Final Review- Microbial Biotechnology
Microbial Genome Project- Pathogen Genome
Prevention
 1918- Spanish flu killed 50 to 100 Million, and half of WW! Soldiers
 Vaccines- part of a pathogen or whole organism that can be given to humans or animals by mouth or injection to
stimulate an immune response against those pathogens
 Subunit Vaccines- part of the microbe
o purified from pathogen
o recombinantly produced
 Attenuated Vaccine- weakened microbe
o 1796 Edward Jenner created 1st vaccine – cowpox
 Inactivated vaccine- killed microbe
 Bacteriophage T4 used against anthrax and the plague, antigen is fused to capsid protein
Immune system & Antibodies
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Antigens- foreign substances that stimulate an immune response (bacterim,fungus,virus,protein,lipid,carbs)
Responds with producing antibodies (antibody-mediated immunity)
o B-cells and T-cells recognize and bind antigen
o B-cells develop to make plasma cells that produce antibody
Antibody Specificity
o Can neutralize antigen or antigen bearing microbe
o Immune cells can recognize antigens coated with antibodies and eat them
o Specificity and affinity can be optimized with protein engineering
Antibody Structure
 Have 2 light and 2 heavy chains
 Have constant and viable regions
 Can tag bacteria or toxins for destruction
Diagnosis
 Disease diagnosis
 Molecular beacons- PCR amplification and fluorescently labeled probe that emit signal wen bound to target. Can detect
as few as 2 cells
 Has central loop region that binds target DNA/RNA
 Involved PCR step to amplify target DNA sequence
 Beacon fluoresces when loop binds to target sequence
 Beacon consists of complementary base pairing nucleotides that can form stem structure
 Food poisoning and outbreaks: track speed and origin
 Bioterrorism- use of biological materials as weapons to harm humans or the plants and animals the depend on
o Microbial forensics- deals with tracing sources of possible terrorist attacks that use microbes as bioweapons.
(Anthrax)
o Potential weapons
 Brucella – infect livestock
 Bascilla anthracis – Anthrax. Skin lesions and pneumonia
 Clostridium botulinum- Botulism. Muscular paralysis
 Rikettsiae- Rocky mountain Fever, Tyhpus
 Yersinia pestis – black plague
 Viruses: Ebola, Marbug, Influenza, Variola (smallpox)
Treatment
 Antibiotics- produced from bacteria or fungus that inhibit other bacterial growth
o 1928- Alexander Flemming discovers penicillin from a fungus and it is effective against S. areus
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 Chemical structure of penicillin is related to ampicillin
 Isolated from the filamentous fungus penicillium notatum
o Methods of inhibition
 Inhibit cell wall synthesis
 Inhibit protein synthesis
 Inhibit nucleic acid replication & transcription
 Inhibit enzymatic activity needed for cell metabolism
o Neosporin
 Has neomycin that interferes with transcription
 OTC- no doctor’s prescription required
 Contains polymyxin B that interferes with plasma membrane integrity
 Contains bacitracin which interferes with peptidoglycan synthesis
Polymyxin B- antibiotic to Gram negative- inserts into cell membrane to make porous
Small molecule antibiotics
o Inhibit peptidoglycan synthesis: B-lactams, Bacitracin
o Inhibit DA replication
 Trimethoprim and methotrexate- nt synthesis
 5-Bromouracin- replaces T – replication errors
 Ciprofloxacin binds DNA Gyrase
o Transcription inhibitor: Acinomysin D, Rifampicin
o Translation inhibitors: macrolides, aminoglycosides, tetracyclines, chloramphenicol
Superbug- bacterium with broad resistance to current antibiotics
o 60% healthcare infections
o MRSA- Methicillin Resistant S. aureus
o ESKAPE pathogens
 Not salmonella
o Caused by misuse and overuse of antibiotics
Antibodies neutralize microbes or toxins
o Antitoxins- polyclonal, purified from serum of immunized animal (diphtheria, tetanus)
o Monoclonal- raxibacumab
Drugs
o Anticancer: Doxorubicin, Bleomycin, Taxol
 Taxol: produced by fungi kills cancers by interfering with microtubule disassembly
o Immunosuppresses (organ rejection): Rapamysin, Cycloporin A
o Hypercholesterolemic
 Statins- drug class to lower cholesterol by inhibiting the enzyme HMG-CoA reductase, the rate limiting
enzyme
o Bacterial Proteins: BOTOX
 Neurotoxin- blocks release of neurotransmitter at muscle-nerve junction
 Approved for treating migraines, comes from Clostridium
 1st microbiocidal toxin used as treatment
 Hyperhidrosis (excessive seating), overactive bladder, lazy eye, facial spasm
Recombinant Microbial Products
Research Agent
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Drug discovery and structured based design
Pharmacokinetics- what the body does to drug (absorb, excrete, metabolize, distribute)
Antigens for capture reagent of affinity based protein purification (protein A binds to IgG) and antibody screening
o Protein A comes from S. areus which is used in chromatography for antibody purification
Biologic Drug Product
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1978- Humulin produced in E. coli was the 1st recombinant drug approved
o Controls blood glucose levels
o That from pig or cow pancreases had different aa and not always effective or got rejected.
