Transcript Document

METAGENOMICS FOR THE
DISCOVERY OF POLLUTANT
DEGRADING ENZYMES
MAIMONA SAEED
Ph. D BOTANY
ORGANIC POLLUTANTS
 Organic
pollutants are synthetic
compounds in the form of herbicides,
dyes, pesticides, plastics and drugs (Rieger
et al., 2002).
 Most
are aromatic molecules, polymers of
ring shaped molecules or planar
molecules,and are therefore among the
most stable and persistent molecules.
TYPES
POLYCYCLIC AROMATIC
HYDROCARBONS (PAH)
 Chlorinated
hydrocarbons,
 Steroids (phenols, phthalates)
 DYES
 Organocyanides nitriles,
 long chain aliphatics plastics
 insulating materials
 polyurethanes, organophosphates
 pyrethroid herbicides and pesticides.
TOXICITY
 Resistance
 Persistent
 Potent
to natural degradation
organic pollutants (POP)
carcinogens or mutagens
 Endocrine
disrupting properties
PHYSICAL AND CHEMICAL
TECHNOLOGIES
 Electrochemical
treatments
 Oxidising agents
 Activation by ultraviolet rays
 Adsorption of pollutants
 Membrane filtration
 Ion exchange
 Electrokinetic coagulation
DRAWBACKS OF PHYSICAL AND
CHEMICAL TECHNOLOGIES
 Formation
 High

of by-products
sludge production
High processing costs
 Lack
of experties
BIOREMEDIATION
 Bioremediation
is a waste management
technique that involves the use of
organisms to remove or neutralize
pollutants from a contaminated site.
 According
to the EPA, bioremediation is
a “treatment that uses naturally occurring
organisms to break down hazardous
substances into less toxic or non toxic
substances”
ADVANTAGES OF BIOREMIDIATION
 Bioremediation
may result in the complete
metabolisation of pollutants.

Considered to be a highly effective and
environmentally friendly strategy
(Colleran,1997).

Microorganisms have aerobic and anaerobic
catabolic strategies to degrade the huge range of
organic compounds present in the ecosystems
they colonise.
MICROBIAL BIODEGRADATION
 Microorganisms
have indeed developed a wide
range of aerobic and anaerobic catabolic
strategies to degrade the huge range of organic
compounds present in the ecosystems they
colonise.
 Because
pollutant molecules are often
structurally similar to natural molecules, one can
assume that there are always some organisms in
contaminated ecosystems that are able to
metabolise pollutants, which serve as their main
carbon source.
Cont…
 During
microbial degradation, all changes in the
chemical structure of pollutants are due to the
action of enzymes, whose specificity is often
broad enough to accommodate several molecules
of similar structures.
 Once
identified and isolated, these enzymes can
therefore be engineered by directed evolution to
improve their stability or efficiency with respect
to a particular compound
‘OMICS’ TECHNOLOGIES.
 Omics
refers to the collective technologies used
to explore the roles, relationships, and actions of
the various types of molecules that make up the
cells of an organism.
technologies include: Genomics, “the
study of genes and their function” (Human
Genome Project (HGP), 2003) Proteomics, the
study of proteins.
 These
DIAGRAM
NEXT GENERATION SEQUENCING
 Next-generation
sequencing (NGS), also
known as highthroughput sequencing, is the
catch-all term used to describe
a number of different
modern sequencing
technologies including:
 Illumina (Solexa) sequencing.
 Roche 454 sequencing.
FUNCTIONAL METAGENOMICS
 Functional
metagenomics, which consists in
assigning functions to proteins encoded by all
genomes of a microbial community with no
isolation and cultivation step.

A highly efficient way to boost the discovery of
novel biocatalysts from the huge diversity of
uncultured microbes.
SCREENING FOR POLLUTANT
DEGRADING ENZYMES
Two complementary approaches can be used to
identify pollutant degrading biocatalysts in
microbial communities.
 Gene sequence analysis and functional
annotation, based on the content of available
sequence databases.

The other is guided by the observation of
pollutant degrading phenotypes, harboured by
recombinant metagenomic clones.
SEQUENCE-BASED APPROACHES
Sequence-based metagenomics relies on,
 whole-genome
DNA extraction from microbial
communities,
 shotgun
 read
sequencing
assembly
Cont…

Another study succeeded in discovering a key functional
operon for naphthalene degradation by using the SIP
method coupled with shotgun sequencing targeted to the
13C-labelled DNA of metabolically active naphthalene
degraders (Wang et al., 2012).

Specific primers were therefore designed from the
operon [13C]DNA sequence to clone it into a biosensorbased genetic transducer system activated by salicylate,
an intermediate metabolite of naphthalene catabolism.

The involvement of the labelled operon in the
degradation of naphthalene was thus experimentally
confirmed through the activation of the biosensor by
salicylate.
DNA SHUFFLING

Another way to exploit the diversity of metagenomic
sequences is metagenomic DNA shuffling.

