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Industrial Microorganism (Lecture 3)
This document discusses strain improvement strategies for developing microorganisms used in industrial fermentation. It describes obtaining strains from environmental screening or culture collections, and then improving strains through natural recombination, mutagenesis, or recombinant DNA techniques. The key strategies discussed are natural genetic exchange between microbes, physical and chemical mutagenesis to generate random mutations, and directed genetic engineering using recombinant DNA technology to develop strains with desired properties like high product yields or utilization of low-cost substrates. The goal is to obtain stable, high-producing industrial microorganisms.
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Industrial Microorganism (Lecture 3)
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- 2. Raw material Microbialstrain Fermentation Product purification ProductEffluent wastes Upstream processing Downstream processing
- 3. Why only microorganismsfor fermentation • Ease of their mass cultivation • Speed of growth • Use of cheap substrate as raw material • Diversity of potential products • Ability to readily undergo genetic manipulations
- 4. Traditional fermentationsused mixed cultures Wild organisms present in the raw materials e.g some food and alcoholic fermentations Pure cultures in fermentations (just 120 years)
- 5. processes inthe first 80 years of 20th century used monocultures Usually obtained from natural environments Processes in the last 20 years used recombinant organisms
- 6. Examples of industrialproducts and their producers
- 7. Products Bacteria Yeastand Fungi Traditional Products Bread, Beer, Wine - Saccharomyces cerevisiae Chees and dairy products Lactic acid Bacteria - Fermented meat, vegitables, Mushrooms Lactic acid Bacteria Agaricus bisporus Vinegar Acetobacter Sp.
- 8. Products Bacteria Yeastand Fungi Agricultural Products Gibberellins Fungicides Insecticides Silage Bacillus thuringenesis Fusarium moniliforme Amino Acids L-Glutamine L-Lysine L-Tryptophan Corynebacterium glutamicum
- 9. Products Bacteria Yeastand Fungi Enzymes Amylases Cellulases Lipases Proteases Bacillus subtilus Streptomyces Bacillus lichniformis Aspergillus niger Kluyveromyces Trichoderma Fuels and Chemicals Acetone Butanol Ethanol Glycerol Methane Clostridium Zymomonas Methanogenic archeans Saccaharomyces cervisiae
- 10. Products Bacteria Yeastand Fungi Organic acids Acetic acid Citric acid Fumaric acid Lactic acid Acetobacter Xylimon Lactobacillus delbrueckii Aspergillus niger Rhizopus Pharmaceuticals Penicillins Cephalosporins Aminoglycosides Macrolides Tetracyclines Polyenens Streptomyces Streptomyces Penicillum Cephalospora
- 11. How to obtainthese industrial organisms There are two sources To screen from the environment To obtain from culture collections
- 12. Isolation from theenvironment There may be two approaches Shotgun approach Objective or targeted screening
- 13. Isolation from environment Inshotgun approach random sampling is done Biofilms or microbial communities from Animal or plant material Sewage, water streams For targeted screening Sampling from more specific sites e.g. Isolation of the organism for degradation or detoxification
- 14. Further steps Selectionof appropriate media Enrichment methods Selection of the strains Purification of the strains Screening for specific activity Screening is good to get one organism but not for a consortia
- 15. Culture collections Goodsource of industrial organisms About 500 culture collection world wide Provide organisms by special agreements UKNCC (made up of several collection, housed in separate institutions) ATCC (centralized collection, holds all types of organisms)
- 16. Prime functions ofa culture collection To maintain the existing collection To collect new useful strains To provide authentic industrial culture
- 17. Culture collections Problems ofculture maintenance can be overcome by Cryopreservation, lypholization Adsorption of cells to glass beads Advantages Getting a required organism is cost effective and time saving Some characterization has already been done Disadvantages Competitors have the same excess to organism
- 18. Examples of theworlds famous culture collections
- 20. STRAIN DEVELOPMENT Regulatory considerationsare of major importance Preference is given to (GRAS) microorganisms Particularly in case of food products Regulations for a new organism are more stringent Regulations for pathogen and GMMs (costly processes) Some time crippled strains are used (cannot grow outside the fermentor)
- 22. Strain development Characteristics ofa good industrial microorganism (expectations) Genetic stability Efficient production of target Compound (pathway characterized) Limited need for additional factors Utilization of low cost C sources Amenability to Genetic manipulations Non pathogenic Easy to harvest Easy for cell disruption Production of limited by products (Reduce Production Cost)
- 23. Targets for strainImprovements
- 24. Strain development Strategies forstrain improvement Natural recombination Mutagenesis Recombinant DNA technology Conventional techniques New techniques
- 25. Strain development Natural recombination BacterialDNA consists of 1- a single chromosome 2- plasmids (autonomous, carries few hundred additional genes, as many as 1000 copies) Exchange of genetic material by three means Conjugation Cell to cell contact Filamentous protein structure (sex pilus)
- 26. Strain development Transduction • Bacteriophagesact as a vector • Bacteriophages can also acquire transposones and can transfer to the new hosts Transformation • Uptake of naked piece of DNA from the surrounding • The cells should be competent In eukaryotes • Recombination occurs by sexual reproduction • Parasexuality in fungi (heterokarion)
- 27. Mutagenesis Physical change tothe DNA of a cell e.g. • Deletions • Insertions • Duplications • Inversions • Organisms are subjected to repeated mutagenesis and screening • Mutation can arise naturally or • may be induced
- 28. Strain development Mutagens (thereare two types) Physical mutagens • UV radiations • Gamma radiations • X-rays etc Chemical mutagens • Ethane Methane Sulphonates (EMS) • Nitrosomethyl Guanidine (NTG) • Nitrous acid, acridine, SDS etc
- 29. Strain development • Mutagenesishave limited application • It’s a random method not specific Site directed mutagenesis May be more useful But ………. More knowledge about the gene e.g. its exact location, size, product etc should be known
- 30. Selection of Mutants Thereare basically two methods of mutants selection Random Screening Selective isolation of mutants
- 31. Strain development Genetic engineering(recombinant DNA technology) • New industrial products e.g. • Insulin • Interferons • Monoclonal antibodies (by fusion of cells) • Enzymes • Advantages (better expression systems) • Strategies ………………………………………