilibration, an aliquot of 0.5 to 1.0 ml

of the cell suspension is dispensed into each
plastic cryovial or glass ampoule.
They are tightly closed, clamped onto labeled
aluminum canes, and placed at -30C for about
1 h or for a few minutes in the gas phase of
liquid nitrogen to achieve a freezing rate of
about 1C/min.
The canes are then placed into canisters, racks,
or drawers and frozen rapidly at -80C or in
liquid nitrogen.

Protocol for Cryopreservation with

Cryoprotectants by a Two-stage
Freezing Process, and Revival of
Culture

For revival of cultures, the frozen ampoules are

removed from the liquid nitrogen.
For thawing, they are immediately immersed to
the neck in a water bath at 37C for a few seconds.
The thawed cell contents of the ampoule or vial
are immediately transferred to membranes to form
a thick layer.
The resulting bacterial membranes with
immobilized cells are used as a biological
component of a biosensor for activity
measurements.

Inoculum Development
The primary purpose of inoculum development is to
provide microbial mass, of predictable phenotype, at a
specific time, and at a reasonable cost for the
productive stage of a microbial activity.
Until now, inoculum development has been more art
than science. There remains a need, especially at the
shake flask or spore-generating stages of the process,
for time and it looks good criteria to be replaced with
biochemical, physiological, or morphological markers
as both descriptors of an optimum inoculum and
indicators for optimum timing of inoculum transfer:
Inoculum Source

Inoculum development
When fungal spores are used as the
inoculum source, it is common for conidia
produced on an agar slant to be dispersed
in sterile distilled water containing 0.01 to
0.1 % Tween 80.
Spore formation of Streptomyces
coelicolor on agar was dependent upon
the type of agar used, the inclusion of
trace elements, the nitrogen source, and a
C/N ration between 40 and 100 (68).

Inoculum development
Nabais and de Fonseca have optimized a medium
for sporulation by Streptomyces clavuligerus.
Spore storage, however, could be a problem,
since the spores lost 72% of their viability after
storage for 1 week in buffer at 4C.
Many strains isolated from nature and often
strains that have been subjected to a mutation
program result in an unstable culture, whose
productivity can be rapidly lost.
For such strains, a single spore selection step or
its equivalent is a necessity for maintenance of
productivity.

Acclimatization
A number of commercial-level
microbiological processes use as the
inoculum, at least in part, culture growth
that has been part of a previous
production phase.
For fermentation processes involved in
the degradation of waste materials, a
very important variable is the extent of
acclimatization of the inoculum source.

Acclimatization
The process lag before initiation of
biodegradation decreases with increased
numbers of competent microorganisms.
High degradation rates are obtained
when acclimated sewage sludge operated
in a plant with low retention times is used
as the inoculum.

Acclimatization h
The use of an acclimatized inoculum
has been reported to result in
significant improvements in operational
efficiencies for xylose conversion to
xylitol by Candida guilliermondii grown
on a sugar cane hemi-cellulosic
hydrolysate.
In the brewery industry, the reuse or
pitching of yeast is a common practice.

 The effect of serial pitching of the

yeast inoculum on subsequent refermentation has not been well
characterized.
The condition of the yeast cell
surface as measured by flocculation
can be predictive before subsequent
fermentation performance.

Seed Media
For the design of media used for the production
of cell mass, the determination of an elemental
material balance is a useful exercise.
For defined media, the determination is a
straightforward calculation from the components.
For complex media, Traders Co. and other
manufacturers of complex nutrients provide the
basic data needed to estimate the contribution of
various components to the sum of an element.

pH
Nutritionally balanced seed media often
result in pH values not far from the optimum
for culture growth.
To prevent pH extremes in shake flasks,
phosphate salts and CaCO3 and/or buffers
such as 2-(N-morpholino) ethanesulfonic acid
(MES) or 3-(N-morpholino) propanesulfonic
acid (MOPS) are often used.
In fermenter inoculum development stages,
buffers are usually replaced with the more
economical online pH control.

Immobilization
The production of microbial inoculum for use
in bioremediation, agricultural applications,
and waste treatment is limited by the ability
of the microorganism to compete in these
environments and to be metabolically
effective.
One of the methods by which microbial
inocula are being improved for these
applications is the use of immobilization
technology.

Immobilization
The unique characteristics of immobilized inocula
include
(i) enhanced inoculum viability,
(ii) protection from stress during manufacture,
(iii) enhanced ecological competence,
(iv) increased metabolite production,
(v) UV resistance,
(vi) the opportunity to use immobilized cells as a
source of continuous inoculum,
(vii) the opportunity to introduce mixed culture
inocula into a process.

Immobilization
Storage of the immobilized inoculum
is enhanced if cells in beads are
incubated in nutrient or
supplemented with nutrient when
prepared.
A protocol for alginate
immobilization is required as
homework?

