3.4.5.6 bio

• Microbial biotechnology


  Exploitation, genetic manipulation and alterations of micro-organisms to make commercially valuable products, involving fermentation and various upstream and downstream processes
• Microbial products


  • Macromolecules (e.g. proteins, nucleic acids, carbohydrate polymers, even cells)
  • Smaller molecules
• Primary metabolites


  Metabolites essential for vegetative growth
• Secondary metabolites


  Metabolites that give advantages over adverse environment
• Recombinant DNA technology


  Also referred to as gene cloning or in vitro genetic manipulation, dramatically broadened the spectrum of microbial genetic manipulations
• Basic steps in DNA cloning

  1. A fragment of DNA is inserted into a carrier DNA molecule, called a vector, to produce a recombinant DNA

  2. The recombinant DNA is then introduced into a host cell, where it can multiply and produce numerous copies of itself within the host

  3. Further amplification of the recombinant DNA is achieved when the host cell divides, carrying the recombinant DNA in their progenies, where further vector replication can occur

  4. After a large number of divisions and replications, a colony or clone of identical host cell is produced, carrying one or more copies of the recombinant DNA

  5. The colony carrying the recombinant DNA of interest is then identified, isolated, analyzed sub-cultured and maintained as a recombinant strain
• Traditional method of strain improvement


  Mutation and screening for higher producing microbial strains
• Targeted mutagenesis


  Introduction of mutations at a specific location in DNA
• Classical genetics


  Backcrossing of overproducing strains with parent strains to improve the vigor of mutant strains, parasexual cycle, protoplast fusion
• Rational selection


  Selecting an improved producer out of a very large population of progeny
• Cloning of the candidate genes


  Using recombinant DNA technology to introduce genes coding for antibiotic synthetases into producers of other antibiotics or into non-producing strains to obtain modified or hybrid antibiotics
• Microbial cell-surface display


  Allows proteins, peptides and other bio macro molecules to be displayed on the surface of microbial cells by fusing them with the anchoring motifs
• Potential applications of genetic manipulation of micro-organisms


  • Production of pharmaceuticals, neutraceuticals
  • Development of diagnostics to detect disease-causing organisms and monitor the safety of food and water quality
  • Genetically modified microorganisms as living sensors to detect chemicals in soil, air or other inorganic or biological specimens
  • Alterations in the genome of the bacterium Deinococcus radiodurans to increase its potential in cleaning up toxic-waste sites
• Investigators are developing systems for identifying pathogens that may be used as biological weapons by rogue nations or even terrorist groups in future
• Bacteria can be genetically altered


  1. Emit a green fluorescent protein visible in ultraviolet light when they metabolize the explosive TNT leaking from land mines

  2. Applied to a tract of land with a crop duster and then be analyzed from a helicopter

  3. Used as a living sensor to detect any particular chemicals in soil, air or other inorganic or biological specimens
• In Microbial Genome Program, alterations in the genome of the bacterium Deinococcus radiodurans are performed to increase its potential in cleaning up toxic-waste sites
• Deinococcus radiodurans


  Microbe with extraordinary DNA-repair processes that enable it to thrive in high-radiation exposed environments
• Using various biotechnological processes


  Genes can be added from other organisms that will confer the ability to degrade toxinogenic chemicals such as toluene, commonly found in chemical and radiation waste sites
• Genetic manipulation of biosynthetic pathways is a useful method for producing analogs of complex bioactive metabolites
• Reconstruction of biosynthetic gene clusters in E. coli could be done for rapid heterologous production of natural products and genetic manipulation of their biosynthetic pathways
• Recombinant technology


  The real challenge lies in the suitable expression of proteins in recombinant microbe system. To obtain enzymes, antibiotics in its native, functional folded structure is the ultimate bottleneck
• Antibiotics


  Small molecular weight compounds that inhibit or kill microorganisms at low concentrations
• Antibiotics are produced by various bacteria, actinomycetes and fungi such as Bacillus, Streptomyces, and Penicillium
• Significance of antibiotic production in microorganisms


  May be for ecological adaptation for the organism in nature
• Engineering Escherichia Coli to Produce Non-Ribosomal Peptide Antibiotics


  1. A monocistronic reconstituted form of the ecm gene cluster from Streptomyces lasaliensis was cloned and expressed in E. coli that directs the biosynthesis of the anti-tumor non-ribosomal peptide echinomycin

  2. Biosynthetic gene function was examined by constructing a set of expression plasmids containing only 15 of the 16 genes required for echinomycin production. The ecm18 gene cassette encoding a methyltransferase was omitted from one of the plasmids

