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 mo’s are used in production of antibiotics, enzymes, amino acids, and other substances.
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.
- 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.
- 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,
- 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
- 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.
- 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 high-yielding strains.
Mutants, which synthesize one component as the main product, are preferable, since they make possible a simplified process for product recovery.
- 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.
- 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.
- 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.
- 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 cost-effective to isolate such mutants for industrial development.
- 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:
- 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.
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).
- The dimers formed between adjacent pyrimidines or between pyrimidines of complementary strands, resulting in cross-links.
- UV radiation mainly induces transitions of GC to AT;
- Transversions (purine/pyrimidine replaces a pyrimidine/purine), frame-shift mutations and deletions are also found.
- Long-wavelength UV radiation: at wave-lengths 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.
- 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 long-wavelength UV, and adduct is formed between the 8-methoxypsoralen and a pyrimidine base.
- 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.
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.
- 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.