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Slide 1 - Industrial Biotechnology Lesson 4 ISOLATION, SCREENING AND STRAIN IMPROVEMENT
Slide 2 - Isolation and Screening of Industrial Strain Isolation of from the environment is by: Collecting samples of free living microorganism from anthropogenic or natural habitats. These isolates are then screened for desirable traits. Or by sampling from specific sites: Mos with desired characteristics are found among the natural microflora After sampling of the organism the next step is of enrichment.
Slide 3 - Enrichment Enrichment in batch or continuous system on a defined growth media and cultivation conditions are performed to encourage the growth of the organism with desired trait. This will increase the quantity of the desired organism prior to isolation and screening.
Slide 4 - Screening Subsequent isolation as pure cultures on solid growth media involves choosing or developing the appropriate selective media and growth conditions. Next step to enrichment and isolation is Screening. The pure cultures must be screened for the desired property; production of a specific enzyme, inhibitory compound, etc. Selected isolates must also be screened for other important features, such as stability and, where necessary, non-toxicity.
Slide 5 - Screening These isolation and screening procedures are more easily, applied to the search for a single microorganism. The industrial microorganism should ideally exhibit: 1. genetic stability 2. efficient production of the target product, whose, route of biosynthesis, should preferably be well characterized.
Slide 6 - Screening 3. limited or no need for vitamins and additional growth factors. 4. utilization of a wide range of low-cost and readily available carbon sources 5. amenability to genetic manipulation; 6. safety, non-pathogenic and should not produce toxic agents, unless there is the target product; 7. ready harvesting from the fermentation; . 8. production of limited byproducts to ease subsequent purification problems.
Slide 7 - Culture Preservation Streptomyces aureofaciens NRRL 2209 was the first microorganism deposited in a culture collection in support of a microbially based patent application. Preservation of microbial cultures was critical for all individuals and firms engaged in the search for patentable products from and patentable processes by microorganisms.
Slide 8 - Culture Preservation Preservation of cultures by freezing, drying, or a combination of the two processes is highly influenced by resistance of the culture to the damage caused by rapid freezing, the dehydrating effects of slow freezing, or damage caused during recovery. To minimize damage, agents have been used that protect against ice formation by causing the formation of glasses upon cooling.
Slide 9 - Culture Preservation Methods to protect against the negative effects of dehydration include adaptation to lower effective water activity by pre-incubation in high osmotic pressure solutions. Damage caused by thawing after freezing can be minimized by rapid melting and by the composition of the medium used for growth after preservation.
Slide 10 - Culture Preservation There are various preservation methods . To date, preservation in liquid nitrogen is still the most successful long-term method.
Slide 11 - Serial Transfer Based upon its ease of use, serial transfer is often the first “preservation” technique used by microbiologists. The disadvantages of relying upon this method for culture maintenance include contamination, loss of genetic and phenotypic characteristics, high labor costs, and loss of productivity.
Slide 12 - Preservation in Distilled Water This method (Castellani method, 50 years ago) was extensively tested on 594 fungal strains: 62% of the strains growing and maintaining their original morphology. In another study, 76% of yeasts, filamentous fungi, and actinomycetes survived storage in distilled water for 10 years.
Slide 13 - Preservation in Distilled Water The pathogen Sporothrix schencki concluded that even though long-term survival was good when this procedure was used, there was a noted loss in virulence. Castellani technique should be considered as one of the options for practical storage of fungal isolates.
Slide 14 - Preservation under Oil One of the earlier preservation methods was the use of mineral oil to prolong the utility of stock cultures. Mineral oil has been found to prevent evaporation from the culture and Decrease the metabolic rate of the culture by limiting the supply of oxygen. This method is more suitable than lyophilization for the preservation of non-sporulating strains.
Slide 15 - Lyophilization One of the best methods for long-term culture preservation of many microorganisms is freeze-drying (lyophilization). The commonly used cryoprotective agents are skim milk (15% [wt/vol] for cultures grown on agar slants and 20% for pelleted broth cultures) or sucrose (12% [wt/vol] final concentration). It should be noted that some plasmid--containing bacteria are successfully preserved by this method.
Slide 16 - Storage over Silica Gel Neurospora has successfully been preserved over silica gel. Preservation on Paper Drying the spores on some inert substrates can preserve spore-forming fungi, actinomycetes, and unicellular bacteria. Fruiting bodies of the myxobacteria, containing myxospores, may be preserved on pieces of sterile filter paper and stored at room temperature or at 6°C for 5 to 15 years. Preservation on Beads The method involving preservation on beads (glass, porcelain) , developed by Lederberg, is successful for many bacteria.
Slide 17 - Liquid Drying To avoid the damage that freezing can cause, a liquid—drying preservation process is applied. It has effectively preserved organisms such as anaerobes that are damaged by or fail to survive freezing. This procedure was preferred over lyophilization for the maintenance of the biodegradation capacity of six gram--negative bacteria capable of degrading toluene. Malik’s liquid-drying method was also found to be markedly superior to lyophilization for the preservation of unicellular algae.
