Lec 12 - Microbes in Biotech

Overview
 Defining microbes
 Finding microbes
 Using microbes
◦ Everyday
◦ Medicines
◦ Energy
◦ Agriculture
◦ Food
https://learn.genetics.utah.edu/content/microbiome/intro/
CS
Copyright © 2009 Pearson Education, Inc. 1
Microbes (microorganisms)
 Tiny organisms that are too
small to be seen individually
by the naked eye and must
be viewed with the help of a
microscope.
 Biotech microbes tend to
be single-celled.
CS
Microbes in all three domains of life
Algae
Fungi
Protozoa
Most of life’s diversity and so mos
of it’s deep evolutionary history i
microbial
Archaea Cyanobacteria
CS 3
Copyright © 2009 Pearson Education, Inc. Bacteria
2/3 of species are bacteria
Remembe
r
this from
Dr. Mark-
Welch’s
talk
Nature Microbiology volume 1, Article number: 16048 (2016) 4

Copyright © 2009 Pearson Education, Inc.
VIRUSES ARE MICROBES too
• Viruses are an important component of various

microbiota.
Can alter the relative abundance of different members of
microbial communities (esp. abundance of bacteria)
• Involved in horizontal transmission of genetic
information
EBOLA BACTERIOPHAGE
CS
Microbial FAQs
 Comprise over 50% of the earth's living matter.
 Bacteria are adapted to all corners of the Earth.
 Terraformers. Microbes are Earth’s OGs (original
geochemists).
 Essential for ecosystem cycles (C, N, P, S).
• Humans have long used microbes.

‘Ur-biotechnology’ – bread, fermentation,
farming, medicine (probiotics), etc.
• Less than 1% of all bacterial species have been
cultured and studied. Imagine the contribution
of microbes to biotechnology in the future.
CS
Bacteria were the first life forms on earth
and have existed for over 3.5 billion years
Macro-
Microbes only scopic
Modified
Copyright from
© 2009Andrew H. Knoll,Inc.
Pearson Education, and Martin A. Nowak Sci Adv 2017;3:e1603076
Microbial FAQs
Bacteria
• Small (1–5 µm)
• No nucleus
DNA is contained in a single, circular
chromosome. Genome sizes range from
0.5-10 Mb
• Many contain plasmids
Important in horizontal gene transfer -
spreading of antibiotic resistance genes
• Capsule
Useful tool for cloning DNA Some bacteria contain an
• Cell wall outer layer of
carbohydrates in a
Surrounds plasma membrane. Contains structure called a capsule
peptidoglycan; impt for protection; infection.
CS
What is Gram positive/negative?
Cell wall differences of bacteria are used to classify & categorize bacteria - the
Gram stain
◦ Purple colored Gram positive (+) bacteria have simple cell walls rich in
peptidoglycan that retain a purple stain (crystal violet)
◦ Red colored Gram negative (–) bacteria have a complex cell wall but the cell
wall has only a thin layer of peptidoglycan so it does not bind crystal violet
tightly. After washes remove the violet stain, the bacteria are counterstained
with a pink stain (safranin).
Gram pos Gram neg
P
G
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Bacteria vary in shape and size
Three common shapes
 Coccus – spherical cells
 Bacillus – rod-shaped cells
 Spiral – corkscrew-shaped cells
Also other shapes!
CS
Bacteria are not
loners
• Cells/colonies can form
extensive microbial biofilms
• Differentiation – specialized
roles
• Social Interactions with
cells/colonies competing and
collaborating
• Neighboring colonies show

oscillatory coordinated
growth and signaling
(cyan = electrical activity due to
changes in K+ ion levels)
CS
J. Liu et
Copyright © al.,
2009Science Vol.Education,
Pearson 356, pp. 638-642.
Inc. (2017). https://doi.org/10.1126/science.aah4204 11
Archaea
 Single-celled prokaryotes - no
nucleus
 Ancient. Some properties of
prokaryotes but many features
much more like eukaryotes.
 Often found in extreme
environments
and have
unique metabolic properties
 Halophiles: salty places
 Thermophiles: hot places
 Psychrophiles: cold places
 Methanogens: produce methane
Some species of Archaea have square cells !