Contamination
Microbial contaminant detection
usually relies upon the use of
differential media and conditions to
encourage the growth of likely
contaminant in the presence of the
inoculated microbe.
It is difficult to detect of
contamination in mixed culture
fermentation.

Contamination
PCR has provided a rapid, effective
technique for the detection of a
contaminant present at low levels in a
sample.
PCR protocols can be applied to mixed
culture fermentations either for the
detection of a particular contaminant of
interest (Listeria monocytogenes)
or for the detection of an indicator
organism, such as the detection of E. coli
as an indicator of fecal contamination..

Phages
Phage contamination is a constant threat
to the productivity of any bacterial
fermentation process, particularly in
fermentations of dairy products.
How to overcome such a problem?
Selection of plasmids that confer phage
resistance ( e. g. for lactic streptococci).
Selection of phage-resistant strains
(preffered).

Phages
The report that alginate-immobilized
streptococci were protected from
attack by phages is potentially an
interesting alternative approach.

Mites
They can devastate a culture source or a
series of culture sources either by eating
the cultures and leaving no viable source or,
more commonly, by causing marked levels
of bacterial and fungal cross contamination.
Often the first indication of a problem is
agar plates with bacterial or fungal tracks
forming in a random-walk pattern across
the plate.

Mites
Treatment of incubators with
acaricides on a preventativemaintenance schedule is also worth
considering.

Strain Improvement
What is the Need?
With the exception of the food
industry, only a few commercial
fermentation processes use wild
strains isolated directly from nature.
Mutated and recombined mos are
used in production of antibiotics,
enzymes, amino acids, and other
substances.

Strain Improvement
What Should We Look for when We
Plan a Strain Improvement Program?
In general economic is the major
motivation.
Metabolite concentrations produced by
the wild types are too low for economical
processes.
For cost effective processes improved
strain should be attained.

Strain Improvement
Depending on the system, it may be
desirable to isolate strains:
Which shows rapid growth
Which shows Genetic stability
Which are non-toxic to humans
Which has large cell size, for easy
removal from the culture fluid.
,

Strain Improvement
Having ability to metabolize inexpensive
substrate.
Do not show catabolite repression
Permeability alterations to improve
product export rates.
which require shorter fermentation times,
which do not produce undesirable
pigments,
which have reduced oxygen needs,

Strain Improvement
with lower viscosity of the culture so
that oxygenation is less of a problem,
which exhibit decreased foaming
during fermentation,
with tolerance to high concentrations
of carbon or nitrogen sources,

Strain Improvement
The success of strain improvement
depends greatly on the target product:
Raising gene dose simply increase the
product, from products involving the
activity of one or a few genes, such as
enzymes.
This may be beneficial if the
fermentation product is cell biomass or
a primary metabolite.

Strain Improvement
However, with secondary metabolites, which
are frequently the end result of complex,
highly regulated biosynthetic processes, a
variety of changes in the genome may be
necessary to permit the selection of highyielding strains.
Mutants, which synthesize one component as
the main product, are preferable, since they
make possible a simplified process for product
recovery.

Methods of Strain
Improvement Up here
(mohamed)
The use of recombinant DNA techniques.

Protoplast fusion,
Site-directed mutagenesis,
Recombinant DNA methods have been
especially useful in the production of
primary metabolites such as amino acids,
but are also finding increasing use in strain
development programs for antibiotics.

1. Mutation
In a balanced strain development program
each method should complement the
other.
Spontaneous and Induced Mutations
Mutations occur in vivo spontaneously or
after induction with mutagenic agents.
Mutations can also be induced in vitro by
the use of genetic engineering techniques.

1. Mutation
The rate of spontaneous mutation
depends on the growth conditions of
the organism.
It is between 10-10 and 10-5 per
generation and per gene; usually the
mutation rate is between 10 -7 and 10-6.
All mutant types are found among
spontaneous mutations, but deletions
are relatively frequent.

Mutation. 1
The causes of spontaneous mutations, which
are thus far understood, include integration
and exclusion of transposons, along with
errors in the functioning of enzymes such as
DNA polymerases, recombinant enzymes,
and DNA repair enzymes.
Because of the low frequency of
spontaneous mutations, it is not costeffective to isolate such mutants for
industrial development.

1. Mutation
The mutation frequency (proportion of
mutants in the population) can be
significantly increased by using mutagenic
agents (mutagens):
It may increase to 10-5-10-3 for the isolation
of improved secondary metabolite producers
or even up to 10-2- 10-1 for the isolation of
auxotrophic mutants.
Spontaneous and induced mutants arise as a
result of structural changes in the genome:

1. Mutation
Genome mutation may cause changes in
the number of chromosomes.
Chromosome mutation may change the
order of the genes within the
chromosome, e.g. by deficiency,
deletion, inversion, duplication, or
translocation.
Gene or point mutations may result from
changes in the base sequence in a gene.