Slide 18 - Cryopreservation Microorganisms may be preserved at - 5 to - 20°C for 1, to 2 years by freezing broth cultures or cell suspensions in suitable vials. Deep freezing of microorganisms requires a cryoprotectant such as glycerol or dimethyl sulfoxide (DMSO) when stored at -70°C or in the liquid nitrogen at -156 to -196°C.
Slide 19 - Cryopreservation Broth cultures taken in the mid--logarithmic to late logarithmic growth phase are mixed with an equal volume of 10 to 20% (vol/vol) glycerol or 5 to 10% (vol/vol) DMSO. Alternatively, a 10% glycerol-sterile broth suspension of growth from agar slants may be prepared.
Slide 20 - Preservation in Liquid Nitrogen Storage in liquid nitrogen is clearly the preferred method for preservation of culture viability.
Slide 21 - Protocol for Cryopreservation with Cryoprotectants by a Two-stage Freezing Process, and Revival of Culture After centrifugation the supernatant is removed and the pellet, consisting of microbial cells, is dissolved in an ice-cold solution containing polyvinyl ethanol (10% [wt/vol]) and glycerol (10% [wt/vol]) in a 1:1 ratio. Due to the presence of polyvinyl ethanol, a viscous thick cell suspension is obtained, which is kept for about 30 minutes in an ice bath for equilibration.
Slide 22 - During equilibration, an aliquot of 0.5 to 1.0 ml of the cell suspension is dispensed into each plastic cryovial or glass ampoule. They are tightly closed, clamped onto labeled aluminum canes, and placed at -30°C for about 1 h or for a few minutes in the gas phase of liquid nitrogen to achieve a freezing rate of about 1°C/min. The canes are then placed into canisters, racks, or drawers and frozen rapidly at -80°C or in liquid nitrogen. Protocol for Cryopreservation with Cryoprotectants by a Two-stage Freezing Process, and Revival of Culture
Slide 23 - For revival of cultures, the frozen ampoules are removed from the liquid nitrogen. For thawing, they are immediately immersed to the neck in a water bath at 37°C for a few seconds. The thawed cell contents of the ampoule or vial are immediately transferred to membranes to form a thick layer. The resulting bacterial membranes with immobilized cells are used as a biological component of a biosensor for activity measurements. Protocol for Cryopreservation with Cryoprotectants by a Two-stage Freezing Process, and Revival of Culture
Slide 24 - Inoculum Development The primary purpose of inoculum development is to provide microbial mass, of predictable phenotype, at a specific time, and at a reasonable cost for the productive stage of a microbial activity. Until now, inoculum development has been more art than science. There remains a need, especially at the shake flask or spore-generating stages of the process, for time and “it looks good” criteria to be replaced with biochemical, physiological, or morphological markers as both descriptors of an optimum inoculum and indicators for optimum timing of inoculum transfer: Inoculum Source
Slide 25 - Inoculum development When fungal spores are used as the inoculum source, it is common for conidia produced on an agar slant to be dispersed in sterile distilled water containing 0.01 to 0.1 % Tween 80. Spore formation of Streptomyces coelicolor on agar was dependent upon the type of agar used, the inclusion of trace elements, the nitrogen source, and a C/N ration between 40 and 100 (68).
Slide 26 - Inoculum development Nabais and de Fonseca have optimized a medium for sporulation by Streptomyces clavuligerus. Spore storage, however, could be a problem, since the spores lost 72% of their viability after storage for 1 week in buffer at 4°C. Many strains isolated from nature and often strains that have been subjected to a mutation program result in an “unstable” culture, whose productivity can be rapidly lost. For such strains, a single spore selection step or its equivalent is a necessity for maintenance of productivity.
Slide 27 - Acclimatization A number of commercial-level microbiological processes use as the inoculum, at least in part, culture growth that has been part of a previous “production phase. For fermentation processes involved in the degradation of waste materials, a very important variable is the extent of acclimatization of the inoculum source.
Slide 28 - Acclimatization The process lag before initiation of biodegradation decreases with increased numbers of competent microorganisms. High degradation rates are obtained when acclimated sewage sludge operated in a plant with low retention times is used as the inoculum.
Slide 29 - Acclimatization h The use of an acclimatized inoculum has been reported to result in significant improvements in operational efficiencies for xylose conversion to xylitol by Candida guilliermondii grown on a sugar cane hemi-cellulosic hydrolysate. In the brewery industry, the reuse or pitching of yeast is a common practice.
Slide 30 - The effect of serial pitching of the yeast inoculum on subsequent re-fermentation has not been well characterized. The condition of the yeast cell surface as measured by flocculation can be predictive before subsequent fermentation performance.
