THE DIVERSITY OF THE MICROBIAL WORLD ...€¦ · ecosystems colonized by a given microbial...

THE DIVERSITY OF THE MICROBIAL WORLD

A CHALLENGE FOR THE FUTUREA CHALLENGE FOR THE FUTURE

Enrica Galli

Department of Biomolecular Sciences and Biotechnology

University of Milano

The Future of Science - Venice, September 20-22, 2009

EVOLUTION OF MICROBIAL WORLDEVOLUTION OF MICROBIAL WORLD

Microorganisms are the most versatile and adaptable forms of life onE th d th h i t d h f 3 5 billi F thEarth, and they have existed here for some 3.5 billions years. - For thefirst 2 billions years of their existence, bacteria alone ruled thebiosphere, colonizing every accessible ecological niche from glacial icep g y g gto the hydrothermal vents of the deep sea bottoms

What they did during this long” microbiological era”?What they did during this long microbiological era ?

Developed the major metabolic pathways characteristic of all living organisms todayliving organisms today

Changed Earth transforming its anaerobic atmosphere to one rich in oxygenyg

Created an environment able to sustain more complex forms of life

Ad t d t h i ld l di t l ti f


Adapted to an even-changing world leading to selection of new metabolic activities

Microbial diversityDiversity can be defined as the number of microbial species and their relative abundance and distribution of information in a community

Microorganisms possess not only the largest share of i i i h l h h bili

y

existing variety, they also have the ability to most rapidly create new diversity.

The microorganisms are the weightiest contributors to our planet biomass and have evolved an incredibly sophisticated way of managing their genetic and p y g g genzymatic contents in response to environmental signals to catalyze processes sustaining all life on earth


earth.

Diversity of soil and extremeDiversity of soil and extreme ecosystems

One gram of soil may harbor up to 10 billions microorganisms of thousands of different species

Most of microorganisms employed in biotechnological processes have been isolated from soil ecosystems but now we have evidence that the majority of microorganisms occurs in oceanic and terrestrial subsurface, representing a great resource for biotechnological exploration of novel organisms products andbiotechnological exploration of novel organisms, products and processes. In 2004 Craig Venter reported on Science the results of g penvironmental genome shotgun sequences in material from the Sargasso Sea near Bermuda. The data derive from at least 1800

i i i l di 148 i l k b i l


genomic species, including 148 previously unknown bacterial phylotypes.

Most of the diversity of microbial world is still unexplored; we can estimate that only 0 5 10 % of the diversity of microbial world is known due to theirthat only 0,5 -10 % of the diversity of microbial world is known due to their unculturability.

Source: De Lorenzo – Env. Microbiol 4, 2002


Sou ce: e o e o v. c ob o , 00

GENOMICS AND DIVERSITYGENOMICS AND DIVERSITYGENOMICS AND DIVERSITYGENOMICS AND DIVERSITYDiversity estimates for natural microbial communities have traditionally depended on cultivable species, leading to a longstanding underestimate ofdepended on cultivable species, leading to a longstanding underestimate of microbial diversity. Genomic analyses imply microbial diversity far greater than was predicted.G i l d d ib th b i f l t iG i l d d ib th b i f l t iGenomics analyzes and describes genomes on the basis of complete genomic Genomics analyzes and describes genomes on the basis of complete genomic sequencessequences

March 31, 2009March 31, 2009Data Release of complete genomes

Bacteria 526

Archaea 42

Viruses 3


Totals 571

FROM GENOMICS TO METAGENOMICSFROM GENOMICS TO METAGENOMICS

Metagenomic libraries are a powerful tool for exploring the fullMetagenomic libraries are a powerful tool for exploring the full extent of microbial diversity ,providing access also to the genetic information of uncultured microorganisms. These libraries, prepared from pools of DNA directly extracted from soil or other ecosystems colonized by a given microbial community, allow us :

•to recreate the gross genetic potential of a given ecosystem

•to know more about the enzymatic diversity of the microbial world

•to discover new reactions for producing new materials and products via environmentally friendly processes.


