Transcript Biotechnology and Genetic Engineering

Chapter 5: Microbial Biotechnology
•Genetic engineering of microbes
•Human pharmaceutical products
•Antibiotics
•Biopolymers
•Bioconversions
•Microbial Cell-Surface Display
•Agriculture
•Bioremediation
•Oil & Mineral Recovery
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
Expressing a foreign protein in a microbe
Bacterial Gene Promoter/Switch
or cDNA
Some recombinant proteins approved for
human use ($50 billion-2008)
Protein
Company
Disorder
Factor VIII
Baxter, Bayer
Hemophilia A
Factor IX
Genetics Institute
Hemophilia B
Tissue plasminogen
activator (TPA)
Genetech
Acute myocardial
infarction
Insulin
Eli Lilly, Novo Nordisk
Diabetes mellitus
Human growth
hormone
Eli Lilly, Genetech, Upjohn,
Novo Nordisk
GH deficiency in children
(dwarfism)
Erythropoietin
Amgen, Ortho Biotech
Anemia
DNase I
Genetech
Cystic fibrosis
Various interferons
(IFN)
Schering, Biogen, Chiron,
Genetech
Hepatitis B and C,
multiple sclerosis
Production of antibiotics
• Antibiotics, novel antibiotics and polyketide antibiotics
• Antibiotics are small metabolites with antimicrobial
activity that are produced by Gram-positive and Gramnegative 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. coli
Blaine A. Pfeifer, Suzanne J. Admiraal, Hugo Gramajo,
David E. Cane, and Chaitan Khosla
Science Mar 2 2001: 1790-1792.
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
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
Microbial Cell-Surface Display
Bioadsorbent
Passenger protein (red)
Carrier protein (black)
Antibody
production
Oral vaccines
Cytosol
Screening peptide
libraries
Mutation detection
Bioconversions
Biosensors
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 2°C
• When the ice gene is deleted from the bacteria,
ice crystal formation (frost damage) does not
occur until -6 to -8°C
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
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
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
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., mercurysee 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: 2442–2445.]
•
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