Slide 31 - Seed Media For the design of media used for the production of cell mass, the determination of an elemental material balance is a useful exercise. For defined media, the determination is a straightforward calculation from the components. For complex media, Traders’ Co. and other manufacturers of complex nutrients provide the basic data needed to estimate the contribution of various components to the sum of an element.
Slide 32 - pH Nutritionally balanced seed media often result in pH values not far from the optimum for culture growth. To prevent pH extremes in shake flasks, phosphate salts and CaCO3 and/or buffers such as 2-(N-morpholino) ethanesulfonic acid (MES) or 3-(N-morpholino) propanesulfonic acid (MOPS) are often used. In fermenter inoculum development stages, buffers are usually replaced with the more economical online pH control.
Slide 33 - Immobilization The production of microbial inoculum for use in bioremediation, agricultural applications, and waste treatment is limited by the ability of the microorganism to compete in these environments and to be metabolically effective. One of the methods by which microbial inocula are being improved for these applications is the use of immobilization technology.
Slide 34 - Immobilization The unique characteristics of immobilized inocula include (i) enhanced inoculum viability, (ii) protection from stress during manufacture, (iii) enhanced ecological competence, (iv) increased metabolite production, (v) UV resistance, (vi) the opportunity to use immobilized cells as a source of continuous inoculum, (vii) the opportunity to introduce mixed culture inocula into a process.
Slide 35 - Immobilization Storage of the immobilized inoculum is enhanced if cells in beads are incubated in nutrient or supplemented with nutrient when prepared. A protocol for alginate immobilization is required as homework?
Slide 36 - Contamination Microbial contaminant detection usually relies upon the use of differential media and conditions to encourage the growth of likely contaminant in the presence of the inoculated microbe. It is difficult to detect of contamination in mixed culture fermentation.
Slide 37 - Contamination PCR has provided a rapid, effective technique for the detection of a contaminant present at low levels in a sample. PCR protocols can be applied to mixed culture fermentations either for the detection of a particular contaminant of interest (Listeria monocytogenes) or for the detection of an indicator organism, such as the detection of E. coli as an indicator of fecal contamination..
Slide 38 - Phages Phage contamination is a constant threat to the productivity of any bacterial fermentation process, particularly in fermentations of dairy products. How to overcome such a problem? Selection of plasmids that confer phage resistance ( e. g. for lactic streptococci). Selection of phage-resistant strains (preffered).
Slide 39 - Phages The report that alginate-immobilized streptococci were protected from attack by phages is potentially an interesting alternative approach.
Slide 40 - Mites They can devastate a culture source or a series of culture sources either by eating the cultures and leaving no viable source or, more commonly, by causing marked levels of bacterial and fungal cross contamination. Often the first indication of a problem is agar plates with bacterial or fungal tracks forming in a random-walk pattern across the plate.
Slide 41 - Mites Treatment of incubators with acaricides on a preventative-maintenance schedule is also worth considering.
Slide 42 - Strain Improvement 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.
Slide 43 - Strain Improvement 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.
Slide 44 - Strain Improvement 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. ·,
Slide 45 - Strain Improvement 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,
Slide 46 - Strain Improvement 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,
Slide 47 - Strain Improvement 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.
Slide 48 - Strain Improvement 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.
Slide 49 - Methods of Strain Improvement Up here (mohamed) 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.
Slide 50 - 1. Mutation 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.
Slide 51 - 1. Mutation 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.
Slide 52 - 1. Mutation 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.
Slide 53 - 1. Mutation 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:
Slide 54 - 1. Mutation 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.
Slide 55 - 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.
Slide 56 - Reaction Mechanisms of Mutagens 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).
Slide 57 - Reaction Mechanisms of Mutagens 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.
Slide 58 - Reaction Mechanisms of Mutagens 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.
Slide 59 - Reaction Mechanisms of Mutagens 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.
Slide 60 - Reaction Mechanisms of Mutagens 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.
Slide 61 - Reaction Mechanisms of Mutagens 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.
Slide 62 - Reaction Mechanisms of 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.
Slide 63 - 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.
Slide 64 - Phenotypic Expression of Mutations This also applies to exponentially growing bacterial cells, which can contain 2-8 chromosomes; not until several steps of reproduction has taken place do pure mutant clones appear. Delays in expression, which are not directly the result of genetic effects, are observed, such as mutations which cause changed ribosome or mutations resulting in the loss of surface receptors.
Slide 65 - Optimizing Mutagenesis The effect of a mutagen on a specific gene or the effect of a mutation on a complex process, such as the biosynthesis of a secondary metabolite can never be predicted. The appearance of mutants depends on several factors. 1) The base sequence of the mutated gene. Mutations are not distributed evenly around the genome;
Slide 66 - Optimizing Mutagenesis There are areas with high mutation frequency, the so-called hot spots. Different mutagens cause hot spots at different sites in the genome. 2) The repair systems of the cell also play a role. In strains with partially defective repair mechanisms, organisms may be killed without having induced mutations, so that specific mutagens can be ineffective.
