Improvement of Strains

The wild strains of microorganisms produce low quantities of commercially important

metabolites, although the yield can be increased by optimizing the fermentation conditions.
The potentiality of the metabolite formation is genetically determined. Therefore, genetic
improvements have to be made and new strains developed for any substantial increase in
product formation in a cost-effective manner.
There are strain development programmes (mutation and recombination) to increase the
product yield by 100 times or even more.
The improved strains should possess the following characteristics:
 Shorter time of fermentation.
 Capable of metabolizing low-cost substrates.
 Reduced O2 demand.
 Decreased foam formation.
 Non-production of undesirable compounds.
 Tolerance to high concentrations of carbon or nitrogen sources.
 Resistant to infections of bacteriophages.

MUTATIONS
Genes are chemically the segments of DNA molecules except in some viruses, as some viruses
are found to contain RNA as genetic material. They are normally transmitted with great
exactness. But sometimes variations may be caused by physicalor chemical agents resulting in
altered phenotype. The heritable changes in the genome of a cell are called mutations. Those
mutations which occur in the somatic cells are called somatic mutations. These are not
transmitted to next generation.
Mutations occurring in the germ cells are called germinal mutations. These mutations influence
the gametes and are passed to next generation, generating new variability and contributing to
the process of evolution.
Mutations have both advantages as well as disadvantages. Increasing the microbial mutation
rates bring out the genetic changes which have been put to many important uses in the
laboratory and industries. For example mutations in some plants like tulips producing colourful
flowers. Mostly mutations effect the normal existence of cells. For example in bacteria,
auxotrophs are developed from wild type because of mutations. In humans mutations leads to
physiological abnormalities like sickle cell anemia.
TYPES OF MUTATIONS
Mutations are classified in different ways on the basis of one or the other criterion. They may
be depending on their origin, depending on the type of change in base composition, on the
basis of type of the cell, on the basis of the nature of their effect,etc. Among all these
classification criteria the significant one‘s are
(A) Depending on their origin.
(B) Depending on the type of change in base composition.
(A) Depending on their origin : Mutations are of two types
 Spontaneous mutations
 Induced mutations.
Spontatneous mutations: Mutations that occur naturally are called spontaneous mutations.
Their origin is indeterminate and unknown.They are generally assumed to be random changes
in the nucleotide sequences of genes. Spontatneous mutations are linked to normal chemical
processes in the organism that alter the structure or the sequences of genes. For example all
the four common bases of DNA have unusual tautomeric forms. Which are,however,
rare.Tautomers are the mutually interconvertable structural isomeric forms. Normally
nitrogenous bases in DNA present in the keto form. As a result of tautomeric rearrangement
they can be transformed into the enol form.
The tautomeric rearrangement changes the hydrogen bonding characteristics of bases.
Normally AU and GT base pairs. The tautomeric changes during replication substitutes
nitrogenous bases with others. If a purine for purine and pyrimidine for pyrimidine are
substituted the type of mutation is called transition mutation. If a purine for pyrimidine and
pyrimidine for purine substituted the mutation is called transversion. The transtition and
transversion mutations are also termed point mutations. Spontaneous mutations also occurs by
frame shifts of DNA.
Once an error is present in the genetic code, it may be reflected in the amino acid composition
of the specified protein. If the changed amino acid is present in a part of the molecule,
determining the structure or biochemical activity, functional alteration can occur. Many
spontaneous mutations are reported. For example albinism and hares lip in man; a tobacco
mutant producing seventy leaves all of a sudden in a normal progeny
producing an average of twenty leaves.
Induced mutations: The mutations resulting from the influence of any artificial factor are
considered to be induced mutations. Muller subjected drosophila to powerful x-rays and
obtained a number of mutations. The chemicals or any other means that induce mu8tations
are called as mutagens or mutagenic agents.The mutagens acts in different ways like
incorporation of base analogs, specific mispairing and intercalation.
Base analogs are structurally similar to nitrogenous bases of DNA, and can be incorporated into
the growing polynucleotide chain during replication. Specific mispairing is caused when a
mutagen changes a bases structure, by that alters its base pairing characteristics.
The different types of mutations changing the nucleotide number or order of DNA are
 Frameshift mutations
 Chromosomal mutations
Frameshift mutations: As pointed out in the third unit the genetic information in DNA is