Reaction Mechanisms of
Mutagens
Mutagens cause mutation directly as a
result of pairing errors and indirectly as
a result of errors during the repair
process.
Mutagenesis through radiation: both
UV radiation and ionizing radiation are
used in mutagenesis studies.
Mechanisms of mutagenesis are quite
different for each type of radiation.

Reaction Mechanisms of
Mutagens
Short-wavelength ultraviolet: is one of
the more effective mutagenic agents.
The wavelengths effective for
mutagenesis are between 200-300 nm,
which is the absorption maximum of
DNA.
The most important products of UV
action are dimmers (thymine-thymine,
thymine-cytosine and cytosine-cytosine).

Reaction Mechanisms of
Mutagens
The dimers formed between adjacent
pyrimidines or between pyrimidines of
complementary strands, resulting in crosslinks.
UV radiation mainly induces transitions of
GC to AT;
Transversions (purine/pyrimidine replaces a
pyrimidine/purine), frame-shift mutations
and deletions are also found.

Reaction Mechanisms of
Mutagens
Long-wavelength UV radiation: at wavelengths of 300-400 nm has less lethal
and mutagenic effects than short
wavelength UV.
Exposure of cells or phages to long
wave-length UV is carried out in the
presence of various dyes, which interact
with DNA, greater depth rates and
increased mutation frequency result.

Reaction Mechanisms of
Mutagens
The psoralen derivatives (effective
activator for long wave length UV
mutation action)
8-Methoxypsoralen intercalates between
the base pairs of double-stranded DNA
and after the absorption of longwavelength UV, and adduct is formed
between the 8-methoxypsoralen and a
pyrimidine base.

Reaction Mechanisms of
Mutagens
Absorption of a second photon causes the
coupling of the pyrimidine-psoralen
monoadduct with an additional pyrimidine.
Biadduct formation between complementary
strands of nucleic acid results in crosslinks.
These lesions cannot be photo-reactivated,
although they are eliminated through
nucleotide excision repair in conjunction with
the mutation-causing SOS repair system.

Reaction Mechanisms of
Mutagens
Ionizing radiation: includes X-rays, gama-rays,
and beta-rays, which act by causing ionization
of the medium through which they pass.
They are usually used for mutagenesis only if
other mutagens cannot be used (e.g. for cell
material impenetrable to ultraviolet rays).
Single- and double-strand breaks occur with a
significantly higher probability than with all
other mutagens.

Reaction Mechanisms of
Mutagens
Ninety percent of the single-strand breaks
are repaired by nucleotide excision.
Double-strand breaks result in major
structural changes, such as translocation,
inversion or similar chromosome mutations.
Therefore, ultraviolet radiation or chemical
agents normally preferable for mutagenesis
in industrial strain development.

Phenotypic Expression of
Mutations
Many mutations which result in increases
formation of metabolites are recessive.
When a recessive mutation takes place a
uninuclear, haploid cell (e.g. bacteria and
actinomycete spores, asexual conidia of
fungi), a heteroduplex results from it; the
mutant phenotype can only be expressed
after a further growth step.

Phenotypic Expression of
Mutations
This also applies to exponentially growing
bacterial cells, which can contain 2-8
chromosomes; not until several steps of
reproduction has taken place do pure mutant
clones appear.
Delays in expression, which are not directly the
result of genetic effects, are observed, such as
mutations which cause changed ribosome or
mutations resulting in the loss of surface
receptors.

Optimizing Mutagenesis
The effect of a mutagen on a specific gene
or the effect of a mutation on a complex
process, such as the biosynthesis of a
secondary metabolite can never be
predicted.
The appearance of mutants depends on
several factors.
1) The base sequence of the mutated gene.
Mutations are not distributed evenly
around the genome;

Optimizing Mutagenesis
There are areas with high mutation
frequency, the so-called hot spots.
Different mutagens cause hot spots at
different sites in the genome.
2) The repair systems of the cell also play
a role. In strains with partially defective
repair mechanisms, organisms may be
killed without having induced mutations,
so that specific mutagens can be
ineffective.

Optimizing Mutagenesis
3) A gene activity, which has become lost
through mutation, can be restored at least
partially through a second mutation, a
suppressor mutation.
Suppressor mutations can occur in the same
gene that already carries the primary mutation
(intragenic suppressors).
The primary missense mutation is compensated
through the exchange of an amino acid or an
additional deletion or insertion, which corrects a
primary frame shift mutation so that the reading
frame remains intact.

Optimizing Mutagenesis
Suppressor mutations which occur in another
gene (extragenic suppressor) compensate the
primary mutation particularly at the level of
translation, by the formation of mutant
transfer RNAs or ribosome.
The treatment conditions have a critical effect
on mutagenesis.
Such factors as the pH, buffer composition,
mutagen concentration, exposure time,
temperature, and growth phase of the
organism may greatly affect the efficiency of
the process.

