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