Slide 67 - Optimizing Mutagenesis 3) A gene activity, which has become lost through mutation, can be restored at least partially through a second mutation, a suppressor mutation. Suppressor mutations can occur in the same gene that already carries the primary mutation (intragenic suppressors). The primary missense mutation is compensated through the exchange of an amino acid or an additional deletion or insertion, which corrects a primary frame shift mutation so that the reading frame remains intact.
Slide 68 - Optimizing Mutagenesis Suppressor mutations which occur in another gene (extragenic suppressor) compensate the primary mutation particularly at the level of translation, by the formation of mutant transfer RNAs or ribosome. The treatment conditions have a critical effect on mutagenesis. Such factors as the pH, buffer composition, mutagen concentration, exposure time, temperature, and growth phase of the organism may greatly affect the efficiency of the process.
Slide 69 - Optimizing Mutagenesis By plotting dose-response curves all of these factors may be optimized. Mutagen effect may be have a lethal effect where in strong exposed may cause more than 99% death. The survived mutations can only be reliably determined by assessing qualitatively or quantitatively changes in the product of the target gene.
Slide 70 - Selection of Mutants Random screening: surviving clones is inspected for ability to produce the product of interest. Inspection is done in model fermentations, which are carefully adapted to the medium and fermentation parameters of the large-scale procedure, in order to maximize the likelihood that the strains will be suitable for industrial production. The best strains from such a mutation cycle are repeatedly mutated and selected.
Slide 71 - Selection of Mutants A gradual increasing in the yield is attained by continuing with these steps. Depending on the capacity of the screening program, the 5-10 best strains of a mutation-selection cycle should be used as parent strains for future mutagenesis. These strains are normally treated with mutagens different from those used in the initial isolation.
Slide 72 - Selection of Mutants Factors which influence the size of the screening program are: · frequency of mutation, · extent of yield increases, · the amount of time required for a mutation-selection cycle, · the available test capacity of the screening program, · and the accuracy of the screening test (e.g. antibiotic assay).
Slide 73 - Selection of Mutants Mutants with high yields are much rarer than those with only slight improvements. The variability of mutagen treated populations is quite high even when mutagenesis is performed under identical conditions. Thus it is usually more economical to screen a small number of survivors (about 20-50) after many different mutagen treatments.
Slide 74 - Selection of Mutants The number of strains, which must be screened to obtain mutants with a yield increase, depends on · The strain, · The conditions of mutagenesis · the biosynthesis pathway · the regulation of the product, which is being optimized. Normally, several hundred to several thousand isolates per mutation cycle must be tested.
Slide 75 - Selection of Mutants The screening capacity determines the speed of the progress to be expected. · In the first stage of mutant screening, only one fermentation sample per isolation is usually assayed, provided that the test error is smaller than the yield increase expected. · The best isolates of the first series (usually 10-30%) are then tested in a second fermentation. · Since the best strains of this second screening are then used in a still further mutation cycle, the yield increase must be statistically significant when compared to the parent strain.
Slide 76 - Selection of Mutants Several industrial companies are developing ways to automate mutant screening procedures to increase the screening capacity. Isolation of Mutants: several examples of the many selective methods used in strain development are mentioned here. Isolation of resistant mutants: A high cell density of a mutagenised population can be plated on a selective medium containing a concentration of a toxic substance that prevents the wild type from growing.
Slide 77 - Selection of Mutants Only the resistant clones can develop. mutants may be isolated which are resistant to antibiotics or anti-metabolites. Mutants isolated may also have increased cell permeability or a protein synthesis with a high turnover, making hem useful for industrial purposes. Anti-metabolite resistance can be used to select mutants, which exhibit defective regulation. Altered regulation may occur in such mutants.
Slide 78 - Selection of Mutants Anti-metabolites, because of their structural similarity to metabolites, may cause feedback inhibition, but are unable to substitute for normal metabolites. Anti-metabolites cause death of normal cells, but analog-resistant mutants can form an excess of metabolites, in some cases through changed regulatory mechanisms (elimination of allosteric inhibition; constitutive product formation). Isolation of auxotrophs: (Auxotrophy is the inability of an organism to synthesize a particular organic compound required for its growth).
Slide 79 - Selection of Mutants The isolation of auxotrophs is done by plating of the mutagenized population on a complete agar medium, on which the biochemically deficient mutants can also grow. By means of Lederberg’s replica plating technique, the clones are transferred to minimal medium where the auxotrophic colonies cannot grow. These mutants are picked up from the master plates and their defect is characterized. Enrichment technique named filtration enrichment method is used to isolate and enrich the mutagenized population.
