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

Factor VIII Factor IX Tissue plasminogen activator (TPA) Insulin Human growth hormone Erythropoietin DNase I Various interferons (IFN)

Company

Baxter, Bayer Genetics Institute Genetech Eli Lilly, Novo Nordisk Eli Lilly, Genetech, Upjohn, Novo Nordisk Amgen, Ortho Biotech Genetech Schering, Biogen, Chiron, Genetech

Disorder

Hemophilia A Hemophilia B Acute myocardial infarction Diabetes mellitus GH deficiency in children (dwarfism) Anemia Cystic fibrosis 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

Bioadsorbent Passenger protein (red) Carrier protein (black) Oral vaccines Antibody production 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 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