Preproinuslin in ER of pancreatic cells
o First 24 aa are signal sequence for secretion
o Sequence cleaved in ER and proinsulin goes to GA for further processing
 Lispro- 1996-lysine and proline reversed (fast acting)
 Glargine- 2001- aspartic acid replaced proline (longer lasting)
 Detemir- fatty acid at lysine (longer lasting)
Immunotoxins
o Antibody fragments fused with peptide toxins (diphtheria, exotoxin A)
 Enzymes that catalyze ADP-ribosylation or EF2
 Toxins with native receptor binding domain deleted and replaced with targeting domain
o Denileukin diftitox: cancer immunotoxin
Antibody portions
o Hybridoma- antibody producing B-cell from mouse immunized with antigen are fused with immortal cell line
o Antibody display libraries- scFU library
 Clone and express antibodies with CHO
Gene Editing
Defects in genes can be caused by me
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Overexpression/weakly expressed genes
Overactive/inactive proteins
Medical Uses
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Knock Out- inactivate gene that causes overexpression of disease
Knock In- gene therapy, insert functional gene in place of defective gene
Tools
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Generation of gene knockouts in animal models if disease
Knock in GOI in mammalian cell line for protein production
Nucleases
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DBD Guided endonuclease
o Zinc-Finger Nuclease (ZFN)
 DNA binding zinc finger motifs are widespread in eukaryotic transcriptional activators
 Designed to target specific sequences in genome
 Meganuclease (dimer) domain combined with zinc finger to cut target DNA
o TALENs: Transcriptional Activator Like Effector Nucleases
 Proteins secreted by pathogenic Xanthomonase spp. Into plant cells
 Activates expression of plant genes that code for proteins involved in increasing plant susceptibility to
infection
 TALE have
 N terminal signals for bacterial type3 secretion
 DNA binding region with variable number of tandem repeats that determine target sequence
 Dimerization of nuclease domain required for dsDNA break
 Nuclear localization signals
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C terminal region (AD) required for transcription activation
Domains with repeats that are ~34aa residues
12th and 13th aa residues are hypervariable- determine nucleotide sequence recognized on
target promoters
Gene Editing (nuclease has)
 DNA recognition of TALE
 Endonuclease domain of FokI in place of transcription activator domain of TALE
 FokI- from Flavobacrium okeanokoites
RNA Guided endonucleases
o Based on CRISPR- Clustered Regulatory Interspersed Palindromic Repeats with CRISPR associated Genes
(Cas9- nuclease)
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CRISPR-cas9
 Adaptive microbial immune system that has resistance against phages and plasmids
 Involved in silencing mRNA of invading phages
 Mechanism
 During infection, viral DNA pieces insert into pager region between repeats
 CRISPR transcribed into long precursor
 tracrRNA binfs to repeated sequence of CRISPR, and then CRISPR is cleaved into smaller
fragments by Rnase 3
 Cas 9 binds cleaved crRNA
 Cas9 guides crRNA to DNA and scans
 crRNA base pairs with viral sequence
 Cas9 nuclease activity causes DNA to break
 Without donor piece of DNA, leads to deletions
 Inserts functional gene in place of defective one
 Cas9-sgRNA: synthetic short guide RNA
 Role: tracrRNA as cas9 handle, crRNA to target DNA
CRISPR-cas9 vs. TALENS
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CRISPR: 20 nt inserted into target DNA
CRISPR: more off target mutations
TALEN: 2 TALENS for 1 DNA
TALEN: change 2aa residue in each of 15 repeats
TALEN: hard to contract with repeats
TALEN: less off target mutations
Ongoing Clinical trials
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AIDS treatment- deletion of receptor used by HIV to attach and infect T-cells
Monogenic genetic disorder (mutation in 1 gene)
o Huntington’s – delete polyQ repeats
o Brain Cancer- KnockOut gene for overactive protein
o Hemophilia- KnockIn normal gene
o Sickle Cell anemia
Microbial bioremediation- use of microbes of their products to remove pollutants from the environment
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200M tons of waste in US- spills, dumping, abandoned industrial sites
Microbes has extremely diverse metabolic pathways and utilize many different compounds for nutrition and energy.