Eukaryotic Microbes
 Yeast (single-celled fungi)
 Molds (fungi that produce hyphae, the
branched tube-like structures, that make
molds fuzzy)
 Algae (photosynthetic – brown, green)
 Protozoa (single-cell)
 Microscopic metazoa (small animals e.g.
nematodes and rotifers)
CS
Overview
 Using microbes
◦ Everyday
◦ Medicines
◦ Energy
◦ Agriculture
◦ Food
https://www.npr.org/sections/thesalt/2018/01/18/578924748/scientists-peek-insi
de-the-black-box-of-soil-microbes-to-learn-their-secrets
Microorganisms play a vital role in growing food and sustaining the planet, but
they do it anonymously. Scientists haven't identified most soil microbes, but
they are learning which are most common.
PeopleImages/Getty Images
CS
Microbe culture
What are
some
variables
that would
affect
ability to
culture
microbes?
CS
Tal Danino bacterial art, http://www.taldaninoart.com/bacteria-gallery-1 15
Microbe culture
Growth affected by
• Nutrients and
minerals
• Temperature
• Atmosphere (O2 is
a poison)
• Social interactions
CS
Tal Danino bacterial art, http://www.taldaninoart.com/bacteria-gallery-1 16
Can characterize microbes
without culturing them
Characterize species by DNA Sequences
◦ 16S rRNA (prokaryotes) and 18S rRNA (eukaryotes) – rDNA
genes are conserved but can discriminate different species
◦ Shotgun sequencing – align small DNA sequences and
assemble a likely genome
Sequencing also useful for:

◦ Epidemiology: identify pathogenic bacteria. Track origin and
spread of disease.
◦ Evolution: study origin/diversity of microbes.
◦ Bioprospecting: find novel genes with commercially useful
traits e.g. for bioremediation, biofuels, medicine.
CS
Human microbiome/microbiota
Different
communities of
microbes and
different roles of
microbes
in different
places on the
body
April 2012
CS
Human microbiome interactions
CS
Plant Microbiome Probiotics for crops
Methylotrophs (M-trophs)
Symbionts
- More sustainable
agriculture
- Improve plant durability,
crop yield.
- fight disease nutrient
uptake.
CS
Microbiome of Mother Earth
90% of the biomass in the oceans is bacteria
– Sorcerer II Expedition (thru 2015): J. Craig
Venter's Institute (JCVI) traveled the globe
by yacht sampling marine microorganisms.
◦ The JCVI has
sequenced billions of
bp of DNA from 400
uncharacterized
microbial species
CS
 Crowd-sourced
 Scientists from 43
countries sent the project's
authors 27,751 DNA
samples of free-living and
host-associated
microorganisms collected
from various
environments.
https://www.nature.com/articles/nature24756 CS
Viral Genomics for everyone
 SEA–PHAGES Project (https://seaphages.org)
The SEA Phage Hunters Advancing Genomics and
Evolutionary Science, or PHAGES, project is built
around a national experiment in bacteriophage
genomics. Students isolate, name, sequence, and
analyze newly-discovered mycobacteriophages.
Students make significant contributions to the field
of genomics as they learn how to think like
scientists.

Build a microbe
◦ Mycoplasma mycoides JCVI-Syn 1.0
◦ Proof of concept that large synthetic genomes
can be synthesized.
CS
Mycoplasma mycoides
"TO LIVE, TO ERR, TO FALL, TO TRIUMPH, TO RECREATE LIFE OUT OF LIFE” (J. Joyce)
JCVI – SYN 1.0 JCVI – SYN 3.0
2010 2016
1.08 M BASES 0.53 M BASES
• JCVI – SYN9013.0 isGENES 473

the smallest genome GENES
of any organism that
can be grown in laboratory media (video)
• 149 of the 473 genes (32%) essential for life in this cell are of
unknown function
• CRAIGVENTER coded as:
TTAACTAGCTAATGTCGTGCAATTGGAGTAGAGAACACAGA
ACGATTAACTAGCTAA
CS
Synthetic E. coli – Syn61
(May 2019, MRC in England)
Genome reduced in size from 4.6 Mb to approx. 4 Mb

