Biotecnologie applicate e

Evoluzione

Sandra Urbanelli - Daniele Porretta

Dipartimento di Biologia Ambientale

Via dei Sardi 70, 4° piano

sandra.urbanelli@uniroma1.it

daniele.porretta@uniroma1.it

We live in an exciting time to study Biotechnology

Technological advances have made data collection easier and cheaper than

we could ever have imagined just 10 years ago.

We can now synthesize and analyze large

data sets containing genomes,

transcriptomes, proteomes, and multivariate

phenotypes.

At the same time, society’s need for the results of biological research has

never been greater.

Solutions to many of the world’s most pressing problems will rely heavily on

biologists, collaborating across disciplines.

curing and preventing

diseases

Challenges for the New Biology for the 21st Century

feeding a global population

preserving

ecosystems and biodiversity,

Qual è la differenza di oggi rispetto a ieri?

Il tasso di estinzione attuale è dalle 100 alle 1000 volte maggiore rispetto al

tasso di estinzione di fondo stimato dai dati fossili

Se in passato la durata mediadella vita di una specie dimammiferi o di uccelli eracompresa tra 1 e 10 milioni dianni, nel XX° secolo essa si èridotta a 10.000 anni

La crescita demografica dell’uomo è alla base dell’impatto che osserviamo

La popolazione umana negli ultimi 200 anni è

passata da circa 500 milioni a oltre 7 miliardi

Food and fiber

HealthEnvironment

What is Biotechnology?

UN Convention on Biological Diversity, “biotechnology is technological

application that uses biological systems, living organisms or derivatives

there of, to make or modify products or processes for specific use”

The American Chemical Society defines biotechnology as the application

of biological organisms, systems, or processes by various industries to

learning about the science of life and the improvement of the value of

materials and organisms such as pharmaceuticals, crops, and livestock.

European Federation of Biotechnology, EFB, biotechnology is the

integration of natural science and organisms, cells, parts there of, and

molecular analogues for products and services.

Biotechnology is based on the basic biological sciences

• Biotecnologie tradizionali

1. trasformazione di prodotti di uso quotidiano grazie a processi

biologici (di cui non si conosceva la causa), in particolare i

processi fermentativi

2. selezione di varietà di piante e animali per uso commerciale o

industriale

• Le biotecnologie moderne

3. applicazione di tecnologie geniche, DNA ricombinante, per

modificare la funzione biologica di un organissmo o sviluppare

un nuovo prodotto

4. sviluppo industriale e tecnologico per la produzione di prodotti

chimici specifici (antibiotici, enzimi, aminoacidi, steroidi,

polisaccaridi)

Percorso formativo

Biotecnologie applicate ed Evoluzione

Though Dobzhansky’s statement is sometimes dismissed by biologists in other fields asself-promotion, recent advances in many areas of biology have shown it to be prophetic.

Our ability to apply evolutionary concepts to a wide range ofproblems has never been greater.

Career opportunities for evolutionary biologists may already be more plentiful outside academia than inside it.

Much of the biotechnology industry is concerned with creating biological molecules thathave specific functions. This goal-oriented enterprise has quickly embraced evolutionaryprinciples to direct the evolution of molecules in test tubes and, in so doing, hasprofoundly expanded the horizon and relevance of evolutionary biology. Evolutionaryprinciples are suddenly the material of multimillion dollar patents, leading industrialbiochemists to new drugs and other commercial molecules.

On a different front, the medical establishment, after long ignoring evolution, is faced withan on slaught of drug-resistant microbes, has seen monkey viruses jump into humans andaccelerate into epidemics, and must now use evolutionary principles to understand theworldwide dynamics of pathogens.

Evolution is socially relevant.

First, public perception of evolutionary biology is not up to date with the discipline.“Evolution” is still a bad word to many people, not only because it is perceived asconflicting with some religious views, but also because it is widely viewed as anirrelevant science with no social value. Acceptance of evolutionary biology is farmore likely when the public realizes that it holds the key to many socialimprovements

A second reason is that historical inertia in the training of evolutionary biologists hasresulted in a lack of exposure to socially relevant applications. Individuals trained asevolutionary biologists will have much to offer in solving these problems, but theyneed to be aware of these applications.