Optimizing Mutagenesis
By plotting dose-response curves all of these
factors may be optimized.
Mutagen effect may be have a lethal effect
where in strong exposed may cause more
than 99% death.
The survived mutations can only be reliably
determined by assessing qualitatively or
quantitatively changes in the product of the
target gene.

Selection of Mutants
Random screening: surviving clones is
inspected for ability to produce the product of
interest.
Inspection is done in model fermentations,
which are carefully adapted to the medium
and fermentation parameters of the largescale procedure, in order to maximize the
likelihood that the strains will be suitable for
industrial production.

The best strains from such a mutation cycle

are repeatedly mutated and selected.

Selection of Mutants
A gradual increasing in the yield is
attained by continuing with these steps.
Depending on the capacity of the
screening program, the 5-10 best strains
of a mutation-selection cycle should be
used as parent strains for future
mutagenesis.
These strains are normally treated with
mutagens different from those used in
the initial isolation.

Selection of Mutants
Factors which influence the size of the
screening program are:
frequency of mutation,
extent of yield increases,
the amount of time required for a
mutation-selection cycle,
the available test capacity of the screening
program,
and the accuracy of the screening test (e.g.
antibiotic assay).

Selection of Mutants
Mutants with high yields are much rarer than
those with only slight improvements.
The variability of mutagen treated
populations is quite high even when
mutagenesis is performed under identical
conditions.
Thus it is usually more economical to screen a
small number of survivors (about 20-50) after
many different mutagen treatments.

Selection of Mutants
The number of strains, which must be
screened to obtain mutants with a yield
increase, depends on
The strain,
The conditions of mutagenesis
the biosynthesis pathway
the regulation of the product, which is
being optimized.
Normally, several hundred to several thousand
isolates per mutation cycle must be tested.

Selection of Mutants
The screening capacity determines the speed of
the progress to be expected.
In the first stage of mutant screening, only one
fermentation sample per isolation is usually
assayed, provided that the test error is smaller
than the yield increase expected.
The best isolates of the first series (usually 1030%) are then tested in a second fermentation.
Since the best strains of this second screening
are then used in a still further mutation cycle,
the yield increase must be statistically
significant when compared to the parent strain.

Selection of Mutants
Several industrial companies are developing
ways to automate mutant screening procedures
to increase the screening capacity.
Isolation of Mutants: several examples of the
many selective methods used in strain
development are mentioned here.
Isolation of resistant mutants:
A high cell density of a mutagenised population
can be plated on a selective medium containing
a concentration of a toxic substance that
prevents the wild type from growing.

Selection of Mutants

Only the resistant clones can develop.

mutants may be isolated which are resistant to
antibiotics or anti-metabolites.
Mutants isolated may also have increased cell
permeability or a protein synthesis with a high
turnover, making hem useful for industrial
purposes.
Anti-metabolite resistance can be used to select
mutants, which exhibit defective regulation.
Altered regulation may occur in such mutants.

Selection of Mutants
Anti-metabolites, because of their structural
similarity to metabolites, may cause feedback
inhibition, but are unable to substitute for normal
metabolites.
Anti-metabolites cause death of normal cells, but
analog-resistant mutants can form an excess of
metabolites, in some cases through changed
regulatory mechanisms (elimination of allosteric
inhibition; constitutive product formation).
Isolation of auxotrophs: (Auxotrophy is the
inability of an organism to synthesize a particular
organic compound required for its growth).

Selection of Mutants
The isolation of auxotrophs is done by plating of
the mutagenized population on a complete agar
medium, on which the biochemically deficient
mutants can also grow.
By means of Lederbergs replica plating technique,
the clones are transferred to minimal medium
where the auxotrophic colonies cannot grow.
These mutants are picked up from the master
plates and their defect is characterized.
Enrichment technique named filtration enrichment
method is used to isolate and enrich the
mutagenized population.

Selection of Mutants
The spores of filamentous organism
(actinomycetes, fungi) are allowed to develop in
a liquid minimal medium.
The developing micro colonies of prototrophs
are then separated by filtration, leaving behind
in the filtrate spores of auxotrophs, which have
been unable to grow.
The filtrate is then plated and the resulting
colonies are checked for auxotrophic
characteristics.

Selection of Mutants
Penicillin selection method for isolation of auxotrophs:
Penicillin kills growing cells but not non-growing cells.
In this procedure, growing cells are selectively killed
by antibiotic treatment, thus enriching for auxotrophs,
which cannot grow on minimal medium.
Several inhibitors other than penicillin can also be
used
in this procedure: dihydrostreptomycin for
Pseudomonas aeruginosa, nalidixic acid for
Salmonella typhimurium, colistin for the penicillinresistant Hydrogenomonas strain H16, and nystatin
for Hansenula polymorpha, P.chrysogenum, A.
nebulas, and S. cerevisiae.