Slide 80 - Selection of Mutants The spores of filamentous organism (actinomycetes, fungi) are allowed to develop in a liquid minimal medium. The developing micro colonies of prototrophs are then separated by filtration, leaving behind in the filtrate spores of auxotrophs, which have been unable to grow. The filtrate is then plated and the resulting colonies are checked for auxotrophic characteristics.
Slide 81 - Selection of Mutants Penicillin selection method for isolation of auxotrophs: Penicillin kills growing cells but not non-growing cells. In this procedure, growing cells are selectively killed by antibiotic treatment, thus enriching for auxotrophs, which cannot grow on minimal medium. Several inhibitors other than penicillin can also be used in this procedure: dihydrostreptomycin for Pseudomonas aeruginosa, nalidixic acid for Salmonella typhimurium, colistin for the penicillin-resistant Hydrogenomonas strain H16, and nystatin for Hansenula polymorpha, P.chrysogenum, A. nebulas, and S. cerevisiae.
Slide 82 - Selection of Mutants Other procedures : The presence or absence of specific enzyme activities can be observed directly in colonies growing on plates by spraying with suitable reagents or by incorporating indicator dyes into culture medium. Detection of amtibiotically active substances may be detected by using agar plug method with antibiotic sensitive organisms producing an inhibition zone. Such a method has some disadvantage where there is only a slight correlation between antibiotic formation in plate culture and the antibiotic production in submerged fermentation.
Slide 83 - Selection of Mutants Strains, which produce at high yields when grown on plates, may produce at only low yields or not at all in liquid culture. The procedure is sufficient suitable for differentiation between productivity and non-productivity, such as for detecting the formation of constitutive enzymes.
Slide 84 - Agar Plug Method
Slide 85 - Recombination The genetic information from two genotypes can be brought together into a new genotype through genetic recombination. The disadvantages of genetic recombination are: In most cases, the productivity of the recombinants usually is intermediate between the values of the parent strains?. During strain development process, there is a frequent decline in the increase in yield is observed. This phenomena is overcome by allowing genetic-cross between unfavorable mutant alleles and alleles of one of the parents. Such a procedure is not available during recombination work.
Slide 86 - Recombination High-yielding strains can actually increase the cost of the fermentation because of changed physiological properties (greater foaming, changed requirements for culture medium, etc.). By crossing back to wild-type strains, high-yielding strains with improved fermentation properties may be formed. An effective strain development approach should involve the use of sister-strain, divergent strain, and ancestral crosses at specific intervals, besides use of carefully mutagenesis to ensure the maintenance of genetic variability.
Slide 87 - Regulation Regulation of metabolism is generally so efficient that excess products are not formed. Strain development and the optimization of fermentation conditions lead to a relaxation of regulation in the producing strains. Strains with less efficient regulation can be selected in a screening process. A broad understanding of biosynthesis, the enzymes involved in these processes, and their regulation is necessary for developing a rational approach to the alteration of the regulation of a fermentation process.
Slide 88 - Regulation Microbial metabolism is controlled by the regulation of both enzyme activity and enzyme synthesis. Regulation of enzyme activity: Feedback inhibition: In an unbranched biosynthetic pathway, the end product inhibits the activity of the first enzyme of the pathway, a process called feedback inhibition. A conformation change and hence inactivation (allosteric effect) occurs when an effector (end product) is attached to a specific site of the enzyme (allosteric site). The end product thus inhibits the activity of the enzyme non-competitively.
Slide 89 - Regulation In a branched biosynthetic pathway, feedback inhibition of the first common enzyme by means of one of the end products would cause more than one end product to be affected. In branched biosynthetic pathways, different kinds of feedback inhibition are found: · The end product inhibits the first enzyme in each case after the branch point. The first step in the common synthesis path is catalyzed by several isoenzymes, each of which can be regulated independently.
Slide 90 - Regulation The first common enzyme in a branched biosynthetic pathway is influenced by each end product only slightly or not at all; there must be an excess of all end products for inhibition to occur (a phenomenon called multivalent inhibition). Each end product of a branched pathway acts as an inhibitor; cumulative inhibition is the effect of all the inhibitors. Breakdown of enzymes: Enzymes, which are no longer needed in metabolism, may be broken down through the action of highly specific proteases. As e.g., tryptophan synthetase in S. cerevisiae is broked down at stationary phase.
Slide 91 - Regulation Modification of enzymes: The activity of some enzymes (such as glutamine synthetase in E. coli) is controlled by conformational changes, such as phosphorylation or adenylylation. Regulation of enzyme synthesis: at least three mechanisms have been detected which regulate synthesis of enzymes. Induction: Some enzymes are formed irrespective of the culture medium; such enzymes are called constitutive. Many catabolic enzymes are induced: they are not formed until the substrate to be metabolized is present in the medium. The product of one enzyme can in turn induce the synthesis of another enzyme (sequential induction).