This is a recent discorvery because:

L’uomo ha applicato i principi della biologia

evolutiva per produrre prodotti biotecnologici

ancor prima di scoprire l’evoluzione e definire le

biotecnologie

Nearly all the common animals and plants we use today were

domesticated thousands of years ago, some (sheep, goats, dogs, wheat,

and rice) at least 9000 years ago.

the first experiments in applied

evolution.

Their impact was so profound as to make civilization possible by enabling

societies to switch from hunting and gathering to agriculture

Domestication

DomesticationBos taurus

Bos primigenius

Uro_Lascaux

The success of artificial selection is evident in its ultimate creation of selected

phenotypes well outside the extremes of the original species.

The major centers of primary domestication and dates for the earliest

domestication of various plant and animal species.

Riso 2.000 B.P.

Miglio 3.000 B.P.

Sorgo 4.000 BP.

Cotone 5.000 B.P.

Patata 4.500 B.P.

Maranta 8.000 BP.

Patata 7.000 B.P.

Quinoa 5.000 B.P.

Mais 9.000 B.P.

Fagioli 4.000 B.P.

Zucca 10.000 BP.

Marshelder 4.400 B.P.

Girasole 4.800 B.P.

Zucca 10.000 BP.

Manioca 8.000 B.P.

Peperoncino 5.000 B.P.

Orzo 10.000 B.P.

Grano 10.000 B.P.

Miglio 8.000 B.P.

Riso 8.000 B.P.

Quinoa 5.000 B.P.

Riso 8.000 B.P.

Nocciole 8.000 B.P.

Banana 7.000 B.P.

Taro 7.000 B.P.

The distribution of prehistoric agriculture, and early agriculturalist

expansion.

EARLY AGRICULTURALISTPOPULATION DIASPORAS ? FARMING, LANGUAGES, AND GENESPeter Bellwood Annu. Rev. Anthropol. 2001. 30:181–207

The origin and dispersal of domestic livestock species in the Fertile

Crescent.

Shaded areas show the general region and the approximate dates in calibrated years B.P. in which initial

domestication is thought to take place. Dates outside of the shaded areas show the approximate date when

the domesticate first appears in a region. Orange, goats (Capra hircus); blue, sheep (Ovis aries); green, cattle

(Bos taurus); fuscia, pigs (Sus scrofa).

M. A. Zeder, PNAS (2008) 105: 11597–11604.

A model of evolution by natural or artificial selection has three components:

(a) variation,

(b) inheritance, and

(c) differential reproductive success

Domestication probably started as a process of taming, then of captive

breeding, and finally of selecting for specific traits.

Seeking Agriculture’s Ancient Roots M. BALTER (2007) SCIENCE 316

Plant Domestication

Historically, artificial selection

The components of variation and inheritance were present but were often not

manipulated from their natural states, except for the occasional introduction of

novel strains or wild relatives into the breeding stock.

manipulation of differential reproductive

success.

Parents closest to the desired phenotype were chosen to produce the next

generation, and individuals that fell short of the ideal were omitted from the

breeding population.

Domestication

Decrease in genetic diversity in modern

crops during domestication due to

bottleneck events

An example of cotton (Gossypium) evolution under human selection and contemporary breeding

programs.

Plant Domestication

Crop wild relatives. These species represent

sources of novel variation for use in crop

improvement. (a) barley (wild barley (Hordeum

spontaneum) growing near Allepo, Syria); (b) coffee

(Coffea brassii), photograph by Australian Tropical

Herbarium; (c) rice (Oryza A genome wild

mpopulations, growing near Mareeba, Australia); (d)

grape (Cissus antarctica).

Pig domestication has been an integral part of the rise of agriculture and the

adoption of the agricultural practices throughout much of the world.

The domestication of plants and animals led to one of the most important

socioeconomic transitions in human history,

yet little is known about whether the process took place

in a limited number of geographic regions

or was a more widespread innovation

involving multiple, independent events.[

The wild boar is naturally occurring in much of Eurasia

and consists of a number of deeply divergent

populations.

