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Their genetic traits are used in genetic exchange, recombination and engineering
Many of them are suitable host for heterologous expression
they grow and multiply rapidly, it is possible to observe several generations in a fairly short time
microbes are endowed of highly useful features
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Without them, biotechnology would not be as advanced as it is, nor would it include such a broad range of applications
they are used to study the common processes to all living organisms
Many microbial products can be useful (e.g. antibiotics, bioinsecticidesbioherbicides, biofungicides, polymers…)
And, indeed, microbes actually are a fundamental element of biotechnology
BIOTECHs
the use of biological
processesorganisms
To obtaingoods
services Principles
scientific engineering
MICROBIAL BIOTECHNOLOGYis the application of:
to the processing of materials by microorganisms
BIOTECHNOLOGY is
GREENAgricultural/environment
WHITEGene-basedindustry
BLUEAquatic
YELLOWFood production
DARKWarfare/Bioterrorism
GRAYEnvironmentprotection
REDmedical
Due to their peculiar metabolic features, microbesintervene in all the colours of biotechnology
How long has been the way to microbial biotechnology?
The first traces of biological preservation methods (fermentation) date back to Neolithic (10000-6000 B.C.)
the Hammurabi ‘s code (1792-1750 B.C.) decreed DEATH
for those who had:
• prepared it out of the established rules
• Sold it without permission
The use of microbes is VERY ancient…
let’s think just of wine, beer, bread, or sauerkraut, cheese and fermented milk
It wasn’t until the mid-nineteenth century, indeed, that Pasteur demonstrated the roleof yeasts in the alchoholic fermentation
While looking for the cause of beetjuice turning sour instead of alcoholic
TODAY, biotechnology influences many aspects of our lives-The basic understanding of microbial genetics and physiology led to
> molecular biology tools
widespread use of microbial biotechnology
FOOD ADDITIVES(aa.; organic acids, fatty acids, vitamins) BIOFUELS
(ethanol, methane, hydrogen..)
ENZYMES(Proteases, amylases, cellulases, lipases
SOLVENTS(acetone, butanol, ethanol..)
AGROCHEMICALS(feed additives, biopesticides..)
WHOLE CELLS(Baker’s yeast, crop inoculants)
FINE CHEMICALS(antibiotics, chemotherapics, nucleic acids, proteins,
biochemicals, optically pure chiral molecules..)
natural isolates may be suitable but..
once taken from their native ecosystems, they could be notsuitable for optimal biotechnologyprocesses:
They could not be able to grow fast under the standard laboratoryconditions
Or fail to produce high levelsof the desired products
Which microbes for biotechnology?
Usually, laboratory screened mutants or genetically modified microorganisms are employed
Faster growing
Better producers
Well characterized strains are available in the international culture collections
But sometimes it’s a good idea to look for well suitedmicroorganisms with specific physiological features
By chasing them in their specific environments
THIS STRATEGY IS CALLED «BIOPROSPECTING»
AND/OR HYPERALINE
HOT
environments may be explored for microbes whose enzymes optimally work in those conditions
We could need enzymes suitable for processes at low temperatures or, on the contrary, able to stand high temperatures, or to function in hyperaline solutions…
COLD
Efficiently cellulase producing microbes will be looked for in cow’s or termites gut
New antimicrobials will be searched in highlycompetitive, stressing environments and so on
BAITING
E.g.: to isolate keratinolytic microorganisms, one could uses metal meshes, filled with wool and placed in the water
Sometimes, it’s possible by using specific substrates, to attract and "capture" the desired microorganisms
Timely, the bait will be checked for the presence of adhering and
multiplying bacteria on it
Or, for cellulose degrading microorganisms the bait could be some straw
Another baiting approach uses half glass-tubes
closed at both ends with filters of about 100 μm, so to stop insects and protozoa but let the bacteria in
a suitable culture medium is poured into the devices
That are tied on the surface of trunks, branches or leaves
Timely, the devices are stamped on the surface of suitablemedia, to isolate the growing bacteria
Each stamp can be roughly regarded as a ten-fold dilution
A very good example of both a successful bioprospecting approach, is the finding of the Penicillium chrysogenum isolate that allowed to produce penicillin on a large scale
The Fleming isolate was a very poor penicillin producer
(<40 units/ml in the best culture conditions)
It was not able to grow in the submerged culture necessary for large scale production
Nor derivatives able to do so could be obtained
The second World war was underway and the ask for penicillin very high
A scientist staff in Peoria (Illinois) started to look for a similar strain able
to grow well in submerged culture
everyone over the world was asked to send samples of blue-green molds
a young woman (Mary Hunt) was employed to scour the markets in Peoria
for blue-green or similar molds
military personnel scooped up soil from exotic locations
She was used to be called "Moldy Mary”
.