Slide 92 - Regulation Repression: Anabolic enzymes are generally present only when the end product is absent. The excess end product suppresses enzyme synthesis, acting as a co-repressor. Attenuation: It is involved in the biosynthesis of amino acids in bacteria, e.g. histidine - in Salmonella typhimurium, tryptophan - in E. coli (In addition to repressor operator mechanism). In attenuation model, the transcription rate of an operon is regulated by a secondary structure of the leader sequence of the newly transcribed mRNA. The structure of this leader sequence determines whether the RNA polymerase continues the transcription of the operon or a termination occurs.
Slide 93 - Regulation If termination occurred the mRNA transcription ceases and the enzyme or enzymes coded for by that mRNA are not made. In the tryptophan situation, repression has a large effect on enzyme synthesis whereas attenuation has a more subtle, although still important, effect.
Slide 94 - Regulation Excess production of primary metabolites (amino acids, vitamins, purine nucleotides) : This has been accomplished primarily by eliminating feedback inhibition. A) The elimination of end product inhibition or repression is achieved by using auxotrophic mutants that can no longer produce the desired end product due to a block in one of the steps in the pathway. By adding the required end product in low amounts, growth occurs but feedback inhibition is avoided. Excretion of the desired intermediate product thus occurs. Both branched and unbranched pathways can be manipulated in this way.
Slide 95 - Regulation B) A second method is the selection of mutants that are resistant to metabolites. In this case either the enzyme structure is changed so that the corresponding enzyme lacks the allosteric control site, or mutations in the operator or regulator gene (Oc-, R--mutants) result in constitutive enzyme production and thus over production. C) In mutants with a block in an allosterically regulatable enzyme, suppressor mutations can lead to restoration of enzyme activity; however, these enzymes are not allosterically controllable.
Slide 96 - Regulation Regulation and overproduction of secondary metabolites: The methods described above, which were used first for primary metabolites, can be successfully applied to secondary metabolites as well. Production of secondary metabolites is controlled by 5 different classes of genes: 1. Structural genes, which code for enzymes involved in secondary metabolite biosynthesis. 2. Regulatory genes, which control secondary metabolite synthesis. 3. Resistance genes, which keep antibiotic-producing strains immune to their own products
Slide 97 - Regulation 4. Permeability genes, which control the uptake and excretion of substances. 5. Regulatory genes, which control primary metabolism and thus indirectly affect the biosynthesis of secondary metabolites. Many genes are involved in the synthesis of secondary metabolites. 300 genes are involved in chlortetracycline biosynthesis and approximately 2000 genes are directly or indirectly involved in neomycin biosynthesis. In such type of systems, a rational approach to increased yield is possible only in rare cases because there is insufficient data
Slide 98 - Regulation Regulatory mechanisms that affect the products of secondary metabolism: Induction: In batch fermentations with readily metabolizable carbon and nitrogen sources, secondary metabolites are formed primarily after growth has ceased. The logarithmic growth phase is called the trophophase, and the subsequent phase, in which the secondary metabolite may be produced, is called the idiophase. Secondary metabolites are referred to as idiolites. The synthesis of enzymes involved in secondary metabolism is repressed during the trophophase.
Slide 99 - Regulation The composition of the culture medium could be arranged so that a significant fraction of a slowly metabolizable substrate is used, the organism thus growing under sub optimal conditions, leading to a situation where growth and secondary metabolite formation occur in parallel. End-product regulation: antibiotics inhibit their own biosynthesis (e.g. penicillin, chloramphenicol, virginiamycin, ristomycin, cycloheximide, puromycin, fungicidine, candihexin, streptomycin). The mechanism of feedback regulation has only been explained in a few cases:
Slide 100 - Regulation chloramphenicol represses arylamine synthetase, which is the first enzyme in the biosynthetic pathway, which branches off from aromatic biosynthesis to chloramphenicol. With chloramphenicol and penicillin, it has been shown that the concentration of the end product, which inhibits corresponds to the production level. Thus, if strains could be isolated which were less sensitive to end-product inhibition by these antibiotics, they might produce higher yields.
Slide 101 - Regulation Catabolite regulation: Catabolite regulation is a general regulatory mechanism in which a key enzyme involved in a catabolic pathway is repressed; inhibited, or inactivated when a commonly used substrate is added. Substrates, which have been found to bring about catabolite repression, include both carbon and nitrogen sources. Carbon sources: Biosynthesis of different secondary metabolites (antibiotics, gibberellins, ergot alkaloids) is inhibited by rapidly fermentable carbon sources, particularly glucose. The mechanism differes according to the organism and metabolite.
Slide 102 - Regulation A well-known carbon catabolite repression found in many bacteria, yeasts and molds, which involve a catabolite activator protein (CAP) that must combine at the promoter site before RNA polymerase can attach. The CAP will only bind if it is first complexed with cyclic adenosine monophosphate, cyclic AMP. Readily utilizable carbon sources such as glucose stimulate an enzyme, which causes the breakdown cyclic AMP, thus rendering CAP inactive. Thus, glucose inhibits the synthesis of the mRNA for any enzyme requiring CAP for its biosynthesis.