This method has been developed for the creation of novel
genes by recombination of the DNA sequence of a gene
of interest with metagenomic DNA fragments is used
from highly complex microbial communities, for
instance those of contaminated soils.

Boubakri et al. used this approach to create a library of
more than one thousand clones harbouring hybrid
sequences that was screened on a solid medium
containing lindane, an organochlorine pesticide. No less
than 23 new genes encoding lindane degrading enzymes
were obtained by using this smart technology (Boubakri
et al., 2006).
ACTIVITY BASED METAGENOMICS FOR THE
DISCOVERY OF NEW TOOLS FOR
BIOREMEDIATION
 This
technology relies on the observation of a
phenotype, linked to the reaction(s) involved in
breakdown of the targeted pollutant.
 It
has three prerequisites:
(i). Cloning of DNA or cDNA fragments between
2 and 200 kbp in length into an expression
vector (plasmids, cosmids or even bacterial
artificial chromosomes) for the creation of
metagenomic or metatranscriptomic libraries,
respectively;
Cont..
(ii). heterologous expression of cloned
genes into a microbial host;
(iii). design of sensitive phenotypic screens
to isolate clones of interest with the
targeted activity, also called screening
‘hits’.
Cont…

Two generic high-throughput strategies are widely used
for primary screening.

One is based on direct detection of colouration or
discolouration, when coloured substrates or chromogenic
substrates are used, or of a reduction in opacity of the
reaction medium when insoluble substrates are used
(Shah et al., 2008).

The other is based on heterologous complementation of
an auxotrophic host by foreign genes, which allows
microbial host growth on selective culture media (Xing
et al., 2012).
Cont..
 activity-based
metagenomics is the only
known way to identify new protein
families
 or
to attribute new functions to already
known protein families.
OXIDOREDUCTASES
Oxygenases
 Oxygenases are mostly sought after for the elimination
of aromatic compounds.
 To break them, bacteria use a typical aerobic degradation
pathway, which can be broken down into two critical
steps:
 ring hydroxylation of adjacent carbon atoms involving
phenol hydroxylases (phenol 2-monooxygenases).


ring cleavage of the resulting catecholic intermediates
involving catechol 1,2- or 2,3- dioxygenases (Silva et al.,
2013).
LACCASES.
 Laccases
are multi-copper oxidoreductases
 which
oxidise a wide variety of phenolic and
non-phenolic compounds, polycyclic aromatic
hydrocarbons like industrial dyes, pesticide
alkenes and recalcitrant biopolymers such as
lignin (Beloqui et al., 2006).
 Due
to their broad substrate specificity, they are
good candidates for bioremediation
Hydrolases
 Esterases

Esterase is a generic term for a hydrolase that catalyses
the cleavage of ester bonds.

Esterases are of particular interest for detoxification of
pesticides and herbicides.

Indeed, organophosphorus, pyrethroids and carbamate
pesticides and herbicides, which are known to affect the
mammalian nervous system (Kuhr and Dorough, 1976;
Sogorb and Vilanova, 2002; Singh andWalker, 2006),
can be hydrolysed and detoxified by these enzymes.
NITRILASES

Nitriles are components of plastics, polymers and
herbicides which can be hydrolysed to their
corresponding carboxylic acids and ammonia by
nitrilases in a one-step reaction (Pace and Brenner,
2001).

In this respect, mention should be made of two
publications describing screening of metagenomic
libraries to search for nitrilases.

The libraries were constructed with bacterial DNA
isolated from environmental samples collected from
terrestrial and aquatic microenvironments worldwide
(Robertson et al., 2004) or from oil-contaminated soil,
wastewater treatment from a refinery and forest soils
(Bayer et al., 2011).
CONCLUSION AND FUTURE GOALS

The rise of meta-omics technologies in the last decade
has enabled to unlock the functional potential of
uncultivable microbial biodiversity.

In particular, many enzymes exclusively produced by
bacteria have been discovered thanks to the activity
based exploration of metagenomes, espically those
originating from highly polluted environments and also
expose to extreme physical conditions.

Represent new tools for environmental biotechnology.
Cont…

Nevertheless, several challenges still have to be faced.

Firstly, although prokaryotic microbial communities are
still the subject of numerous studies, the functions of
uncultured eukaryotes are rarely explored using activity
based metatrancriptomic approaches. However,
eukaryotes play a fundamental role in many
ecosystemand some cultivable fungi are valuable
bioremedation tool.
Cont…

Secondly, the literature is very rich in functional
metagenomics studies targeting oxidases and esterases
with wide range of specifications. But references to the
discovery of proteases, which are likely to be effective
for the hydrolysis amide bond are almost inexistent.

Finally, whatever their origin, using enzymes for
bioremediation processes at low cost requires breaking
the locks of enzyme expression in appropriate host and
for some applications in open environment.
References

Ufarte, L., E. Laville, S. Duquesne, G. Potocki-Veronese.
2105. Metagenomics for the discovery of pollutant
degrading enzymes. Journal of Biotechnology
Advances,06982:10.