What are the grand issues thatWhat are the grand issues that microbial diversity might helpmicrobial diversity might help

us to solve?


EnvironmentBioremediation

Monitoring i t l lit

Pollution of water and soil by xenobiotic and recalcitrant compounds

environmental quality

Replacement of chemical pesticides by

CHALLENGESCHALLENGES

chemical pesticides by environmentally-friendly substitutes

CHALLENGESCHALLENGES Energy

Deleterious effects of f il f l

Novel biocatalysts(Green Chemistry)

R l t f f ilfossil fuels

Human health Novel therapeutic agents

Replacement of fossilfuels with biofuels

Human health

Antibiotic resistance and reemerging

Novel therapeutic agents (drugs, antibiotics)


g gdiseases

RIndustry

Wastes

L OR

Fuels

Solvents

PharmaceuticalsPolymers

AgrochemicalsGlues

Industry Living Organisms

RO H

O H

RO H

O H

Fuels

Explosives

Agrochemicals

Food

O H

Microbial Activities

O H


BIOREMEDIATIONBIOREMEDIATIONBIOREMEDIATIONBIOREMEDIATION

Bioremediation is the use of biological systems (mainly microoganisms) for the removal ofpollutants from aquatic or terrestrial systems. It is based on the extremely diverse metabolicpollutants from aquatic or terrestrial systems. It is based on the extremely diverse metabolicpotential of natural microbial communities. A challenge in this area is the existence ofxenobiotics, i.e. compounds produced by chemical synthesis for industrial or agriculturalpurposes and having no counterparts in the natural world.purposes and having no counterparts in the natural world.

The U.S. Environmental Protection Agency’s list of “priority pollutants”includes:industrial solventsbuilding blocks of plastics

l hl bi h lpolychlorobiphenylspesticides (halogenated and nitroaromatics, polycyclic aromatic compounds)

“In situ”: polluted soil is treated in its original place“In situ”: polluted soil is treated in its original place“Ex situ”: polluted soil is removed and treated somewhere else

Bioremediation may include the introduction of microorganisms (bioaugmentation),


y g ( g )ventilation and/or, adding nutrient solutions (biostimulation)

QuestionsQuestionsQuestionsQuestions

1. Is there the microbiological potential for bioremediation?

2. Is it possible to stimulate the indigenous microflora?2. Is it possible to stimulate the indigenous microflora?

Microbiological characterization of contaminated site:

- metagenomic analysisg y

- catabolic diversity of microbial population


CASE STUDY – Exxon Valdez – Alaska 1989 – Oil spill


CASE STUDY TRECATE 1994CASE STUDY – TRECATE 1994

12.6 thousands cube meters of oil contaminated thousands of hectares of cultivated soil The soilsthousands of hectares of cultivated soil . The soils with levels of contamination between 50 and 10000

ppm of TPH have been treated through “landfarming” h l Th fi l d htechnology.The final target was to reduce the

concentration of TPH to less than 50 ppm. After two years of treatment , the soils have been again used

for agricultural purposes.

13 h t f il ith hi h l l f13 hectares of soil with a higher level of contamination (20000 ppm) have been treated

through the biopile technology . The biodegradation reached the rate of 95% after 18 months of

treatment.


Source: ENI


Source: Lovley Nature Rev. 1, 2003







p yenvironmentally-friendly substitutes







Human health




g gdiseases

BiocatalysisBi t l t ff l ti f th d ti f i t f hi lBiocatalysts offer green solutions for the production of a variety of chiral chemicals that can be used as basic buildings blocks for the synthesis of pharmaceuticals, agrochemicals, pesticides.

