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 to natural degradation Persistent organic pollutants (POP) Potent 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 of by-products High 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. These technologies include: Genomics, “the study of genes and their function” (Human Genome Project (HGP), 2003) Proteomics, the study of proteins.

D IAGRAM

NEXT GENERATION SEQUENCING Next-generation sequencing (NGS), also known as high- throughput 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 sequencing read 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 biosensor- based 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 Metagenomics for the discovery of pollutant degrading enzymes. Journal of Biotechnology Advances,06982:10.