It is the progenitor of the domestic pig, and thought to have

been domesticated on at least two occasions, once in the

Middle East and once in China. More domestications have

been suggested based on mtDNA phylogeography (e.g.

Larson et al. 2005),

L’uomo ha applicato i principi della biologia evolutiva per

produrre prodotti biotecnologici ancor prima di scoprire

l’evoluzione e definire le biotecnologie

Tale applicazione ha innescato effetti a catena sull’uomo stesso

e sull’ambiente alla base dei quali ci sono, ancora una volta, i

processi evolutivi

Dietary shifts

Switch from hunting and gathering to

agriculture

Health consequences

The human genome encompasses only a fraction of the total genetic

diversity found within humans.

The collective microbial communities inhabiting the human body,

contain a vast amount of genetic and functional diversity far

exceeding that of our own nuclear and mitochondrial genomes.

Human microbiome

The human microbiome: an astounding number of bacteria.

the number of bacterial cells (∼1014) in and on the human body

exceeds the number of human cells (∼1013) by at least an order of

magnitude.

the estimated number of unique bacterial genes in our ‘accessory

genome’ (∼3,300,000) exceeds the number of our own genes (∼22,000)

by a factor of 150

the number of cells:

the number of genes:

Despite being 1,000 times smaller than human cells, bacteria still make up

about 2% of adult body mass (1.5 kg), making them collectively equivalent

in size to the human brain (1.4 kg) or liver (1.6 kg), leading some to refer to

our resident microbes as an additional human organ.

The human–microbial relationship has also been compared to that of a

superorganism, like a colony of bees, or that of a holobiont,

like a coral reef.

The human microbiome: an astounding number of bacteria.

the body mass:

Human microbiome: function and dysfunction

Health Human Microbiome Project (HMP) in the United States

Metagenomics of the Human Intestinal Tract (MetaHIT) project in Europe.

Since the early 2000s, numerous studies have investigated the structure

and function of the human microbiome.

2008

previously viewed at best as passive

commensal or nuisances to be scrubbed or

flossed away, we now recognize that the

human oral, gut, skin, and uritogenital

microbiota play critical roles in maintaining

host health by performing essential

functions

in digestion and metabolism

vitamin production

immune system education and maintenance

by restricting the colonization, growth, reproduction, and virulence expression

of exogenous bacterial pathogens through resource competition.

The oral, gut, skin, and uritogenital microbiota play essential functions for

human health

On the other hand, poor diet, illness, stress, antimicrobial drugs, and other

environmental disruptions,

obesity,

type II diabetes,

irritable bowel disease,

colon cancer,

periodontal disease and dental

decay, atherosclerosis and

endocarditis,

autism, anxiety, and depression.

the ecology of the human

microbiome can transition

from a mutualistic to a

dysbiotic state,

The healthy human microbiome also plays host to a number of endemic,

but potentially acute, opportunistic pathogens.

microbiome plays a critical role in making us human, in keeping us

healthy, and in making us sick,

Streptococcus pneumoniae,

Haemophilus influenzae,

Neisseria meningitidis,

Clostridium difficile,

Propionibacterium acnes

Staphylococcus aureus

implicated in hospital and community-

acquired infections and pose particular risk

for the elderly and immunocompromised.

Alarmingly, multiple antibiotic resistant strains are increasingly being

detected in the normal oral and gastrointestinal microbiota of healthy

individuals.

This suggests that the use of antibiotic therapy, either through direct clinical

application or through indirect growth stimulating or prophylactic application in

livestock, impacts non-clinical targets and can result in long-term endogenous

reservoirs of antibiotic resistance.

Antibacterial therapies can themselves be disruptive to healthy bacterial

communities, leading to further complications. Therapeutic courses of broad-

spectrum antibiotics, for example, are known to disrupt gut and uritogenital

microbiota, where they may induce antibiotic-associated colitis and bacterial

vaginosis, respectively.

