Strain Improvement Natural isolates usually produce commercially important products in - PowerPoint Presentation

1. Strain Improvement

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3. Natural isolates usually produce commercially important products in very low concentrations and therefore every attempt is made to increase the productivity of the chosen organism.In nature, metabolism is carefully controlled to avoid wasteful expenditure of energy and the accumulation of intermediates and enzymes needed for their biosynthesis.The potential productivity of the organism is controlled by its genome and, therefore, the genome must be modified to increase the potential yield.Genetic modification may be achieved by selecting natural variants, by selecting induced mutants and by selecting recombinants.Introduction

4. Irrespective of the origins of an industrial microorganism, it should ideally exhibit:Genetic stabilityEfficient production of the target product, whose route of biosynthesis should preferably be well characterizedLimited or no need for vitamins and additional growth factorsUtilization of a wide range of low-cost and readily available carbon sources Amenability to genetic manipulationSafety, non-pathogenicity and should not produce toxic agents, unless this is the target productReady harvesting from the fermentationReady breakage, if the target product is intracellularProduction of limited byproducts to ease subsequent purification problems.Attributes of Improved Strain

5. In many cases strain improvement has been accomplished using natural methods of genetic recombination, which bring together genetic elements from two different genomes into one unit to form new genotypes.An alternative strategy is via mutagenesis. Those recombinants and mutants are then subjected to screening and selection to obtain strains whose characteristics are more specifically suited to the industrial fermentation process.However, such strains are unlikely to surviveStrain Improvement

6. Several procedures are employed to improve microbial strains. All bring about changes in DNA sequence. These changes are achieved by mutation, genetic recombination, or the modern DNA splicing techniques of “genetic engineering.” Each procedure has distinct advantages. In some cases, a combination of one or more techniques is employed to attain maximum strain improvement.Strategies for Strain Improvement

7. Microbes, generation after generation, generally inherit identical characteristics as their parents.However, when changes occur in the DNA, they too are passed on to daughter cells and inherited in future generations. This permanent alteration of one or more nucleotides at a specific site along the DNA strand is called a genetic mutation.Mutation

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9. On the basis of the method of screening and selection chosen, there are basically two methods of improving microbial strains through random mutation: (1) random selection and (2) rationalized selection.

10. Random mutagenesis and selection is also referred to as the classic approach or non-recombinant strain improvement procedure.After inducing mutations in the culture, the survivors from the population are randomly picked and tested for their ability to produce the metabolite of interest.it offers a significant advantage over the genetic engineering route alone by yielding gains with minimal start-up and sustaining such gains over years despite a lack of scientific knowledge of the biosynthetic pathway, physiology, or genetics of the producing microbe.One drawback to the random selection approach is that it relies on non-targeted, non-specific gene mutations, so many strains need to be screened to isolate the improved mutant in the mixed population.Random Selection

11. This process of strain improvement involvesrepeated applications of three basic principles:

12. Typical steps in mutation and random strain selection process.

13. Although the procedure is repetitive and labor intensive, this empirical approach has a long history of success and has given dramatic increases in titer improvement, as best exemplified by the improvements achieved for penicillin production in which titers over 50 g/liter are reported—a 4000-fold improvement over the original parent strain.To increase the efficiency of random selection, ways by which the key steps in the process can drive the throughput higher without adding labor are typically sought. In some instances high throughput screens have been automated with robotics technology.

14. An alternate approach to random screening requires a basic understanding of product formation and the fermentation pathway; this can be acquired through radioisotope feeding studies and isolation of mutants blocked in various pathways.These observations can shed light on the metabolic checkpoints, and suggest ways to isolate specific mutants. For example, environmental conditions (pH, temperature, aeration) can be manipulated, or chemicals can be incorporated in the culture media to select mutants with desired traits.Rationalized selection for strain improvement does not generally require a sophisticated understanding of molecular biology to manipulate environmental or cultural conditions.Rationalized Selection