Selection of Mutants
Other procedures :
The presence or absence of specific enzyme activities
can be observed directly in colonies growing on plates
by spraying with suitable reagents or by incorporating
indicator dyes into culture medium.
Detection of amtibiotically active substances may be
detected by using agar plug method with antibiotic
sensitive organisms producing an inhibition zone.
Such a method has some disadvantage where there
is only a slight correlation between antibiotic
formation in plate culture and the antibiotic
production in submerged fermentation.

Selection of Mutants
Strains, which produce at high yields when
grown on plates, may produce at only low yields
or not at all in liquid culture.
The procedure is sufficient suitable for
differentiation between productivity and nonproductivity, such as for detecting the
formation of constitutive enzymes.

Agar Plug Method

Recombination
The genetic information from two genotypes can be
brought together into a new genotype through
genetic recombination.
The disadvantages of genetic recombination are:
In most cases, the productivity of the recombinants
usually is intermediate between the values of the
parent strains?.
During strain development process, there is a
frequent decline in the increase in yield is
observed. This phenomena is overcome by allowing
genetic-cross between unfavorable mutant alleles
and alleles of one of the parents. Such a procedure
is not available during recombination work.

Recombination
High-yielding strains can actually increase the
cost of the fermentation because of changed
physiological properties (greater foaming,
changed requirements for culture medium, etc.).
By crossing back to wild-type strains, highyielding strains with improved fermentation
properties may be formed.
An effective strain development approach should
involve the use of sister-strain, divergent strain,
and ancestral crosses at specific intervals,
besides use of carefully mutagenesis to ensure
the maintenance of genetic variability.

Regulation
Regulation of metabolism is generally so
efficient that excess products are not formed.
Strain development and the optimization of
fermentation conditions lead to a relaxation of
regulation in the producing strains.
Strains with less efficient regulation can be
selected in a screening process.
A broad understanding of biosynthesis, the
enzymes involved in these processes, and their
regulation is necessary for developing a rational
approach to the alteration of the regulation of a
fermentation process.

Regulation
Microbial metabolism is controlled by the regulation
of both enzyme activity and enzyme synthesis.
Regulation of enzyme activity:
Feedback inhibition: In an unbranched biosynthetic
pathway, the end product inhibits the activity of the
first enzyme of the pathway, a process called
feedback inhibition.
A conformation change and hence inactivation
(allosteric effect) occurs when an effector (end
product) is attached to a specific site of the enzyme
(allosteric site).
The end product thus inhibits the activity of the
enzyme non-competitively.

Regulation
In a branched biosynthetic pathway, feedback
inhibition of the first common enzyme by
means of one of the end products would cause
more than one end product to be affected.
In branched biosynthetic pathways, different
kinds of feedback inhibition are found:
The end product inhibits the first enzyme in
each case after the branch point.
The first step in the common synthesis path is
catalyzed by several isoenzymes, each of which
can be regulated independently.

Regulation
The first common enzyme in a branched
biosynthetic pathway is influenced by each end
product only slightly or not at all; there must be an
excess of all end products for inhibition to occur (a
phenomenon called multivalent inhibition).
Each end product of a branched pathway acts as
an inhibitor; cumulative inhibition is the effect of
all the inhibitors.
Breakdown of enzymes: Enzymes, which are no
longer needed in metabolism, may be broken
down through the action of highly specific
proteases. As e.g., tryptophan synthetase in S.
cerevisiae is broked down at stationary phase.

Regulation

Modification of enzymes: The activity of some

enzymes (such as glutamine synthetase in E. coli) is
controlled by conformational changes, such as
phosphorylation or adenylylation.
Regulation of enzyme synthesis: at least three
mechanisms have been detected which regulate
synthesis of enzymes.
Induction: Some enzymes are formed irrespective of
the culture medium; such enzymes are called
constitutive.
Many catabolic enzymes are induced: they are not
formed until the substrate to be metabolized is
present in the medium.
The product of one enzyme can in turn induce the
synthesis of another enzyme (sequential induction).

Regulation
Repression: Anabolic enzymes are generally present
only when the end product is absent. The excess
end product suppresses enzyme synthesis, acting
as a co-repressor.
Attenuation: It is involved in the biosynthesis of
amino acids in bacteria, e.g. histidine - in
Salmonella typhimurium, tryptophan - in E. coli (In
addition to repressor operator mechanism).
In attenuation model, the transcription rate of an
operon is regulated by a secondary structure of the
leader sequence of the newly transcribed mRNA.
The structure of this leader sequence determines
whether the RNA polymerase continues the
transcription of the operon or a termination occurs.