At last, it was a moldy cantaloupe covered with a “pretty, goldy mold”,
to provide the desired strain
That mold turned out to be a highly productive strain of Penicillium chrysogeum
Its yield, in submerged culture reached up to 70-80 units/ml
From this strain, an industrial highly producing mutant was obtained
up to 250 units/ml
But microbial strains, can be improved by
In microbial biotechnology, indeed, production is ultimately limited by the genetic and physiological characteristic of the microbial strains
modifying the metabolic characteristics of the strain
introducing new genes altering the expression of existing ones
the Peoria strain, as many other industrial microorganisms has been obtained with in vivo random mutagenesis
With UV or X rays and/or DNA-damaging chemicals
In vivo chemical mutagenesis induce changes across the entire genome
It has the advantage of selecting for non-lethal mutations, as the cells must replicate for the changes to be observed
A drawback is that we do not actually know how many mutations have beenintroduced and where, so that some unpredictable consequences could occurr
e.g. The Lenape potato, developed in the 1960s for the snack business, made a very fine potato chip. Unfortunately, it was also toxic..
1) Random Mutagenesis
2) Site directed Mutagenesis
Besides the in vivo mutagenesis, performed on living cells, molecular biology has provided us with powerful tools for in vitro mutagenesis
To obtained casual mutations, and screen for favourable ones
To introduce a specific, previously projectedchange in a known sequence
.
In vitro Random Mutagenesis
Whenever the encoding gene is known, it’s possible to apply an in vitro approach to obtain several mutants of an enzyme. Several techniques can
be applied to achieve this goal
amplify the gene
create a “library” of thousands of versions of the gene
Introduce the randomly mutations
Tranform in a suitable host for the expression
screen
CHEMICAL MODIFICATIONIn 2004 Lai et al. described the use of the alkylating agent ethyl methane
sulfonate (EMS) for in vitro mutagenesis in the coding region of a gene
EMS results in G-T mismatches through introduction of AT to GC and
GC to AT transition mutations
Nitrous acid is another mutagenic agent which acts by deaminating adenine and cytosine residues and causing
transversion point mutations (A/T to G/C and vice versa)
The extent of mutagenesis can be altered by changing the reactions conditions
incubation time and temperature pH
length and amount of the target gene
Mutagen concentration
ERROR-PRONE PCRa “sloppy” version of PCR, in which the polymerase has a fairly high
error rate (up to 2%). This can be done by
increasing MgCl2
using unequal concentrations of each nucleotide
adding MnCl2
The process can be repeated through many rounds of selection
point mutations ( ) are the most common ones in EP-PCR, but deletions and frameshift mutations are also possible
EP-PCR EP-PCR
Chemical methods avoid the bias that PCR-based mutagenesis has toward AT to GC transversions, but the last one is easier and does not employ dangerous substances
After amplification, the library of mutant coding sequences must be cloned into a suitable plasmid
The efficiency of the cloning stepLimits the library size
excessively altered conditions poor amplification and undesired amplicons
Possible drawbacks
Taq polymerase has a bias toward inducing mutations in A and T bases
.
ERROR-PRONE ROLLING CIRCLE AMPLIFICATION (RCA)is a variant of EP-PCR
The wild-type sequence is cloned into a plasmid
the whole plasmid is amplified under error-prone conditions
The EP-RCA eliminates the ligation step that limits library size in conventional error-prone PCR
Giving rise to plasmid concatemers
Once trasformed, the concatemers circularize in the cell by intramolecular homologous recombination in vivo and a mutants library is obtained in the host strain
EP-RCA Transformation
Only improved variants survive the selection
WT gene coding the Taq polymerase
large gene library obtained by mutagenesis (~ 108 random variants)
The gene variants are compartmentalized and
expressed in emulsion and then screened
The commercial KAPA SYBR DNA Polymerase (KAPA biosystems) was obtained with a DE approach
DIRECTED EVOLUTION
Employs repeated steps of mutagenesis and selection, to mimic the process of natural selection and evolve proteins or nucleic acids toward a user-defined goal
water-in-oil (w/o) emulsions to mimic cellular compartments that link genotype and phenotype..
Emulsions of cell-like compartments were formed by adding in vitro transcription/ translation reaction mixture to stirred mineral oil containing surfactants
For details on this technique see Liisa D. van Vliet, Pierre-Yves Colin and Florian Hollfelder(2015) http://dx.doi.org/10.1098/rsfs.2015.0035
MUTATOR STRAINS
A commercial available mutator strain, is the E. coli “XL1-Red” (Stratagene)
Are deficient for One or more genes of the primary
DNA repair pathways
So that they make errors while replicating their DNA, including
the cloned plasmid
XL1-red is deficient mutS (mismatch repair)mutD ( 3’-5’ exonuclease of DNA polymerase III) &
mutT (hydrolyses 8‐oxo dGTP)
MUTATOR STRAINS
W-T sequence cloned into a plasmid
transformed into the mutator strain,
In a mutator strains a wide variety of mutations can be obtained including substitutions, deletions and frame-shifts
BUT..As more and more mutations
accumulate in their genome, many cells stop growing or transforming
MOREOVERTo keep stock cultures of a mutator
strain becomes by and by more difficult
each copy of the plasmid is potentially different from the
wild-type
TEMPORARY MUTATOR STRAINS
Are not commercially available but could be built “in the lab”
mutD5 is a dominant negative version of mutD which limits the cell’s ability to repair DNA lesions
allow the cells to cycle
between:
the overexpressing of mut5D from an inducible promoter
Mutator(mutD5 ON)
Normal (mutD5 OFF)
During the normal growth the cells can recover, differently from conventional mutator strains
mutD5
Inducible promoter
MOREOVER..