Slide 103 - Regulation Nitrogen sources: In several antibiotic fermentations it has been observed that ammonia or other rapidly utilizable nitrogen sources act as inhibitors. The fundamentals of this regulation have not yet been completely understood, although glutamine synthetase and glutamic dehydrogenase are considered key enzymes. In enteric bacteria it has been established that glutamine synthetase has a regulatory function in the synthesis of additional enzymes, which are involved in nitrogen assimilation.
Slide 104 - Regulation Phosphate regulation: In a culture medium inorganic phosphate (Pi) is required within a range of 0.3-300 mM for the growth of prokaryotes and eucaryotes. A much lower phosphate concentration inhibits the production of many secondary metabolites. In a number of systems studies, the highest Pi concentration, which allows unimpeded production of secondary metabolites, is about 1 mM; complete inhibition of production occurs at about 10 mM Pi. Phosphate regulation has been observed in the production of alkaloids, gibberellins and particularly in several antibiotics.
Slide 105 - Regulation The phosphate regulation mechanism is not yet fully understood. Pi controls the metabolic pathways, which precede the first stage of secondary metabolite formation, but also affects the biosynthesis of secondary metabolites themselves. It has been shown that phosphate restricts the induction of secondary metabolite production. For instance, dimethyl allultryptophan synthetase, the first specific enzyme of ergot alkaloid biosynthesis, is not produced in the presence of high Pi concentrations.
Slide 106 - Regulation Auto regulation: In some actinomycetes it has been possible to show that differentiation and secondary metabolism are subjected to a type of “self-regulation” from low-molecular weight substances. For instance, in Streptomyces griseus and S. bikiniensis the formation of streptomycin, the development of streptomycin resistance, and spore-formation are all affected by factor A, a substance produced by the streptomyces themselves. It has been shown that the streptomycin resistance property is due to the increased transcription of the gene for the enzyme, streptomycin phosphotransferase, induced by the factor A.
Slide 107 - Regulation The effect on streptomycin formation is thought to be due to a shift in the metabolism of the carbohydrate source: although the activity of the enzyme glucose-6-phosphate dehydrogenase is high in factor A-deficient mutants, this enzyme cannot be demonstrated in high-yielding strains. Addition of factor A to mutants leads to a strong decrease in enzyme activity. It is assumed that when the pentose phosphate cycle is blocked through the absence of glucose-6-phosphate dehydrogenase, glucose is channeled into pathways involved in the formation of streptomycin units.
Slide 108 - Regulation In a sense, factor A can be considered analogous to a hormone. Auto-regulatory mechanisms similar to that of factor A have been found in other actinomycetes. For instance, a factor is hypothesized in S. virginiae, which stimulates the formation of the antibiotic virginiamycin. In rifamycin-producing Nocardia mediterranei butyryl phospho-adenosine has been characterized as a regulatory factor. Two g-lactones (L factors) have been shown to be auto-regulatory agents in leukaemomycin producing S. griseus.
Slide 109 - Gene Technology Gene technology includes in vitro recombination, gene cloning, gene manipulation, and genetic engineering. Gene technology permits introduction of specific DNA sequences into prokaryotic or eucaryotic organisms and the replication of these sequences; that is, to clone them. To carry out these procedures, the following steps are necessary: The DNA sequence to be cloned must be available. The sequence must be incorporated into a vector. The vector with the DNA insert must be introduced by transformation into a host cell, where the vector must replicate the insert in a stable manner. The clone, which contains the foreign DNA, must be selectable in some manner.
Slide 110 - Isolation of DNA Sequences for Cloning Genome fragments: Restriction endonucleases are used to cut DNA. Endonucleses belong to specific restriction and modification systems and are used by the cell to protect itself from foreign DNA. They split double-stranded DNA at specific sites, 4-11 nucleotides in length. More than 600 of those enzymes are known in bacteria. If the sequence of the DNA to be cloned is unknown, it is possible to use a so-called “shot-gun” approach.
Slide 111 - Isolation of DNA Sequences for Cloning With this procedure, a gene bank is produced by using suitable restriction enzymes to fragment the total genome of the organism into pieces of about 20 kilo bases in length. The DNA fragments is linked to a vector (generally a phage or cosmid) and cloned into a suitable host. By applying screening methods the cloned organism could be then isolated. It is preferable to carry out the initial cloning with enriched DNA fragments. Enrichment is done by use of sucrose gradient centrifugation, agarose-gel electrophoresis, column chromatography, or by use of specific gene probes.
