Mutant Selection in Classical Mutagenesis

Classical mutagenesis is a random procedure to generate aberrations in the genetic material, and hence, the selection of the mutant is largely based on change in morphology or colour, nutritional

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Fig. 10.2 Strain improve-ment of Acremonium chrysogenum by UV mutagenesis programme for cephalosporin production

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requirements (biochemical mutants) and screening of enhanced production of the end product.

10.4.1 Morphological Mutants

In morphological mutants, the production ability of the end product is improved; however, the exact mechanism of this improvement remains uncertain. Gibberellic acid production by Gibberella fujikuroi is commonly carried out by aerobic fermentation. A variety of morphological mutants possessing different mycelial and soluble pigmentation have been generated from Gibberella fujikuroi upon exposure to UV radiation. It was observed that the unpigmented morphological mutant of Gibberella resulted in the lowering of the viscosity of the fermentation broth as well as resulted in an increase in the production of gibberellic acid (Lale et al. 2006 ).

Streptomyces species have been designated as the most prolifi c producers of antibiotics in acti-nomycetes which fi nd use in agriculture, medi-cine and veterinary purpose. Bald colonies have been exhibited by Streptomyces coeruleorubi-dus which produces higher titre of daunorubi-cin (Blumauerova et al. 1978 ). Bald mutants in Streptomyces coelicolor are found to be defective in carbon utilisation, morphogenesis and cell signalling.

In Aspergillus nidulans , mutagenesis leads to the formation of mutants which lack conidia apart from being coloured differently. Wet, white- coloured mutant of Aspergillus nidulans was obtained by random mutagenesis using nitrous acid. In these mutants, the conidia are colourless and become brown and accumulate water droplets. Abacus is an aconidial mutant of A. nidulans generally produced by the treatment of nitrous acid, UV radiation and NNTG. It bears rod-like structures with swellings at intervals in place of conidia (Clutterbuck 1969 ). Actinoplanes teichomyceticus is a teicoplanin-producing strain that when mutated exhibits pink instead of brown mycelia and leads to 25-fold higher pro-duction of teicoplanin than the parent strain (Lee et al. 2003 ).

10.4.2 Auxotrophic Mutants

These microorganisms possess specifi c nutritional requirements as a result of mutation. Mutant auxotrophs of Corynebacterium glutamicum have been developed which possess high rates of amino acid production (Nakayama 1985 ). The development of these auxotrophs has been done through the iterative process of mutagenesis and selection. Optimisation of the mutant strains has been further carried out by chemical mutagenesis and UV irradiation to release enzymes which help in the regulation of metabolite production by feedback inhibition (Shiio et al. 1990 ).

Streptomyces has been studied using various aerial mycelium negative (amy ^- ) mutants or by biochemical analyses during their exploitation in the industry (Hopwood 1978 ). During the produc-tion of bicozamycin (previously bicyclomycin) by Streptomyces griseofulvin , a mutant which was an arginine auxotroph could grow with citrulline and ornithine and could produce maximum aerial mycelium with citrulline (Godfrey 1973 ).

α -Ketoglutarate has applications in agrochem-ical, food industry, pharmaceuticals and cosmet-ics. A Yarrowia lipolytica isolate producing a very high titre was isolated when production of single-cell protein was carried out using petro-leum as the raw material (Finogenova et al.

2005 ). Torulopsis glabrata and Y. lipolytica are the only thiamine-auxotrophic species which have been studied as α- ketoglutarate producers (Liu et al. 2007 ; Zhang et al. 2009 ). Thiamine limitation essentially deactivates pyruvate decar-boxylase and α-ketoglutarate dehydrogenase complex leading to overfl ow of α- ketoglutarate (Morgunov et al. 2004 ; Nosaka et al. 2008 ).

10.4.3 Mutants Exhibiting Resistance to Antimetabolites

Compounds that resemble structurally to natural metabolites are the metabolic analogues and interfere with the normal function of the cells, thus referred as antimetabolites. The simplest

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example of antimetabolite is sulfa drugs.

N -fl ourophenylalanine is an antimetabolite of tyrosine, and it was observed that E. coli mutants resistant to p -fl uorophenylalanine secreted high amount of tyrosine (Adelberg 1958 ). Canavanine- resistant mutants of E. coli and S. cerevisiae and arginine hydroxamate-resistant mutants of Bacillus subtilis have been recognised to excrete or accumulate L -arginine (Kubota et al. 1973 ).