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Break down contaminants to harmless byproducts
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More efficient an cost effective
More environmentally friendly
Uses
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Eliminate toxic chemicals from spills, waste
Sewage treatment
o Decompose organic matter
o Remove nitrogen and phosphorus so no plant overgrowth
Remove metals – mercury and arsenic- and radioactive material from soil and groundwater
Microbial biosensors to detect chemicals and pollutants
Petroleum Degradation
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Pseudomaonas putidia – 1st GM microbe with patent.
o In the presence of tolulene as inducer, transcriptional regulator, XyIR, is able to bind to the promoter, Pm, to
activate expression of xyIDLEGFKIH operon
Different strains mated that grow on different components of crude oil- not successful
Organophosporous Pesticides
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Potent nerve agents- poisoning common in developing countries
Bacterial genes with OP degradation
o Opd- organophosphorus parathion degradation
 OPH- organophosphorus hydrolase- end products are dietheylphospahse and 4-nitrophenol
o Mpd- methyl parathion degradation
 MPH- methyl parathion hydrolase- dimethyl thiophosphate and 4-nitrophenol
Strain Engineering
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Both wih opd and 4-nitrophenol degradation pathways
Lpp-OmpA-Oph on outer surface = 7 fold higher degradation
Periplasmic mph and OP displayed oph
Wastewater treatment
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Accumulibacter phosphatis – microbe that removes phosphate and stores inside cells in energy rich phosphate
Nitrosomas europea- oxidizes ammonium to nitrite
RidX
o Cellulase- TP, veggies, some food
o Lipase- fat, oil, grease
o Protease- proteins
o Amylase- starches
Methylmercury- neurotoxin that bioaccumulates in food web
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E.coli engineered to accumulate mercury
o EC20 (glu-cys)- synthetic, high affinity, metal binding proteins
o Lpp-PmpA-EC20 fusion- anchors EC20 to cell surface
o MaleE-EC20 fusion- localizes EC20 into periplasm
o MerT and MerP- transport mercury
Microbial biosensors- genetically engineered microbes that have transcriptional fusions of chemical or pollutant- responsive
gene promoters that drives reporter gene expression.
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Constitutive- used to track changes in cell numbers
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Pseudomonas fluroescens HK44- 1st GE microbial biosensor released
Polycyclic aromatic hydrocarbon -PAH- inducible promoters control expression of lux reporter- fiber optics detect light
Energy and Biofuels
Microbial enhanced oil recovery- biosurfactants increase oil wettability, pyrolase
Biofuels- energy sources derived from organic matter other than fossil fuels
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Renewable, less toxic, earth friendly (lower CO2 emission)
Biogas- methane
o From anaerobic digestion of waste
Bioalchohols- ethanol, butanol
o First gen: from microbial fermentation of sugar from food sources (corn, sugarcane, beets)
 Controversial because it used same products as humans
o Second gen: produced by microbial fermentation of non-food sources (switchgrass and agricultural waste)
Biodesil- lipid derivatives
o From algea oil, veg oil, animal fat, waste cooking oil, tall oil
Biohydrogen- anaerobic conversion of glucose
o Clean energy, low yield
Metagenomics- study of DNA of microbial communities without isolating and culturing in a lab
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99% of microbes unculturable in lab
Sequence Based Screen limitations
o Constrained by protein with known sequences
o Some enzymes with similar catalytic activity may not exhibit enough conserved aa sequence
Functional / Activity based screens
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Assays
 Enzyme assay
 Growth inhibition- antimicrobials
 Ability to grow/use/degrade compounds
Limitations
 Not all genes expressed
 Not all enzymes active at certain conditions
Finds
 Thermostable DNA polymers
 Antibiotic resistance genes
 Biocatalysts
 Plant cell wall degrading enzymes
Steps
 Positive screening
 Re-cline GOI in vectors or host to optimize expression
 Computational modeling and protein engineering to improve properties
 Techniques: (directed evolution)
o Random and site-directed mutagenesis
o DNA shuffling
iCHIP
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Allows activation of up to 50% microbes
Led to teixobactin discovery
o Gram+ effected, but not Gram-
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Interfered with peptidoglycan production