Slide 112 - Isolation of DNA sequences Synthetic DNA: In order to produce a specific DNA fragment containing the coding region of a protein, the DNA sequence is deduced by “reverse translation” from the amino acid sequence of this protein. Automated DNA synthetic machine can be used for production of DNA fragments of 20-100 bases, which can be connected together to make longer sequences. Example of the use of this technique are the artificial synthesis of the gene for somatostatin, a peptide hormone with 14 amino acid residues and the synthesis of the A and B chains of insulin, which were cloned and expressed in E. coli.
Slide 113 - Isolation of DNA sequences It is also possible to produce sequences in which one or more bases have been changed, making possible the production of highly specific mutations. Production of complementary DNA (cDNA): Specific mRNA molecules, are used as templates in vitro with the enzyme reverse transcriptase, to produce complementary DNA. Analysis of recombinant clones: To select transformed cells, the marker inactivation technique can be used.
Slide 114 - Isolation of DNA fragments Vectors are used containing two selectable markers (for instance, antibiotic resistance) one of which contains the recognition site for restriction enzyme used in the cloning process. If the foreign DNA becomes integrated into this antibiotic resistance gene, the activity of that gene is lost (insertional inactivation). Host cells that lack the vector are sensitive to both antibiotics, host cells containing a vector lacking the foreign DNA are resistant to both antibiotics, whereas vectors with inserted foreign DNA are sensitive to the one antibiotic into whose resistance gene the foreign DNA has been inserted.
Slide 115 - Colony hybridization (Colony Hybridization is the screening of a library with a labeled probe (radioactive, bioluminescent, etc.) to identify a specific sequence of DNA, RNA, enzyme, protein, or antibody). and Southern blotting (DNA blot) are used for detection of cloned DNA in the cell. A different procedure for detecting the cloned DNA involves seeking for expression of the cloned DNA. Since the expression efficiency is often quite low, a sensitive method is applied, e. g. using immunological methods, in which an antibody (marked by radioactivity or enzyme) is used as a probe. Isolation of DNA fragments
Slide 116 - Production of Recombinant DNA
Slide 117 - Use of genetic methods High-yielding strains can be produced by: · Isolation of mutants resistant to inhibitors of protein synthesis, which often overproduced proteins; · Manipulation of regulatory signals to increase transcription or translation by cloning the gene on an expression vector or inserting the gene into a transposon which has a strong promoter; · Modification of the gene by use of site-directed mutagenesis.
Slide 118 - Use of genetic methods The yield may be increased, by increasing the gene dosage (gene amplification), which can be done by: · Increasing the number of DNA replication sites in growing bacterial cells causes amplification of the genes situated near the origin of replication. · Diploidization of fungi increases gene dosage, although the strains are usually unstable. · Isolation of hyper induced strains, which have been cultivated under selective conditions over a long period. These strains are extremely unstable, however, and are usually not suitable for commercial processes.
Slide 119 - Use of genetic methods The greatest success is likely by use of genetic engineering methods, for example, cloning and amplification of the gene by means of a multicopy plasmid or a phage vector. For instance, by use of a cosmid system the formation of the enzyme penicillin acylase in E. coli has been markedly increased when compared to the wild type. A whole series of industrial enzymes have been optimized in this way. Difficulties faced the goal to increase in yield of a multi-gene product such as a primary or secondary metabolite, although some successes have been achieved. Amino acid production has been increased by cloning the whole genome, first in E. coli, later in production strains such as Corynebacterium, Brevibacterium, or Serratia..
Slide 120 - Use of genetic methods For secondary metabolites such as antibiotics, cloning and amplification of the rate-limiting enzyme of the biosynthetic pathway can be done. As a first step in this direction, the genes for a number of antibiotics have been isolated, cloned, and in few cases expressed. These include actinorhodin, methylenomycin, and undecylprodigiosin (Streptomyces coelicolor), cephalosporin (Cephalosporium acremonium), erythromycin (S. erythreus), oxytetracylcine (S. glaucescens) and tylosin (S. fradiae).
Slide 121 - Stability of the Strain An important consideration in strain improvement is the stability of the strain. An important aspect of this is the means of preservation and storage of stock cultures so that their carefully selected attributes are not lost. This may involve storage in liquid nitrogen or lyophilization. Strains transformed by plasmids must be maintained under continual selection to ensure that plasmid stability is retained. Instability may result from deletion and rearrangements of recombinant plasmids, which is referred to as structural instability, or complete loss of a plasmid, termed segregational stability.
Slide 122 - Stability of the Strain Some of these problems can be overcome by careful construction of the plasmid and the placement of essential genes within it. Segregational instability can also be overcome by constructing so-called suicidal strains that require specific markers on the plasmid for survival. Consequently, plasmid-free cells die and do not accumulate in the culture. These strains are constructed with a lethal marker in the chromosome and a repressor of this marker is located on the plasmid. Cells express the repressor as long as they possess the plasmid, but if it is lost the cells express the lethal gene. However, integration of a gene into the chromosome is normally the best solution, as it overcomes many of these instability problems.