It’s possible to remove the mutagenic plasmid, restoring the normal repair systems
By cloning mutD5 in a plasmid with a temperature-sensitive origin of replication…
Making easier to grow, analyze and screen the mutants obtained during the mutator
phase
Mutagenesis directed evolution
Temperature shift loss of the plasmid
Olga Selifonova et al. Appl. Environ. Microbiol.
2001;67:3645-3649
TransformationWith pmut
Growth, mutagenesis
selection curing
Acceleration of the evolution of a microorganism by using a mutator plasmid
multiple rounds of growth and selection
DNA SHUFFLING
copies of same gene, with different mutations are digested with DNAseI
the fragments are then randomly re-joined by self-priming PCR
And amplified again with external primers
Shuffling allows to test the effects of different
combinations of mutations
PRE-DETERMINED, NON CAUSAL MUTATIONS CAN BE INTRODUCED BY SITE DIRECTED MUTAGENESIS
First introduced by Michael Smith (Canada, late 70s)
The techniques can be divided in two main groups
enzymatic extension of a mutagenic oligonucleotide annealed to a DNA template
cassette mutagenesis
In cassette mutagenesis a gene is constructed by ligating a series of synthetic oligonucleotides, cloned and transformed
Anneal two synthetic complementary oligonucleotides
Ligate with the plasmid transform
Cut and remove the WT fragment
Unfortunately, unique, conveniently spaced restriction endonuclease recognition sites in the desired place are often not present
ssDNA cloning vector+ source DNA
transformation
This procedure derived from a series of experiments demonstrating that:
a single-stranded DNA template could be converted to a double-stranded molecule in vitro
ssDNA
annealing of the mutagenic primer:
extensionHeteroduplex
ds DNA
Mutantsselection
the DNA polymerase needed a short oligonucleotide primer to initiate DNA synthesis
This method does not yield an high frequence of mutants
enzymatic extension of a mutagenic oligonucleotide
Another method is the “megaprimer PCR”
The first PCR round (PCR1) is performed with
an external fwd Low Tm primer (a)
The mutagenic primer (m)
The mutagenic product (megaprimer - Mp) acts as the reverse primer in the second step
By adding another external (High Tm) primer “b”
Which uses three primers and two PCR rounds
PCR1 (low annealing temp)a
m
Mp
b
Mp
Final product to be cloned
PCR2 (high annealing temp)
A further modification is the technique that employs DsDNA, overlapping primers and template degradation
Two identical complementary mutagenic primers (~ 40-50 mer) are designed, and
annealed to the region to be modified
The primers are extended with an high fidelity DNA polimerase; the two
complementary amplimers anneal by homology
DpnI digests the parental methylated and hemimethylated DNA
After transforming, the E. coli DNA ligase repairs the nicks
A further modification employs just one modifying primer and a rapidly acting mix of kinase, ligase and DpnI to obtain substitutions, insertions or deletions
1) PCR amplification with High-Fidelity DNA Polymerase, according the modification project
2
2) Quick multiple enzymatic reaction (RT, 5 min)
phosphorilation ligation Template removal
1
3) Transformation
SUBSTITUTIONS: the desired nucleotide change(s) are incorporated in the center of the Fwd primer, with at least 10 complementary nts at the 3’ side of the mutation(s)
The reverse primer is designed to anneal back to back with the fwd one
DELETIONS are simply created with a standard couple of primers flanking the region to be deleted
≥ 10 nts
Several effects can be obtained by muddling around the primers
SMALL INSERTIONS: up to 6 nts can be added to the 5’ end of the fwd primer
≥ 10 nts
The reverse primer is designed to anneal back to back with the 5’ end of the complementary region of the fwd one
LARGE INSERTIONS: are obtained by incorporating half of the nts to be added to the 5’ end of each primer
≥ 10 nts
≥ 10 nts
One example of Site directed mutagenesis has been the adding of 3 sulfide bonds to the T4 lysozime, now produced in E. coli, that is
largely used as a food additive
Of course, it’s necessary to know the three-dimensional structure of the peptide to be modified, so to target the aminoacids to be substituted, amongthose located in a suitable position and not critical for the normal functioning