The field covers all scales of chemical production and catalytic intervention:a) identification of new enzymes in metagenomes of different ecosystems, including extreme environments (hot springs,antarctic ice etc)b) the experimental evolution of such enzymes for endowing them at a highest level of performance of catalytic properties, also adapted to highlevel of performance of catalytic properties, also adapted to high salinity,elevated pressure and temperature and anoxic conditionsc) the application of Systems and Synthetic Biology approaches for metabolic engineeringengineeringd) the creation of designed enzymes “a la carte”e)the success of biocatalysis depends finally on the economics of specific processes


processes

PROCESSPROCESS

BiocatalystBiocatalystProductProduct

ProductsProductsReactantsReactants

Economics selectionselection

Screening

recoveryrecovery

Downstream

Enzyme orCell?

In situ recovery

BiocatalystBiocatalyst

ApplicationApplicationBiocatalystBiocatalyst

characterizationcharacterization

engineeringengineering

Cell engineering

Stability

Immobilization

Kinetics

Reaction conditions

Process engineering

Enzyme engineeringNew

Reactions

Cofactorregeneration

Multiphaset

Structural information


systems

BIOCATALYSIS GREEN CHEMISTRYBIOCATALYSIS GREEN CHEMISTRY

What is it?Chemical modifications of organic moleculesperformed by microorganisms and enzymes

Why are they useful ?y y

substrate specificity

…they allow:

substrate specificityenantioselectivityregioselectivityregioselectivitymild process conditionsenvironmental friendly processes


environmental-friendly processes

Stereoisomery and PropertiesStereoisomery and PropertiesStereoisomery and PropertiesStereoisomery and PropertiesFood sector

(R) Asparagine(S) AsparagineH2NCOCH2 CO2HF CH2CONH22HOOC F (R)- Asparagine

sweet taste(S) – Asparagine

bitter tasteNH2 H

F

NH2H

F

O O

(R)-CarvoneFlavour Mint

(S)-CarvoneFlavour Cumin

O O

Agrochemicalsector

(S)-Fluazifop butyl

CF3C

N

O O

HMe

BuOOCS

F3C

N

OO

HMe

CO2BuS

(R)-Fluazifop butyl herbicide(S) Fluazifop butyl

no activity

OH

t

OH

t

(2S,3S)-Paclobutrazol (2R 3R) Paclobutrazol

N

NN

But

Cl

R

N

NN

But

Cl

R

D


Plant growth regulator(2R,3R)-Paclobutrazol

Fungicide

Stereoisomery and PropertiesStereoisomery and Properties

Ph ti l(R)-Thalidomide(S)-Thalidomide

Stereoisomery and PropertiesStereoisomery and Properties

Pharmaceuticalsector

(R)-ThalidomideTeratogenic

(S)-ThalidomideSedative

Anti-emeticN

H

NH

O

O

DN

H

NH

O

O

O

D

OO

OO

OH HO

(S S) Chl h i l i i(R,R)-Cloramfenicolo

tibi ti

OH

O2N NHCOCHCl22

HO

NO22Cl2CHCONH2

(S,S)-Chloramphenicol no activityantibiotic

O

HO

HO

OH

H

(R)-Propranol(S)-Propranolol

OPr'NHCH2R

OCH2NHPr'R


100 times less active than isomer (S) β-blocker












Human health

Replacement of fossilfuels with biofuels:bioethanol and biodiesel

Human health

Antibiotic resistance and reemerging Novel therapeutic agents


g gdiseases

p g(drugs, antibiotics)

