Biotechnology and Genetic Engineering-PBIO 450/550

igocheddarBiotechnology

Dec 14, 2012 (4 years and 11 months ago)

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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

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

or cDNA

Bacterial Gene Promoter/Switch

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 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.

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

Cytosol

Bioadsorbent

Oral vaccines

Screening peptide

libraries

Mutation detection

Biosensors

Bioconversions

Antibody

production

Passenger protein (red)

Carrier protein (black)

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 p
tet

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., 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: 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