Regulation
If termination occurred the mRNA transcription
ceases and the enzyme or enzymes coded for
by that mRNA are not made.
In the tryptophan situation, repression has a
large effect on enzyme synthesis whereas
attenuation has a more subtle, although still
important, effect.

Regulation
Excess production of primary metabolites (amino
acids, vitamins, purine nucleotides) : This has been
accomplished primarily by eliminating feedback
inhibition.
A) The elimination of end product inhibition or
repression is achieved by using auxotrophic mutants
that can no longer produce the desired end product
due to a block in one of the steps in the pathway.
By adding the required end product in low amounts,
growth occurs but feedback inhibition is avoided.
Excretion of the desired intermediate product thus
occurs.
Both branched and unbranched pathways can be
manipulated in this way.

Regulation
B) A second method is the selection of mutants
that are resistant to metabolites.
In this case either the enzyme structure is
changed so that the corresponding enzyme lacks
the allosteric control site, or mutations in the
operator or regulator gene (Oc-, R--mutants)
result in constitutive enzyme production and
thus over production.
C) In mutants with a block in an allosterically
regulatable enzyme, suppressor mutations can
lead to restoration of enzyme activity; however,
these enzymes are not allosterically controllable.

Regulation
Regulation and overproduction of secondary
metabolites:
The methods described above, which were used
first for primary metabolites, can be successfully
applied to secondary metabolites as well.
Production of secondary metabolites is controlled
by 5 different classes of genes:
1. Structural genes, which code for enzymes
involved in secondary metabolite biosynthesis.
2. Regulatory genes, which control secondary
metabolite synthesis.
3. Resistance genes, which keep antibioticproducing strains immune to their own products

Regulation
4. Permeability genes, which control the uptake
and excretion of substances.
5. Regulatory genes, which control primary
metabolism and thus indirectly affect the
biosynthesis of secondary metabolites.
Many genes are involved in the synthesis of
secondary metabolites. 300 genes are involved
in chlortetracycline biosynthesis and
approximately 2000 genes are directly or
indirectly involved in neomycin biosynthesis.
In such type of systems, a rational approach to
increased yield is possible only in rare cases
because there is insufficient data

Regulation
Regulatory mechanisms that affect the products of
secondary metabolism:
Induction: In batch fermentations with readily
metabolizable carbon and nitrogen sources,
secondary metabolites are formed primarily after
growth has ceased.
The logarithmic growth phase is called the
trophophase, and the subsequent phase, in which
the secondary metabolite may be produced, is
called the idiophase.
Secondary metabolites are referred to as idiolites.
The synthesis of enzymes involved in secondary
metabolism is repressed during the trophophase.

Regulation
The composition of the culture medium could be
arranged so that a significant fraction of a slowly
metabolizable substrate is used, the organism thus
growing under sub optimal conditions, leading to a
situation where growth and secondary metabolite
formation occur in parallel.
End-product regulation: antibiotics inhibit their
own biosynthesis (e.g. penicillin, chloramphenicol,
virginiamycin, ristomycin, cycloheximide,
puromycin, fungicidine, candihexin, streptomycin).
The mechanism of feedback regulation has only
been explained in a few cases:

Regulation
chloramphenicol represses arylamine
synthetase, which is the first enzyme in the
biosynthetic pathway, which branches off from
aromatic biosynthesis to chloramphenicol.
With chloramphenicol and penicillin, it has been
shown that the concentration of the end
product, which inhibits corresponds to the
production level.
Thus, if strains could be isolated which were
less sensitive to end-product inhibition by these
antibiotics, they might produce higher yields.

Regulation
Catabolite regulation: Catabolite regulation is a
general regulatory mechanism in which a key
enzyme involved in a catabolic pathway is
repressed; inhibited, or inactivated when a
commonly used substrate is added.
Substrates, which have been found to bring about
catabolite repression, include both carbon and
nitrogen sources.
Carbon sources: Biosynthesis of different secondary
metabolites (antibiotics, gibberellins, ergot
alkaloids) is inhibited by rapidly fermentable carbon
sources, particularly glucose. The mechanism
differes according to the organism and metabolite.

Regulation
A well-known carbon catabolite repression found
in many bacteria, yeasts and molds, which involve
a catabolite activator protein (CAP) that must
combine at the promoter site before RNA
polymerase can attach.
The CAP will only bind if it is first complexed with
cyclic adenosine monophosphate, cyclic AMP.
Readily utilizable carbon sources such as glucose
stimulate an enzyme, which causes the
breakdown cyclic AMP, thus rendering CAP
inactive.
Thus, glucose inhibits the synthesis of the mRNA
for any enzyme requiring CAP for its biosynthesis.