Università degli Studi di MilanoDipartimento di Scienze Biomolecolari e Milanop

Biotecnologie


Replacement of fossil fuels with Biofuels


SOURCE: ENI 2009


SOURCE: ENI 2009

Microbial production of biofuels from lignocellulose has severalMicrobial production of biofuels from lignocellulose has several advantages: 1) the production is not reliant on agricultural resources2) lignocellulose is the most abundant biopolymer on earth in the form of2) lignocellulose is the most abundant biopolymer on earth, in the form of plant materials (grasse, wood and crop residues); is a renewable, geographically distributed source of sugars that can be converted to ethanol

d h li id f land other liquid fuels3) new biosynthetic pathways can be engineered to produce fossil-fuel replacements4) a variety of micro organisms either bacteria or fungi have evolved the ability to use this nutrient source5) recent genome sequencing of cellulolityc organisms has espanded our ) g q g y g prepertoire of known or potential cellulolytic enzymes6) recent advances in synthetic biology and metabolic engineering will allow to overcome the limitations and engineer microorganisms for the cost


to overcome the limitations and engineer microorganisms for the cost effective production of biofuels from cellulose biomass.

Biodiesel from microbial source

Generation of Biodiesel from many microorganisms

Biodiesel from microbial source

Generation of Biodiesel from many microorganisms including bacteria, yeasts, algae and fungi have the ability to accumulate oils through the transesterification of oilsto accumulate oils through the transesterification of oils with short chain alcohols and can thus become feedstocks for biodiesel production. Bacteria can be engineered also p gto produce diesel type fuels.

The use of microorganisms as cell factories forThe use of microorganisms as cell factories for production of biodiesel and other types of fuel is today under intense development.


under intense development.


SOURCE: ENI 2009














Human health




g gdiseases

Problems

HHUMAN UMAN HHEALTHEALTH

•Widespread and misuse of antibiotics•Use of antibiotics to promote growth andprevent diseases in animals

Reemerging infectious diseases(i.e. tubercolosis) in growingpopulation groups with AIDS,cancer or other conditionsprevent diseases in animals

•Release of pharmaceuticals in theenvironment

cancer, or other conditionsleading to immunosuppression

ANTIBIOTIC RESISTANCE

CHALLENGEThe need for new antibiotic agents

Screening for new antibioticproducing microorganismsbased on biodiversity of

Combinatorial approach to create new antimicrobial agentsthrough the combination of cloned genes for the productionof new antibiotics and the screening of combinatorial


microbial worldg

libraries for affinity to defined target structures

Most of the antibiotics in use today are derivatives of natural products of Actinomycetes mainly and fungi. p y y g

Antibiotics produced by actinomycetes have been evolving for 1 billion of years acquiring the ability to penetrate otherfor 1 billion of years acquiring the ability to penetrate other microorganisms and inhibit the target enzymes, macromolecules or macromolecular structures.


The need for novel antibiotics

New antibacterial agents approved in the United States, 1983–2007, per 5‐year period (CLIN INFECT DIS 48(1):1-12, © 2008 the Infectious Diseases Society of America)

but only two new chemical classes!


but only two new chemical classes!

Antibiotics R&D

Discovery: longer Lead Optimization stage


scove y: o ge ead Opt at o stage

Approaches to revitalize antibiotic discovery from actinomycetes

High-throughput miniaturized fermentation and screening

Enrichments and selections for uncommon terrestrial and marine actinomycetes

Mining actinomycete genomes for cryptic antibiotic pathways

Accelerated evolution of new biosynthetic pathways by combinatorial biosynthesis

Optimizing natural product pharmacological properties with medicinal chemistry

Integrating a multidisciplinary BIG SCIENCE approach to take natural


product discovery to a higher level

Wh b t i bi l di it ?Wh b t i bi l di it ?Why care about microbial diversity?Why care about microbial diversity?

Learning more about these organisms allows us to explorethe frontiers of knowledge about the strategies and limitsof life.of life.

Microorganisms are of central importance to biosphere g p psustainability.

Microbial resources have value, particularly for biotechnology.

Microorganisms will give us the tools to improve the quality of life on our Planet.


quality of life on our Planet.

THE DIVERSITY OF THE MICROBIAL WORLD ...€¦ · ecosystems colonized by a given microbial...

Documents

Transcript of THE DIVERSITY OF THE MICROBIAL WORLD ...€¦ · ecosystems colonized by a given microbial...