61 codons instead of 64. Eliminated 2 Ser codons and
one stop codons. Recoded >18,000 codons. Allowed
elimination of a previously essential tRNA. Grows slower
and cells longer.
CS
Copyright © 2009https://www.nature.com/articles/d41586-019-01584-x
Pearson Education, Inc. 26
Overview
 Using microbes
◦ Medicines
◦ Everyday
◦ Energy
◦ Agriculture
◦ Food
https://www.npr.org/sections/thesalt/2018/01/18/578924748/scientists-peek-inside-the
-black-box-of-soil-microbes-to-learn-their-secrets
Microorganisms play a vital role in growing food and sustaining the planet, but they
do it anonymously. Scientists haven't identified most soil microbes, but they are
learning which are most common.
PeopleImages/Getty Images
CS
Why use microbes for biotech?
 Grow and divide rapidly
◦ Some bacteria like E. coli can divide every 30 minutes.
In 12hrs, 224 rounds of division =
1 cell  4 million cells (1 cell  colony)
◦ Can be grown (cultured) in liquid media OR on solid
surfaces such as plates of agar.
◦ Scalable  industrial fermentation vats.
 Easy to modify bacteria (add genes, mutate genes) –
e.g. molecular/genetic studies, protein production
 Microbes have figured out how to do some
interesting things that are hard to do synthetically
CS
Biotech tools from microbes
 Name some
 Biosorption
 CRISPR
 Plasmids
 Taq Polymerase
 Biofilms
 Fermentation
CS
 Cloning/recombinant DNA
(rDNA) technology
components from microbes
◦ Plasmid (with ori)
◦ Restriction enzyme
◦ Ligation (DNA ligase)
◦ Antibiotic resistance genes (for
selection)
◦ Reverse transcriptase
◦ Many others
CS
 CRISPR
◦ Cas9 enzyme (esp. from
Streptococcus pyogenes)
◦ tracrRNA
◦ Other Cas enzymes are
being tested
• PCR
heat stable DNA polymerases
from archaea and thermophilic
bacteria (e.g. hot springs,
thermal vents).
CS
Microbes for recombinant proteins
Therapeutic Proteins
Bacteria and yeast are used to produce
medically important proteins
Insulin, the first recombinant molecule to get
FDA approval in 1982, expressed in bacteria for
use in humans
Industrial Proteins
rBGH (Bovine Growth hormone—increases milk
production)
- Created by Genentech in 1970s (published in 1981)
- FDA approved in 1993 (despite proven safe in 1986)

Synthesis of Recombinant Insulin

Therapeutic Proteins

Bacterial fusion proteins
 Bacteria can be used to mass-produce proteins
◦ Gene for protein of interest is inserted into a plasmid
containing a gene for a well-known protein that serves as
a “tag”
 The tag protein allows for studying temporal and spatial
regulation of gene expression
 The tag protein allows for the isolation and purification of the
recombinant protein as a fusion protein
 Plasmid vectors used are often called expression vectors
 Incorporate prokaryotic promoter sequences
 Recombine DNA for the tag protein with the cloned gene

Fusion proteins – ease of Purification

Microbial Proteins as Reporters
 Bioluminescence – method of producing light used by
marine organisms
◦ Created by bacteria such as Vibrio fisheri that use marine
organism as a host
 Create light through action of lux genes
 Encode for luciferase that acts on luciferin and generates light when
luciferin gets oxidized to oxyluciferin
◦ Lux genes have been cloned and used to study gene expression
 Clone lux genes into plasmid
 If inserted into animal or plant cells, will produce luciferase and
will fluoresce, providing a visual indicator of gene expression
- Used in the TB bioassay (M. tuberculosis)

Yeast expression systems
◦ Saccharomyces cerevisiae
 Has been used for ages in fermentation
 Model system for studying cell biology (a eukaryotic version of “E. coli” for the lab)
 Was the 1st eucaryotic organism to be sequenced (12 Mbp)
◦ Pichia pastoris
 Soy leghemoglobin used for Impossible Meat
 Grows to a higher density in liquid culture than other yeast strains
 Strong promoters that can be used for production of proteins
◦ Kluyveromyces lactis
 Preferred over other yeast for food products. GRAS status (FDA says K. lactis enzymes
are safe to eat). Used for:
 chymosin (used for making cheese)
 Lactase (used to make milk lactose free)
 “Super secreter” mutants.
CS
What is an advantage of using a eukaryotic expression host?
 post-translational modifications (e.g,. complicated

folding, glycosylation*)
 Secretion (easier to purify if secreted)
* BUT NON-HUMAN GLYCOSYLATION

Microbes - name some other uses
CS
Uses of microbes/microbial products
LAB TOOLS FOOD

 Cloning/editing genes • Fermentation
 Synthesis of proteins, RNAs, • Modification
carbohydrates, lipids • Dietary/nutrient
BIOPHARMACEUTICALS supplementation (vitamins,
 Drug production lipids,)
 Bioprospecting EVERYDAY PRODUCTS
CHEMICAL SYNTHESIS • Laundry and cleaning
 Work under extreme products
conditions (hot, cold, salty, • Clothes
etc.)
CS
Microbe Applications - Antibiotics
◦ 1928 discovery of penicillin by Alexander Fleming
◦ Majority are produced by soil bacteria, and inhibit
the growth of other bacteria.
Beta – lactam
group
DO NOT NEED TO KNOW THIS STUCTURE FOR QUIZ CS
Antibiotics – modes of action