Regulation
Nitrogen sources: In several antibiotic
fermentations it has been observed that
ammonia or other rapidly utilizable nitrogen
sources act as inhibitors.
The fundamentals of this regulation have not
yet been completely understood, although
glutamine synthetase and glutamic
dehydrogenase are considered key enzymes.
In enteric bacteria it has been established that
glutamine synthetase has a regulatory function
in the synthesis of additional enzymes, which
are involved in nitrogen assimilation.

Regulation
Phosphate regulation: In a culture medium
inorganic phosphate (Pi) is required within a range
of 0.3-300 mM for the growth of prokaryotes and
eucaryotes.
A much lower phosphate concentration inhibits the
production of many secondary metabolites.
In a number of systems studies, the highest Pi
concentration, which allows unimpeded production
of secondary metabolites, is about 1 mM; complete
inhibition of production occurs at about 10 mM Pi.
Phosphate regulation has been observed in the
production of alkaloids, gibberellins and particularly
in several antibiotics.

Regulation
The phosphate regulation mechanism is not yet
fully understood. Pi controls the metabolic
pathways, which precede the first stage of
secondary metabolite formation, but also
affects the biosynthesis of secondary
metabolites themselves.
It has been shown that phosphate restricts the
induction of secondary metabolite production.
For instance, dimethyl allultryptophan
synthetase, the first specific enzyme of ergot
alkaloid biosynthesis, is not produced in the
presence of high Pi concentrations.

Regulation
Auto regulation: In some actinomycetes it has been
possible to show that differentiation and secondary
metabolism are subjected to a type of selfregulation from low-molecular weight substances.
For instance, in Streptomyces griseus and S.
bikiniensis the formation of streptomycin, the
development of streptomycin resistance, and sporeformation are all affected by factor A, a substance
produced by the streptomyces themselves.
It has been shown that the streptomycin resistance
property is due to the increased transcription of the
gene for the enzyme, streptomycin
phosphotransferase, induced by the factor A.

Regulation
The effect on streptomycin formation is thought to
be due to a shift in the metabolism of the
carbohydrate source: although the activity of the
enzyme glucose-6-phosphate dehydrogenase is
high in factor A-deficient mutants, this enzyme
cannot be demonstrated in high-yielding strains.
Addition of factor A to mutants leads to a strong
decrease in enzyme activity.
It is assumed that when the pentose phosphate
cycle is blocked through the absence of glucose-6phosphate dehydrogenase, glucose is channeled
into pathways involved in the formation of
streptomycin units.

Regulation
In a sense, factor A can be considered analogous
to a hormone.
Auto-regulatory mechanisms similar to that of
factor A have been found in other actinomycetes.
For instance, a factor is hypothesized in S.
virginiae, which stimulates the formation of the
antibiotic virginiamycin.
In rifamycin-producing Nocardia mediterranei
butyryl phospho-adenosine has been
characterized as a regulatory factor.
Two g-lactones (L factors) have been shown to be
auto-regulatory agents in leukaemomycin
producing S. griseus.

Gene Technology

Gene technology includes in vitro recombination,

gene cloning, gene manipulation, and genetic
engineering.
Gene technology permits introduction of specific
DNA sequences into prokaryotic or eucaryotic
organisms and the replication of these sequences;
that is, to clone them.
To carry out these procedures, the following steps
are necessary:
The DNA sequence to be cloned must be available.
The sequence must be incorporated into a vector.
The vector with the DNA insert must be introduced
by transformation into a host cell, where the vector
must replicate the insert in a stable manner.
The clone, which contains the foreign DNA, must be
selectable in some manner.

Isolation of DNA Sequences

for Cloning

Genome fragments: Restriction endonucleases

are used to cut DNA.
Endonucleses belong to specific restriction and
modification systems and are used by the cell
to protect itself from foreign DNA.
They split double-stranded DNA at specific
sites, 4-11 nucleotides in length.
More than 600 of those enzymes are known in
bacteria.
If the sequence of the DNA to be cloned is
unknown, it is possible to use a so-called shotgun approach.

Isolation of DNA Sequences

for Cloning

With this procedure, a gene bank is produced by

using suitable restriction enzymes to fragment the
total genome of the organism into pieces of about
20 kilo bases in length.
The DNA fragments is linked to a vector (generally
a phage or cosmid) and cloned into a suitable host.
By applying screening methods the cloned
organism could be then isolated.
It is preferable to carry out the initial cloning with
enriched DNA fragments.
Enrichment is done by use of sucrose gradient
centrifugation, agarose-gel electrophoresis, column
chromatography, or by use of specific gene probes.

Isolation of DNA sequences

Synthetic DNA: In order to produce a specific DNA
fragment containing the coding region of a protein,
the DNA sequence is deduced by reverse
translation from the amino acid sequence of this
protein.
Automated DNA synthetic machine can be used for
production of DNA fragments of 20-100 bases,
which can be connected together to make longer
sequences.
Example of the use of this technique are the
artificial synthesis of the gene for somatostatin, a
peptide hormone with 14 amino acid residues and
the synthesis of the A and B chains of insulin,
which were cloned and expressed in E. coli.