As a consequence, there is growing interest in probiotic and prebiotic therapies

for treating disrupted microbiomes, but lack of basic knowledge on what

constitutes a healthy microbiome, as well as a clearer understanding of the

transmission and formation of healthy microbiota, are limiting factors in the

development of these therapies.

Need for paleomicrobiology data

Although considerable effort has been invested in characterizing healthy gut and

oral microbiomes, recent investigations of rural, non-Western populations have

raised questions about whether the microbiota we currently define as normal

have been shaped by recent influences of modern Western diet, hygiene,

antibiotic exposure, and lifestyle.

When and how did our bacterial communities become distinctly human?

And what does this mean for our microbiomes today and in the future?

How do we acquire and transmit microbiomes and to what degree is this affected

by our cultural practices and built environments?

How have modern Western diets, hygiene practices, and antibiotic exposure

impacted ‘normal’ microbiome function?

Are we still in mutualistic symbiosis with our microbiomes, or are the so-called

‘diseases of civilization’—heart disease, obesity, type II diabetes, asthma,

allergies, osteoporosis—evidence that our microbiomes are out of ecological

balance and teetering on dysbiosis?

At an even more fundamental level, who are the members of the human

microbiome, how did they come to inhabit us, and how long have they been

there?

Who is ‘our microbial self’?

we know remarkably little about the diversity, variation, and evolution of the human

microbiome both today and in the past.

We are left with many questions:

Even the most extensive sampling of modern microbiota will provide

limited insight into Pre-Industrial microbiomes.

By contrast, the direct investigation of ancient microbiomes

from discrete locations and time points in the past would

provide a unique view into the coevolution of microbes and

hosts, host microbial ecology, and changing human health

states through time.

Ancient microbiome research

Upon death, the ecology of the human microbiome transforms

dramatically through the process of soft tissue decomposition.

With the exception of frozen and mummified remains, only two

microbiomes routinely produce substrates that, under favorable

conditions, persist after death in archaeological contexts: fecal

material of the gut microbiome may desiccate or mineralize to

produce coprolites, and dental plaque of the oral microbiome

calcifies in situ during life such that by the time of death it is already

in a semi-fossilized state known as dental calculus that resists

decomposition and thus continues to preserve after death.

Ancient microbiome research

Dental calculus

Dental calculus is a calcified bacterial biofilm that forms on the

surfaces of teeth, and it is found in all human populations, as

well as Miocene apes (12.5–8.5 Ma), Neanderthals, wild

chimpanzees, and a range of animals.

Among humans, both in the past and today,

the incidence of dental calculus is near-

ubiquitous among adults by age 30 and it is

not uncommon to observe dental calculus

deposits in excess of 100 mg in

archaeological assemblages of agricultural

populations.

a rich source of ancient

biomolecules,

Dental calculus: a rich source of ancient biomolecules,

Extracted DNA yields up to three orders of magnitude greater than from bone or dentine

of the same individual.

Importantly, dental calculus is unique among

ancient microbiome sources in that it does

not shed, remodel, or turnover; rather, it

forms incrementally through serial deposition

and mineralization in situ, making it a

layered record of human life history specific

to each person.

Two of the greatest dietary shifts in human evolution involved

the adoption of carbohydrate-rich Neolithic (farming) diets

(beginning ~10,000 years before the present) and the more

recent advent of industrially processed flour and sugar (in

~1850).

Sampling

34 prehistoric European

human skeletons:

11 males,

11 females

12 of unknown sex,

Dating from before the

Mesolithic period (before

farming) to the medieval

period.

The ancient samples were compared to modern calculus (n = 6)

and plaque (n = 13) samples.

Experimental design

We extracted bacterial DNA from sterilized ancient calculus samples (n = 34) and

generated PCR amplicon libraries of the 16S rRNA gene, targeting three

hypervariable regions (V1, V3 and V6) with barcoded primers

In addition, primers specific to Streptococcus mutans and Porphyromonas

gingivalis were used to detect oral pathogens in ancient dental calculus

Amplicons generated from

extracted samples and

multiple extraction blanks

were sequenced using both

conventional and

pyrosequencing

technology.