Common Antibiotics

Antibiotics
◦ Improper use of antibiotics has lead to dramatic increase in
antibiotic-resistant bacteria
 http://www.fda.gov/animalveterinary/guidancecomplianceenforcement/guidancefori
ndustry/ucm216939.htm#question6
◦ Since we can see that known antibiotics attack a bacterial

cell in a limited number of ways, resistance to one antibiotic
often leads to resistance to many other drugs.
◦ New antimicrobial drugs that act in unique ways need to be
developed
◦ Microbiologists are bioprospecting in diverse habitats to
identify sources of new anti microbial substances
© 2013 Pearson Education,

Copyright © 2009 Pearson Education, Inc. Inc.
New antibiotic discovery using iCHIP
Typically, only about 1%

of microbes in a soil
sample are able to grow
in the lab. The iChip
expands that fraction to
50%

Microbes - Everyday Applications
 Microbial Enzymes
◦ Cellulase
 Convert wood and plant cellulose into biofuels (ethanol, methane)
 Stone-washed jeans (break down fibers)
◦ Proteases (subtilisin), lipases (lipid breakdown), amylases
(starch breakdown)
 Laundry detergents
Microbes in a termite gut
convert 95% of cellulose
into simple sugars, in less
than a day.
200 species of microbes in
a termite gut!
CS
Using Microbes in Food Products
Fermentation Lab
 Fermentation - process of deriving energy
from sugars in the absence of oxygen

 Examples
 Lactic acid fermentation: (e.g. Lactobacilli,
Lactococci)
 Acetic acid fermentation (e.g. Acetobacter)
 Alcohol fermentation: (e.g. Saccharomyces)

Aerobic respiration vs Fermentation
respiration = full
oxidation of
organic
molecules to CO2
fermentation =
partial oxidation
of organic
molecules
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Energetics
Aerobic respiration  more ATP
CS
Fermentation – without oxygen
• Recycles NAD
• Allows for some
ATP production
• Buildup of
fermentation
products
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Fermentation products
makes the Dr. Gilbert Based on the

holes in Swiss mentioned
cheese butyrate
organism doing the
(via CO2 production by fermenting, a variety
bubbles) our microbiota of different short chain
acids can be produced,
giving a distinctive
flavor to the product.CS
52
Microbes and Food
◦ Bread, yogurt, alcoholic beverages and CHEESE!

◦ The first recombinant DNA food ingredient approved
by the Food and Drug Administration (FDA) is a
recombinant form of rennin, called chymosin. Use to
make cheese
◦ Chymosin genes cloned and expressed in
molds/fungi (Aspergillus niger, Kluyveromyces
lactis).
◦ 90% of US cheese is produced using chymosin. Only
a small percentage of cheese in Europe produced
using chymosin. In Europe, traditional stomach
derived rennin used.
◦ Fun Fact: cloned camel chymosin works best!
CS
Microbes in the manufacture of dairy products
A variety of microbes are utilized in the manufacture of cheese,
butter, sour cream, yogurt and other fermented dairy products.
Streptococcus - Italian cheese

Lactobacillus - Swiss cheese, yogurt
Lactococcus - Cheddar, Colby, sour cream,
cottage cheese, cultured butter
Propionibacter - Swiss cheese
Penicillium - Roquefort, Blue, Camembert

Field Applications
◦ 1987 Steven Lindow at University of California
 First field application using a GMO (on strawberry fields to
prevent frost-bite)
 Creation of “ice-minus” bacteria would provide protection
from frost, the spray was called “Frostban”
 Pseudomonas syringae, a normal soil strain has surface
bacterial proteins called the ice nucleation-active protein, that
initiates ice crystal formation. Creating a genetic ice minus
variant would prevent frost formation.
◦ 1980s - Genetically altered strain of bacteria that would
protect plants against root-eating insects on cotton and corn
 Pseudomonas fluorescens with cloned Bacillus thuringiensis toxin
gene that kills pests

Coordinated Framework for Regulation
 Coordinated Framework for Regulation of Biotechnology
◦ Proposed by White House Office of Science and Technology Policy
◦ Passed in 1986
◦ Based on the source of funding, the organism being researched and the
application, different agencies work together – NIH, FDA, USDA (EPA & S&E)
 http://www.aphis.usda.gov/brs/fedregister/coordinated_framework.pdf
◦ Allowed for continuing Biotech development without immense social burdens
 FDA in 1992 passed a policy whereby genetically engineered
plant foods referring to food derived from GM crops were
considered as food additives and were subject to existing food
additive regulation – GRAS (generally recognized as safe)