Isolation of DNA sequences

It is also possible to produce sequences in
which one or more bases have been changed,
making possible the production of highly
specific mutations.
Production of complementary DNA (cDNA):
Specific mRNA molecules, are used as
templates in vitro with the enzyme reverse
transcriptase, to produce complementary DNA.
Analysis of recombinant clones:
To select transformed cells, the marker
inactivation technique can be used.

Isolation of DNA fragments

Vectors are used containing two selectable
markers (for instance, antibiotic resistance) one of
which contains the recognition site for restriction
enzyme used in the cloning process.
If the foreign DNA becomes integrated into this
antibiotic resistance gene, the activity of that gene
is lost (insertional inactivation).
Host cells that lack the vector are sensitive to both
antibiotics, host cells containing a vector lacking
the foreign DNA are resistant to both antibiotics,
whereas vectors with inserted foreign DNA are
sensitive to the one antibiotic into whose
resistance gene the foreign DNA has been
inserted.

Isolation of DNA fragments

Colony hybridization (Colony Hybridization is the
screening of a library with a labeled probe
(radioactive, bioluminescent, etc.) to identify a
specific sequence of DNA, RNA, enzyme, protein, or
antibody).
and Southern blotting (DNA blot) are used for
detection of cloned DNA in the cell.
A different procedure for detecting the cloned DNA
involves seeking for expression of the cloned DNA.
Since the expression efficiency is often quite low, a
sensitive method is applied, e. g. using
immunological methods, in which an antibody
(marked by radioactivity or enzyme) is used as a
probe.

Production of Recombinant DNA

Use of genetic methods

High-yielding strains can be produced by:
Isolation of mutants resistant to inhibitors of
protein synthesis, which often overproduced
proteins;
Manipulation of regulatory signals to increase
transcription or translation by cloning the gene
on an expression vector or inserting the gene
into a transposon which has a strong promoter;
Modification of the gene by use of sitedirected mutagenesis.

Use of genetic methods

The yield may be increased, by increasing the
gene dosage (gene amplification), which can be
done by:
Increasing the number of DNA replication sites in
growing bacterial cells causes amplification of the
genes situated near the origin of replication.
Diploidization of fungi increases gene dosage,
although the strains are usually unstable.
Isolation of hyper induced strains, which have
been cultivated under selective conditions over a
long period. These strains are extremely unstable,
however, and are usually not suitable for
commercial processes.

Use of genetic methods

The greatest success is likely by use of genetic
engineering methods, for example, cloning and
amplification of the gene by means of a multicopy
plasmid or a phage vector.
For instance, by use of a cosmid system the formation of
the enzyme penicillin acylase in E. coli has been
markedly increased when compared to the wild type. A
whole series of industrial enzymes have been optimized
in this way.
Difficulties faced the goal to increase in yield of a multigene product such as a primary or secondary metabolite,
although some successes have been achieved.
Amino acid production has been increased by cloning the
whole genome, first in E. coli, later in production strains
such as Corynebacterium, Brevibacterium, or Serratia..

Use of genetic methods

For secondary metabolites such as antibiotics,
cloning and amplification of the rate-limiting
enzyme of the biosynthetic pathway can be
done.
As a first step in this direction, the genes for a
number of antibiotics have been isolated,
cloned, and in few cases expressed.
These include actinorhodin, methylenomycin,
and undecylprodigiosin (Streptomyces
coelicolor), cephalosporin (Cephalosporium
acremonium), erythromycin (S. erythreus),
oxytetracylcine (S. glaucescens) and tylosin (S.
fradiae).

Stability of the Strain

An important consideration in strain improvement
is the stability of the strain.
An important aspect of this is the means of
preservation and storage of stock cultures so that
their carefully selected attributes are not lost.
This may involve storage in liquid nitrogen or
lyophilization.
Strains transformed by plasmids must be
maintained under continual selection to ensure
that plasmid stability is retained.
Instability may result from deletion and
rearrangements of recombinant plasmids, which is
referred to as structural instability, or complete
loss of a plasmid, termed segregational stability.

Stability of the Strain

Some of these problems can be overcome by careful
construction of the plasmid and the placement of essential
genes within it.
Segregational instability can also be overcome by
constructing so-called suicidal strains that require specific
markers on the plasmid for survival.
Consequently, plasmid-free cells die and do not
accumulate in the culture.
These strains are constructed with a lethal marker in the
chromosome and a repressor of this marker is located on
the plasmid.
Cells express the repressor as long as they possess the
plasmid, but if it is lost the cells express the lethal gene.
However, integration of a gene into the chromosome is
normally the best solution, as it overcomes many of these
instability problems.