Strain Improvement PDF

STRAIN IMPROVEMENT Criteria - in the choice of organism: The nutritional characteristics of the organism. It is frequently required that a process be carried out using a very cheap medium or a pre-determined one, e.g. the use of methanol as an energy source. These requirements may be met by...

STRAIN IMPROVEMENT Criteria - in the choice of organism: The nutritional characteristics of the organism. It is frequently required that a process be carried out using a very cheap medium or a pre-determined one, e.g. the use of methanol as an energy source. These requirements may be met by the suitable design of the isolation medium. 2. The optimum temperature of the organism. The use of an organism having an optimum temperature above 40° C considerably reduces the cooling costs of a large-scale fermentation and, therefore, the use of such a temperature in the isolation procedure may be beneficial. 3. The reaction of the organism with the equipment to be employed and the suitability of the organism to the type of process to be used. 4. The stability of the organism and its amenability to genetic manipulation. 5. The productivity of the organism, measured in its ability to convert substrate into product and to give a high yield of product per unit time. 6. The ease of product recovery from the culture. Strain improvement The selection of induced mutants synthesizing improved levels of primary metabolites The isolation of induced mutants producing improved yields of secondary metabolites where directed selection is difficult to apply The use of recombination systems for the improvement of industrial micro-organisms The selection of induced mutants synthesizing improved levels of primary metabolites Feedback Inhibition Feedback Repression 1. The organism may be modified such that the end products which control the key enzymes of the pathway are lost from the cell due to some abnormality in the permeability of the cell membrane. 2. The organism may be modified such that it does not produce the end products which control the key enzymes of the pathway. 3. The organism may be modified such that it does not recognize the presence of inhibiting or repressing levels of the normal control metabolites.  Modification of permeability Corynebacterium glutamicum THE ISOLATION OF MUTANTS WHICH DO NOT PRODUCE FEEDBACK INHIBITORS OR REPRESSORS The isolation of auxotrophic mutants may result in the isolation of high-producing strains, provided that the mutation for auxotrophy occurs at the correct site 1. Overlay of agar 2. Enrichment culture Visual Identification Replica Plating Sandwich technique  EXAMPLES OF THE USE OF AUXOTROPHS FOR THE PRODUCTION OF PRIMARY METABOLITES  Isolation of mutants which do not recognise the feedback inhibitors or repressors 1. Isolation of analogue resistant mutants 2. Isolation of revertants  Biosynthesis of P where P* is inhibitory due to its mimicing the control properties of P; a mutant may be isolated which may be capable of growing in the presence of P* due to the fact that the first enzyme in the pathway is no longer susceptible to inhibition by the analogue. The modified enzyme of the resistant mutant may not only be resistant to inhibition by the analogue but may also be resistant to the control effects of the natural end product, P, resulting in the uninhibited production of P.  analogue resistant mutants may be expected to overproduce the end product to which the analogue is analogous provided that: (i) The toxicity of the analogue is due to its mimicing the control properties of the natural product. (ii) The site of resistance of the resistant mutant is the site of control by the end product.  Lysine analogue-resistant mutants of Brevibacterium flauum for the production of lysine. S-(2 aminoethyI) cysteine (AEC) –Analogue of Lysine the inhibition by AEC and threonine could be reversed by the addition of lysine. This evidence suggested that the inhibitory effect of ABC was due to its mimicing lysine in the concerted inhibition of aspartokinase. Gradient plate technique  The isolation of induced mutants producing improved yields of secondary metabolites where directed selection is difficult to apply Isolation of Auxotrophic mutants THE ISOLATION OF RESISTANT MUTANTS (i)Mutants may be isolated which are resistant to the analogues of primary metabolic precursors of the secondary metabolite, thus increasing the availability of the precursor.  isolation of mutants of Pseudomonas aureofaciens overproducing the antibiotic pyrrolnitrin. Tryptophan is a precursor of pyrrolnitrin and Elander et al., isolated mutants resistant to tryptophan analogue using the gradient plate technique A strain was eventually isolated which produced two to three times more antibiotic than the parent and was resistant to feedback inhibition ii. Mutants may be isolated which are resistant to the feedback effects of the secondary metabolite. The mechanism of the control of its own synthesis by chloramphenicol appears to be the repression of arylamine synthetase (the first enzyme in the pathway from chorismic acid to chloramphenicol) by chloramphenicol. (iii) Mutants may be selected which are resistant to the toxic effects of the secondary metabolite when added to the trophophase of the producing organism. (iv) Mutants may be isolated which are resistant to the toxic effects of a compound due to the production of the secondary metabolite. A potentially toxic compound may be made harmless by an organism converting it to a secondary metabolite or a secondary metabolite complexing it. Ions of heavy metals such as Hg2 +, Cu2 + related organometallic ions are known to complex beta -lactam antibiotics Isolation of revertants (i) The isolation of revertants of mutants auxotrophic for primary metabolites which may influence the production of a secondary metabolite. (ii) The reversion of mutants which have lost the ability to produce the secondary metabolite Recombination Parasexual Cycle Protoplast Fusion Transformation Conjugation Transduction rDNA Technique Parasexual Cycle Application of Protoplast Fusion Protoplast Sphaeroplast  Transformation Conjugation Transduction conjugation rDNA Technique (j) A 'vector' DNA molecule (plasmid or phage) capable of entering the host cell and replicating within it. Ideally the vector should be small, easily prepared and must contain at least one site where integration of foreign DNA will not destroy an essential function. (ij) A method of splicing foreign genetic information into the vector. (iii) A method of introducing the vector foreign DNA recombinants into the host cell and selecting for their presence. Commonly used simple characteristics include drug resistance, immunity, plaque formation, or an inserted gene recognizable by its ability to complement a known auxotroph. (iv) A method of assaying for the 'foreign' gene product of choice from the population of recombinants created.  The production of heterologous proteins The use of recombinant DNA technology for the improvement of native microbial products The improvement of industrial strains by modifying properties other than the yield of product Stable strains Strains resistant to infection Non foaming strains