  3. Expression of the reconstructed cluster in E. coli results in production of echinomycin, whereas expression of the reconstructed cluster minus the ecm18 gene results in production of triostin A

  4. Ecm18 functions as a S-adenosylmethionine dependent methyltransferases (SAM-MTs) that catalyses the conversion of triostin A to echinomycin in vitro using the purified recombinant enzyme
• Metabolic engineering


  • Considers metabolic and cellular system as an entity and accordingly allows manipulation of the system with consideration of the efficiency of the overall bioprocess, which distinguishes itself from simple genetic engineering
  • Allows defined engineering of the cell, thus avoiding unnecessary changes to the cell and allowing further engineering if necessary
• Various synthetic drug derivatives, their host and modification strategy


  • A novel amidated polyketide - Streptomyces coelicolor, heterologous co-expression of amidotransferase OxyD with minimal oxytetracycline polyketide synthase
  • Clavulanic acid - Streptomyces clavuligerus, knockout of gap1 and gap2 and addition of arginine in the medium
  • Daptomycin - Streptomyces lividans, heterologous production, inactivation of actinorhodin, and optimization of the medium
  • Daptomycin derivatives - Streptomyces roseosporus, use of recombination to exchange single or several modules in the subunit of the non-ribosomal peptide synthase
  • Erythromycin A - Saccharopolyspora erythraea, overexpression of eryK and eryG with copy number ratio of 3:2
  • Macrolide 6-deoxyerythromycin D - Escherichia coli, heterologous production and several generations of activity-based screening assay for further evolution
• Enzymes


  Produced by microorganisms, may be intracellular or secreted into the extracellular medium
• Types of commercially important enzymes


  • Enzymes of industrial importance: Amylases, Proteases, Chymosin, Catalases, Isomerases, recombinant Lipases
  • Enzymes used for analytical purposes: Glucose oxidase, Alcohol dehydrogenase, Hexokinase, Cholesterol oxidases, Horseradish peroxidase, Alkaline phosphatase
  • Enzymes of medicinal importance: Trypsin, Asparaginase, Proteases, Lipases
• Industrial enzymes have now reached an annual market of US$1.6 billion. Recombinant therapeutic enzymes already have a market value of over US$2 billion
• Recombinant DNA techniques


  Used to engineer microorganisms to over-express the desired enzymes, including heterologous enzymes derived from other species
• Genetic engineering


  1. Could be used to give ethanol fermenting microbes the metabolic pathways needed to utilize sugar sources such as the 5-carbon xyloses and other pentoses that are released from hydrolysis of woody biomass

  2. Splice genes encoding the enzymes making up the ethanol fermentation pathway into an organism lacking that trait but having the ability to digest the complex cellulosic components

  3. Create and use engineered microbes to manufacture novel or improved industrial enzymes, which can then be used as a catalyst in fuel fermentations to enhance or accelerate biofuel production processes
• Calf chymosin (prochymosin) was cloned and expressed in E. coli (first genetically engineered protein approved for human consumption, 1990)
• Synthetic biology and metabolic engineering


  Used to create novel or synthetic microorganisms possessing enzymatic capabilities not found in the original host organism
• Industrially important enzymes produced in recombinant microorganisms


  • Phytase, Chymosin, Lipase, Pectinase, Alpha-Amylases, Glucose oxidases, Alcohol dehydrogenase, Cholesterol oxidases, Horseradish peroxidase, Alkaline phosphatases, Trypsin, Asparaginase
• Genetic engineering in Methylophilus methylotrophus


  1. The glutamate dehydrogenase gene of E. coli has been cloned into broad host range plasmid and can complement glutamate synthase mutants of Methylophilus methylotrophus

  2. Assimilation of ammonia via glutamate dehydrogenase is more energy efficient than via glutamate synthase, allowing the recombinant microorganism to convert more growth-substrate, methanol into cellular carbon

  3. Trans-conjugants were selected for the antibiotic resistance encoded by the vector, and the GDH enzyme activity measured to confirm the presence of the GDH gene

  4. The strain constructed was able to convert methanol to single-cell protein more efficiently than the original parent strain
• dehydrogenase


  More energy efficient than via glutamate synthase
• The recombinant microorganism can convert more growth-substrate, methanol into cellular carbon
• Trans-conjugants selection


  1. Selected for the antibiotic resistance encoded by the vector

  2. GDH enzyme activity measured to confirm the presence of the GDH gene
• The strain constructed by these manipulations was able to convert methanol to single-cell protein more efficiently than the original parent strain