15. It can be done byOptimizing environmental conditionOptimizing nutrition of micro-organismMethods not involving foreign DNA-mutagenesisOptimization of microbial activity

16. The best example of the modification of the permeability of a micro-organism is provided by the glutamic acid fermentation. Kinoshita et al. (l957a) isolated a biotin-requiring, glutamate-producing organism, subsequently named Corynebacterium glutamicum, the permeability of which could be modified by the level of biotin.MODIFICATION OF THE PERMEABILITY

17. Given figure is a branched pathway controlled by the concerted inhibition of the first enzyme in the pathway by the combined effects of E and G. The mutant illustrated is auxotrophic for E due to an inability to convert C to D, resulting in the removal of the concerted control of the first enzyme. Provided that E is included in the medium at a level sufficient to allow growth but insufficient to cause inhibition then C will be accumulated due to the control of the end product G on the conversion of C to F.Auxotrophic mutants

18. Isolating strains relaxed in regulation can usually be accomplished by selecting strains desensitized to feedback inhibition (enzyme activities) or feedback repression (enzyme synthesis) involved in the pathway.The use of auxotrophic mutant is useless when the end product controls its own synthesis independently.Regulatory mutants

19. In many microbes, the end products of metabolism, when accumulated in the microbial cell, inhibit the enzyme activities of many pathways. The end product causes conformational changes by binding to a specific (allosteric) site on the enzyme, and inhibits activity. The binding is usually noncompetitive.Mutation in the structural gene, however, can alter the enzyme binding site and prevent these inhibitory effects.Studying the interaction of various analogs of end products and their resistance, improved strains can be selected that lack feedback inhibition and thus overproduce metabolites of interest.Mutants Resistant to Feedback Inhibition

20. Here intermediates, products of catabolism (derived from breakdown of compounds containing carbon, nitrogen, or phosphorus), or end products regulate the amount of biosynthetic enzymes synthesized and, therefore, the amount of final product formed.However, mutations at the operator site or other regulatory sites on the gene relieve such end-product repression and allow overproduction of the biosynthetic enzyme.Mutants Resistant to Repression

21. Protoplast fusionFusing two closely related protoplasts (cells whose walls are removed by enzyme treatment) is a versatile technique that combines the entire genetic material from two cells to generate recombinants with desired traits that cannot be obtained through a single mutation.TransformationTransformation is the process involving the direct uptake of purified, exogenously supplied DNA by recipient cells or protoplasts.ConjugationConjugation introduces mutational changes in microbes through unidirectional transfer of genetic material from one strain to the other; it is mediated by plasmid sex factors.Genetic recombination

22. In vitro recombinant DNA technologyBy employing restriction endonucleases and ligases, investigators can cut and splice DNA at specific sites.Isolate the desired gene (DNA fragment) from the donor cells.Isolate the vector (a plasmid or a phage).Cleave the vector, align the donor DNA with the vector, and insert the gene into the vector.Introduce the new plasmid into the host cell by transformation or, if a viral vector is used, by infection.Select the new recombinant strains that express the desired characteristics.Cloning and genetic engineering

23. So far the mutations and the modifications of the strains discussed have been randomly directed at the level of the genome of the culture. The application of recombinant technology and the use of synthetic DNA now make it possible to induce specific mutations in specific genes. This procedure of carrying out mutagenesis at a targeted site in the genome is called site-directed mutagenesis.It involves the isolation of the DNA of the specific gene and the determination of the DNA sequence.It is then possible to construct a modified version of this gene in which specific bases or a series of bases are changed. The modified DNA can now be reinserted into the recipient cells and the mutants selected.

24. Increase the productivitiesRegulating the activity of the enzymesIntroducing the new genetic properties into the organism by recombinant DNA technology/Genetic engineering.Purpose

25. Rapid growthGenetic stabilityNon-toxicity to humansAbility to use cheaper substratesElimination of production of compounds that may interfere with downstream processing.To improve the use of carbon and nitrogen sources.Reduction of cultivation cost.Shorter fermentation time.IDEAL CHARACTERISTICS OF STRAIN