expressed first into mRNA by the transcription. mRNA is translated to proteins on reading
triplet code from a fixed starting codon. If a single nucleotide is deleted or inserted in the
normal sequence then the reading frame changes.
The mutations leading to the change in reading frame are called frame shift
mutation. These are two types.
 Deletion mutations
 Insertion mutations
Deletion mutations: The reading frame of mRNA does not have any punctuation. So if
nucleotides deleted it changes the amino acid sequence of protein expressed by it.
Normal sequence :
DNA AAA GCT ACC TAT CGG TTA
mRNA UUU CGA UGG AUA GCC AAU
Protein Phe Arg Trp IIe Ala Asn
Addition mutation :
DNA AAA GCT ACC ATA TCG GTT
mRNA UUU CGA TGG TAT AGC CAA
Protein Phe Arg Trp Tyr Ser Gin
Deletion mutation :
DNA AAA GCT CCT ATC GGT
mRNA UUU CGA GGA UAG
Protein Phe Arg Gly Stop
Deletion mutations are of variable length ranging in deletion of the number of nucleotides.
Deletion of three successive nucleotides will not effect all the protein composition. It is with
one amino acid less only, as the codon is triplet code.
Dyes like acridines can bring about deletion mutations. In heterozygous diploid eukaryotes, a
deletion involving the dominant alleles amy result in the expression of the recessive phenotype.
Insertion mutations: Inserting nucleotides into a normal gene results in a mRNA,
in which the reading frame is altered. This type of mutations causing insertion
of nucleotides are called insertion mutations.
Chromosomal mutations: The mutations effecting the number, size, shape and gene
complements are chromosomal mutations. These are of different types like a chromosomal
segment may be lost by deletion, or it may undergo inversion or it may be translocated to a
different site or may be duplicated to tandem repeats.
MUTAGENS :
Mutations inducing agents are called mutagens. They create mutations in different ways.
Depending on the nature of mutagens they are of two types
Physical mutagens
Chemical mutagens
Physical mutagens: Mutations can be naturally or artificially induced by a variety of physical
mutagens. H.J.Muller, founder of genetics, demonstrated in 1927 that mutations can be
artificially induced by treating flies with x-rays. Similarly L.J.Stadler in 1928 demonstrated and
increase in the rate of mutations due to x-rays in barely and maize. Besides x-rays gamma rays
can also induce mutations.
The physical agents are broadly divided into two types :
 Ionizing radiation
 Non-Ionionizing radiation
Ionizing radiation: X-rays and gamma (Y) rays are ionizing radiations. They have short
wavelength and high penetration power. They can penetrate into deeper tissues causing
ionization of the molecules along their way. When X-rays penetrate into cells, electrons are
ejected from the atoms of molecules encountered by the radiation. As a result the stable
molecules and atoms change into free radicals and reactive irons. The radicals and ions can
initiate a variety of chemical reactions, which can affect the genetic material, resulting in point
mutations. i.e., affecting only one base pair in a given location. The rate of mutation increases
with the increasing dose of X-rays administered.
Nonionizing radiation: Ultra Violet (UV) rays are nonionizing radiations. They have long
wavelength and low penetration power. The purines and pyrimidines absorb UV radiation most
intensely at about 260 nm.This property has been useful in the detection and analysis of nucleic
acids.In 1934 it was discovered that UV radiation is mutagenic. The major effect of UV radiation
is formation of pyrimidine dimmers, particularly between two thymines. Cytosine-cytosine and
cytosine-thymine dimmers are less prevalent.
The dimers damage the DNA structure and effects normal replication.
Chemical mutagens: Charlotte Auerbach, author of Science of Genetics‖ was the first to find
that mutatins can also be induced due to certain chemicals. Chemical mutagens can remove,
replace or modify DNA bases.
Alkylating agents: Alkylatin of nitrogenous bases by the alkylating agent either removes the
base or modifies it. Guanine residues can be alkylated by the methyl methane sulfonate and
ethyl methane sulfonate. These agents alkylates guanine at N7 and weakens the purine-
deoxyribose linkage. This leads to deppurination creating ga at that site. N-methyl-N1-nitro –
N –nitrosoguanidine CH3-N(NOC(NH)-NH-NO2 is a powerful mutagen in E.coli. Some
alkylating agents change the GC positin ina nucleotide to AT.
Intercalating agents: Intercalating agents produces frame shift mutation in bacteriophages like
T4. For example acridines are mutagenic to bacteriophphages but not to bacteria. As the
acridines are unable to enter bacterial cell.
Base analogs: Base analogs are structurally similar to normal nitrogenous bases and can be
incorporated into the growing polynucleotide chain during replication. These analogs will have
base pairing properties direrent from the bases they replace. One of the first base analog
formed to induce mutations in phage T2 is 5 bromouracil (BU) an analog of thymine. In the
normal keto form BU base pairs with adenine.But its tautomeric enol form pairs with guanine
like cytosine.