Phosphate has been a key regulator of biosyn-thesis of peptide, polyene and tetracycline class of antibiotics. Candicidin synthetases in Streptomyces griseus are regulated by phosphate, and therefore, mutants which can resist a phos-phate concentration higher than 10 mM are of great industrial interest, suggesting the loss of regulatory control by phosphate in candicidin biosynthesis (Martin et al. 1979 ). Wild strains of Penicillium chrysogenum produce low titres of penicillin V in medium containing excess glu-cose; however, GRI (glucose repression insensi-tive) mutants have been reported to produce high penicillin V than the wild-type strain consisting of lactose as the main carbon source in the medium (Chang et al. 1990 ). Thus, GRI selection mutants can be used as selective markers for higher penicillin-producing strains.

High astaxanthin-producing strains of Phaffi a rhodozyma were screened using diphenylamine, and the mutants did not exhibit variation in size or numbers but appeared as salmon-coloured colonies against pink salmon of wild type.

Diphenylamine-resistant mutant exhibited a two-fold increase in astaxanthin production over wild type (Chumpolkulwong et al. 1997 ). Ashbya gos-sypii , a hemiascomycete fungus, is characterised as a natural ribofl avin producer and has been uti-lised for industrial production of ribofl avin.

Isocitrate lyase was identifi ed as the key enzyme responsible for ribofl avin production using soy-bean oil as sole carbon source. Isocitrate lyase gets strongly inhibited by oxalate or itaconate.

An oxalate-resistant Ashbya gossypii has been recently reported to produce threefold higher ribofl avin production when compared to the wild strain (Sugimoto et al. 2010 ). Roseofl avin has been used successfully for screening mutants of Bacillus subtilis for high ribofl avin production.

Coenzyme Q 10 is an isoprenylated benzoquinone which is a well-known component of electron transport chain in eukaryotes and is used as an oral nutritional supplement in several disorders like cardiomyopathy, diabetes and neurodegen-erative diseases associated with CoQ 10 defi ciency.

Sporidiobolus johnsonii is the only heterobasid-iomycete yeast strain known to produce CoQ10 under submerged fermentation conditions. UV and EMS mutagenesis has been attempted for strain improvement of S. johnsonii which has been screened using atorvastatin resistance marker. EMS induced the atorvastatin- resistant mutant strain EA22 generated from S. johnsonii ATCC 20490 which exhibited a twofold increase in the CoQ 10 titre (Ranadive et al. 2011 ).

10.4.4 Enhanced Production of the End Product: Agar Zone Mutants

Agar zone assay is a screening method of mutants based on their fermentation performance and high yield of the desired end product. Lovastatin is an inhibitor of HMG-CoA reductase thereby limiting cholesterol biosynthesis for the treatment of hypercholesterolemia. Mutant Aspergillus terreus colonies were screened by preparing indi-vidual agar plug of each colony and then extracted with ethyl acetate for 15 min at 50 °C and subse-quently the supernatant removed and tested by transferring on a 6-mm paper disc and placed on a 90-mm Candida albicans plate. The positive control comprised of a known concentration of lovastatin loaded on a 6-mm paper disc.

Subsequently, the inhibition zones of the mutants were compared with positive control for selection of mutants with high titre of lovastatin (Ferron et al. 2005 ). Starch iodine assay has been used to screen and select high glucoamylase-producing deregulated mutants of Thermomucor indicae-seudaticae after sequential exposure to nitrous acid and γ-radiation (Kumar and Satyanarayana 2009 ). Zone diameter assay has also been used in screening and selection of UV and NTG mutants of Trichoderma reesei MTCC 3929 for higher alkaline protease production (Zambare 2010 ).

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Methylene blue agar zone assay has been used for screening and selection of mutants of Streptomyces diastatochromogenes with enhanced εpoly L -lysine (Wang et al. 2012 ).

Streptoverticillium rimofaciens produces mildiomycin nucleoside antibiotic which is potentially effective against powdery mildew disease on different crops. Enhanced production by an improved strain can be determined by ana-lysing extracts from agar plugs by HPLC/TLC and diffusion assays. A bioassay method has been developed to report improved strains by assessing the mildiomycin content in complex broth using a strain of Rhodotorula rubra AS 2.166 as an indicator organism. Mildiomycin gives a clear inhibition zone with sharp edges when the mutant colonies were punched into individual agar plugs and then placed on the test agar medium seeded with the test microorgan-isms. The zone formation was directly related to higher production of mildiomycin by the mutant S. rimofaciens (Xie et al. 2005 ) .