At the phylum level, the bacterial composition of ancient calculus was similar to

that of modern oral samples and sequences from the Human Oral Microbiome

Database (HOMD)30 but markedly distinct from the compositions identified for

laboratory reagents (extraction blanks) and environmental samples (soils,

sediments and water) and within the ancient teeth themselves

Results

The archaeological calculus was dominated by Firmicutes (33%), which was

found at a frequency comparable to those in both the HOMD (37%), and modern

oral samples (average 50%). In addition to Firmicutes, the ancient dental calculus

samples contained all 15 phyla commonly found in the modern human oral cavity,

with high percentages of Actinobacteria (19%), as is observed in modern

calculus deposits (7%).

Results

PCA plots of PC1 and PC2 (c) and PC2 and PC3 (d) that only include ancient and

modern oral pyrosequencing samples separated the hunter-gatherer (Mesolithic)

samples from modern, medieval and Neolithic samples.

Changes in the diversity and composition of oral microbiota.

The composition of oral microbiota underwent a distinct shift with the

introduction of farming in the early Neolithic period, with the earlier hunter-

gatherer groups having fewer caries- and periodontal disease–associated

taxa.

Overall, it is clear that

modern Europeans have

much lower oral microbial

diversity than either

Mesolithic or preindustrial

Neolithic groups

(P < 0.001, including fewer

bacteria associated with good

health, Ruminococcaceae).

After the transition to agriculture in the early Neolithic period, there was a

notable consistency in the composition of bacteria through the medieval period

(~400 years before the present), in parallel with the broad similarity of food-

processing technologies during these times. In contrast, today’s oral

environment is much less biodiverse and is dominated by potentially cariogenic

bacteria.

Major changes in carbohydrate intake in human history seem to have affected the

ecosystem of the mouth, opening up pathological niches for periodontal disease in

the early Neolithic period and caries in the recent past.

Overall, it is clear that modern Europeans have periodontal disease–associated

taxa (for example, Porphyromonas gingivalis similar to early agriculturists.

This is consistent with skeletal evidence showing marked increases in

periodontal disease following the transition to an agricultural diet,

suggesting a major impact on the human oral ecosystem around this time.

This is thought to be caused by increased amounts of soft carbohydrate

foods compared with hunter-gatherer diets.

Notably, the frequency of Streptococcus mutans is significantly higher in

modern samples than in preindustrial agricultural samples (P < 0.0001),

indicating that caries-associated bacteria have only become dominant after

medieval times.

This change is most likely associated with the onset of the Industrial Revolution,

which began some 200 years ago and represents the largest change in food

production and processing technology since the shift to farming. The Industrial

Revolution saw the production of refined grain and concentrated sugar from

processed sugar beet and cane, generating mono- and disaccharides, which are

the main substrates for the microbial fermentation that lowers plaque pH and

causes enamel demineralization.

Conclusions

Biotecnologie applicate ed Evoluzione

Scopo del corso: studio dei meccanismi genetici ed evolutivi alla

base delle risposte degli organismi all’ambiente

Approccio: genetico-ecologico

Analisi dei principi di base della genetica ecologica, condotta a diversi livelli

(molecolare, di organismo e di popolazione), attraverso la prospettiva

evolutiva

Obiettivi formativi:

- comprendere i meccanismi che sono alla base della formazione e

mantenimento della diversità genetica

- capire l’importanza dei meccanismi adattativi in ambito biotecnologico

- comprendere il destino dei biotech nei sistemi naturali, recettori ultimi di

tali prodotti

- rilevare costi e benefici dell’applicazione delle biotecnologie.

Prima parte:

comprensione dei processi microevolutivi alla base dell’origine e mantenimento

della diversità biologica

The Modern Synthesis

Prima parte:

comprensione dei processi microevolutivi alla base dell’origine e mantenimento

della diversità biologica

Seconda parte:

applicazione dei principi dell’evoluzione nello sviluppo ed applicazione delle

biotecnologie

Food and fiber

HealthEnvironment

Genetic markers

Genetic population

models

Strumenti e Attività pratiche

Results

Raw data

Input files

Data analyses

QUESTION

Experimental

design

ANSWER