Microbial Genomes
 1994 Microbial Genome Program (MGP) by U.S Dept. of
Energy
◦ To sequence the entire genomes of microorganisms that have
potential applications in environmental biology, research, industry,
and health as well as genomes of protozoan pathogens
 2007 NIH announced plans for the Human Microbiome
Project (HMP)
◦ (2007 – 2012) - 5-year project to sequence 600 genomes of
microorganisms that live on and inside humans
◦ (2013 – 2015) – Integrated biological dataset of microbes and
host to study microbiome associated disease

Microbial Diagnostics
 Microbial Diagnostics – techniques used to detect
and track microbes
 Bacterial Detection Strategies
◦ RFLP analysis, PCR and DNA sequencing
◦ Databases are available for comparison of clinical samples
◦ Used to detect and track bacterial contamination of food
 Pulse-Net database (CDC, USDA, State health dept.)
 Pulsed field gel electrophoresis patterns of food borne disease
isolates
 CDC Currently monitors E. coli O157, Shigella, Salmonella and
Listeria


 Microarrays for Tracking Contagious Diseases
◦ Microarrays have created new approaches for detecting
and identifying pathogens and for examining host
responses to infectious diseases
 Contains ~30,000 probes representing the entire viral genome
◦ Microarrays also used to find "signature" changes in gene
expression for a particular pathogen
◦ Affymetrics developed the SARS CoV Gene Chip which
can be used to detect SARS


Combating Bioterrorism
 Bioterrorism – the use of biological materials as
weapons to harm humans or the animals and
plants we depend on for food
 Only 12 or so organisms could feasibly be
cultured, refined, and used in bioterrorism

 Delivered by aerosols, crop duster planes, or
water supplies

Potential Agents of Bioterrorism

 Using Biotechnology Against Bioweapons
◦ Field test detecting air and water borne pathogens are
essential for detection of an attack
◦ Some involving ELISAs used at Pentagon during anthrax
scare, Gulf War, Afganistan and Iraq wars
 Flawed
◦ Need more sensitive and accurate biosensors.
 PCR based
 Protein Microarrays

 Using Biotechnology Against Bioweapons
◦ Should an attack occur, treatment drugs such as antibiotics will be
needed
◦ Countries must stockpile
◦ Vaccines must be administered before exposure to bioweapons
◦ Even drugs and vaccines could be ineffective if attack involves
organisms engineered against conventional treatments, or an
unknown organisms is used


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Overview

Nature Microbiology volume 1, Article number: 16048 (2016) 4

• Viruses are an important component of various

• Humans have long used microbes.

Also other shapes!

• Neighboring colonies show

Some species of Archaea have square cells !

Sequencing also useful for:

Copyright © 2009 Pearson Education, Inc.

• JCVI – SYN9013.0 isGENES 473

Genome reduced in size from 4.6 Mb to approx. 4 Mb

Copyright © 2009 Pearson Education, Inc.

Copyright © 2009 Pearson Education, Inc. 33

Copyright © 2009 Pearson Education, Inc. 34

Copyright © 2009 Pearson Education, Inc. 35

Copyright © 2009 Pearson Education, Inc. 36

Copyright © 2009 Pearson Education, Inc. 37

 post-translational modifications (e.g,. complicated

* BUT NON-HUMAN GLYCOSYLATION

LAB TOOLS FOOD

Copyright © 2009 Pearson Education, Inc. 43

Copyright © 2009 Pearson Education, Inc. 44

◦ Since we can see that known antibiotics attack a bacterial

© 2013 Pearson Education,

Typically, only about 1%

Copyright © 2009 Pearson Education, Inc.

from sugars in the absence of oxygen

Copyright © 2009 Pearson Education, Inc. 48

makes the Dr. Gilbert Based on the

◦ Bread, yogurt, alcoholic beverages and CHEESE!

Streptococcus - Italian cheese

Copyright © 2009 Pearson Education, Inc.

Copyright © 2009 Pearson Education, Inc. 56

Copyright © 2009 Pearson Education, Inc. 57

Copyright © 2009 Pearson Education, Inc. 58

Copyright © 2009 Pearson Education, Inc. 59

Copyright © 2009 Pearson Education, Inc. 60

© 2013 Pearson Education,

Copyright © 2009 Pearson Education, Inc. 62

cultured, refined, and used in bioterrorism

Copyright © 2009 Pearson Education, Inc. 63

Copyright © 2009 Pearson Education, Inc. 64

© 2013 Pearson Education,

© 2013 Pearson Education,

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