Chapter 5: Microbial Biotechnology

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Chapter 5 Microbial Biotechnology

• Genetic engineering of microbes
• Human pharmaceutical products
• Antibiotics
• Biopolymers
• Bioconversions
• Microbial Cell-Surface Display
• Agriculture
• Bioremediation
• Oil Mineral Recovery

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Producing a foreign protein in a microbe

• Identify the gene you wish to express in the
  microbe (is it prokaryotic or eukaryotic?)
• Make sure no introns are present (cDNA?)
• Attach the gene (or cDNA) to an appropriate
  microbial promoter and add a Shine-Delgarno
  sequence (ribosome binding sequence)
• Add an appropriate transcriptional termination
  sequence at the 3 end of the gene
• Introduce the engineered gene in an appropriate
  vector into the microbe

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Expressing a foreign protein in a microbe
Bacterial Gene Promoter/Switch
or cDNA
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Some recombinant proteins approved for human use
(50 billion-2008)
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Production of antibiotics

• Antibiotics, novel antibiotics and polyketide
  antibiotics
• Antibiotics are small metabolites with
  antimicrobial activity that are produced by
  Gram-positive and Gram-negative bacteria as well
  as by fungi
• Antibiotics act by 1) disrupting the plasma
  membranes of microbes, 2) by inhibiting cell wall
  synthesis or 3) by inhibiting the synthesis of of
  metabolites such as proteins, nucleic acid and
  folic acid
• See Biosynthesis of Complex Polyketides in a
  Metabolically Engineered Strain of E. coliBlaine
  A. Pfeifer, Suzanne J. Admiraal, Hugo Gramajo,
  David E. Cane, and Chaitan KhoslaScience Mar 2
  2001 1790-1792.

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Production of biopolymers

• Production of biodegradable plastics (PHAs),
  spider silk and adhesives from barnacles
• See Biochemistry 1995,34, 10879-10885 10879
  Construction, Cloning, and Expression of
  Synthetic Genes Encoding Spider Dragline Silk by
  John T. Prince, Kevin P. McGrath, J Carla M.
  DiGirolamo, and David L. Kaplana

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Biopolymers are great products for recombinant
microbes

• Animal adhesive proteins (from the blue mussel)
• Rubber (from the rubber plant Hevea brasiliensis)
• Biodegradable plastics (polyhydroxyalkanoates or
  PHAs)
• Note that in all of these cases, one needs to
  clone the genes encoding enzymes in order to
  create or alter a biochemical pathway

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Microbial Cell-Surface Display
Bioadsorbent
Passenger protein (red) Carrier protein (black)
Oral vaccines
Screening peptide libraries
Antibody production
Mutation detection
Bioconversions
Biosensors
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Microorganisms and Agriculture

• Ice-nucleating bacteria story
• Plant frost damage is caused by the presence of
  ice-nucleating bacteria (Pseudomonas, Erwinia,
  Xanthomonas) on plants
• The ice gene on the bacterial chromosome encodes
  an ice-nucleating protein which allows for ice
  crystal formation at 0 to 2C
• When the ice gene is deleted from the bacteria,
  ice crystal formation (frost damage) does not
  occur until -6 to -8C

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Microbes and Agriculture

• The Bt toxin story
• B. thuringiensis is a soil bacterium that
  produces a toxin (Bt toxin or Cry) that kills
  certain insects
• The Bt toxin or Cry is produced when the bacteria
  sporulates and is present in the parasporal
  crystal
• Several different strains and subspecies of B.
  thuringiensis exist and produce different toxins
  that kill specific insects

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The Cry protein mode of action

• The Cry protein is made as an inactive protoxin
• Conversion of the protoxin (e.g., 130 kDa) into
  the active toxin (e.g., 68 kDa) requires the
  combination of a slightly alkaline pH (7.5-8) and
  the action of a specific protease(s) found in the
  insect gut
• The active toxin binds to protein receptors on
  the insect gut epithelial cell membrane
• The toxin forms an ion channel between the cell
  cytoplasm and the external environment, leading
  to loss of cellular ATP and insect death

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Isolation genetic engineering of Cry genes

• The Cry (or protoxin) genes are encoded by
  plasmid DNA, not by chromosomal DNA in B.
  thuringiensis
• Cry genes were expressed in B. thuringiensis
  under the control of the ptet promoter (rather
  than its sporulation-specific promoter) and
  provided increase yield
• Constructs have also been produced to enhance
  toxin action and/or expand its specificity

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Bioremediation

• The process of cleaning up contaminated sites
  using microorganisms to remove or degrade toxic
  wastes or pollutants
• Can encourage natural microbe populations or add
  genetically engineered microbes
• Oil spills, toxic chemicals, heavy metals e.g.,
  mercury- see S. Chen and D. B. Wilson (1997)
  Construction and characterization of Escherichia
  coli genetically engineered for bioremediation of
  Hg2-contaminated environments. APPLIED AND
  ENVIRONMENTAL MICROBIOLOGY 63 24422445.

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Oil and Mineral Recovery

• Oil recovery (MEOR-microbial enhanced oil
  recovery)-secreted polysaccharides loosen oil
  from rocks
• Metal extraction (biomining)-nickel, cooper,
  zinc, colbalt, lead, cadmium,gold can stick to
  the negatively charged or anionic bacterial cell
  surfaces loaded with polysaccharides