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Spontaneous mutations
Mutation rate depends on growth conditions: 10-10 to 10-5 per generation and per gene; usually 10-7 to 10-6. All mutant types are found, although deletions are relatively frequent. Causes include integration and excision of transposons, errors in the functioning of enzymes such as DNA polymerases, recombination enzymes, and DNA repair enzymes. Not cost-effective to isolate mutants for industrial strain development. (low frequency of mutation) The mutation frequency (proportion of mutants in the population) can be significantly increased by using mutagenic agents (mutagens): may increase to 10-510-3 for the isolation of improved secondary metabolite producers or even up to 10-2-10-1 for the isolation of auxotrophic mutants.
Alkylating agents
Most potent mutagen for practical application, except UV Compounds frequently used: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), diethylsulfate (DES), diepoxybutane (DEB), N-methyl-N'-nitro-N-nitrosoguanidine (NTG, the most effective chemical mutagens, but its use in a mutation program is difficult because of its carcinogenic effects), N-methyl-N-nitroso-urea and mustard gas Formation of alkylated bases in DNA, along with phosphotriester, purine-free sites and single-strand breaks / 7alkylguanine is the most common product but it does not result in mutations / O6-alkyl-guanine and O4-alkylthymine are the most important premutational lesions, and as a result of pairing errors, mainly ATGC transitions are elicited (direct mutagenesis). Transitions, transversions, deletions and frameshift mutations A second process results in mutation is the induction of error-prone SOS repair when relatively high doses of mutagen are used.
NTG
A large proportion of mutants under optimal conditions with a low killing rate. 8-10% of the survival Streptomyces coelicolor are auxotrophs, ~ 50% of the survival E. coli population consists of mutants 90% mutations are GCAT transitions; also small extent deletions and frameshift after the deletion of GC pairs Easily decomposed in vivo; formed nitrous acid in acidic solutions (not effective as mutagen in pH 6-9 where NTG is active; diazomethane (a strongly methylating agent) is formed under alkaline conditions Alkylation of nonreplicating DNA and the main point of action of at the replication point of DNA, through a change in DNA polymerase III during DNA replication (there is incorrect duplication in a short segment of the DNA until the defective polymerase is replaced by an intact molecule). This explains that NTG mutations frequently occur in gene clusters.
IS-Elements
DNA sequences of variable length (800-1400 base pairs), can incorporated in different sites of the genome and released again ( recA-independent recombination) Destroy the function of the gene at the site of their integration. IS2 bears a promotor, which, when incorporated in the appropriate orientation, results in the constitutive expression of genes located downstream. In particular IS1 causes deletions, whereas IS2 causes duplications.
Transposons
Genetic elements containing flanking IS-elements in inverse orientation, often with antibiotic-resistance genes Available for a wide variety of purposes in gene technology: Tn5 contains an aminoglycoside antibiotic-resistance gene which can be expressed in a wide variety of both procaryotes and eucaryotes. Several transposons have been integrated into plasmids (e.g. Tn1 and Tn3), others in either plasmids or chromosomes (e.g. Tn5). Transposon mutagenesis offers a wide variety of advantages: (1) can obtained a mutant phenotype with a very low reversion rate, (2) relatively easy to isolate insertion mutations, (3) the site at which the transposon has been integrated can be readily determined
Optimizing mutagenesis
Dose-response (killing) curves A high death rate alone is no guarantee of the occurrence of mutations in specific genes. Easily detectable changes such as mutations for resistance or re-version to auxotrophy are frequently used to optimize conditions for mutagenesis.
Metabolic engineering
Complete sequencing of many microbial genomes of food grade organisms Relatively minor metabolic engineering has already been implemented to improve the production of existing metabolites, allow the production of new metabolites, impart new catabolic activities and improve fermentation performance Whole metabolic networks within a microorganism may be restructured and such extensive metabolic engineering has major implications within industrial microbiology
5 (10%)
5-8
E. coliPichia pastoris Antimetabolite-resistant mutantsDirected selection of mutants Metabolic engineeringProtoplast fusion Suicidal strains