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Tuesday, August 14, 2012

Strain Improvement - Methods and Applications


STRAIN IMPROVEMENT
Several options are open to an industrial microbiology organization seeking to maximize its profits in the face of its competitors‘ race for the same market. The organization may undertake more aggressive marketing tactics, including more attractive packaging while leaving its technical procedures unchanged. It may use its human resources more efficiently and hence reduce costs, or it may adopt a more efficient extraction system for obtaining the material from the fermentation broth.

The operations in the fermentor may also be improved by its use of a more productive medium, better environmental conditions, better engineering control of the fermentor processes, or it may genetically improve the productivity of the microbial strain it is using. Of all the above options, strain improvement appears to be the one single factor with the greatest potential for contributing to  greater profitability. While realizing the
importance of strain improvement, it must be borne in mind that an improved strain could bring with it previously non-existent problems.

For example, amore highly yielding strain may require greater aeration or need more intensive foam control; the products may pose new extraction challenges, or may even require an entirely new fermentation medium. The use of a more productive strain must therefore be weighed against possible increased costs resulting from higher investments in extraction, richer media, more expensive fermentor operations and other hitherto non existent problems. This possibility not withstanding, strain improvement is usually part of the program of an industrial microbiology organization. To appreciate the basis of strain improvement it is important to remember that the ability of any organism to make any particular product is predicated on its capability for the secretion of a particular set of enzymes. The production of the enzymes, themselves  depends ultimately on the genetic make-up of the organisms. Improvement of strains can therefore be put down in simple term as follows:

  1. Regulating the activity of the enzymes secreted by the organisms.
  2. In the case of metabolites secreted extra-cellularly, increasing the permeability of the organism so that the microbial products can find these way more easily outside the cell.
  3. Selecting suitable producing strains from a natural population.
  4. Manipulation of the existing genetic apparatus in a producing organism.
  5. Introducing new genetic properties into the organism by recombinant DNA technology or genetic engineering. 
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
  1. Spontaneous mutations
  2. 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
  1. Frameshift mutations
  2. 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.
  1. Deletion mutations
  2. 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 :
  1. Ionizing radiation
  2. 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.

References
Food Biotechnology Course material,by K V Anand Raj