A detailed PowerPoint presentation on "Synthetic Biology Approaches to Identify Enzymes From Metagenome" mainly extracted from the article that can be accessed at: Appl Biochem Biotechnol (2017) 183:636–651 DOI 10.1007/s12010-017-2568-3 CONTENTS Synthetic Biology and Metagenomics Metagenomic Technology Metagenomic Approaches to identify enzymes and biomolecules Function-based metagenomics Synthetic Biology overcome limiting steps in activity-based metagenomic library screening Enzymes discovered by function-based metagenomics Tools for function-based metagenomic analysis Sequenced-based screening of metagenomic libraries High-Throughput Metagenomic Sequencing and Screening Strategies Omic Technologies Integrated with Metagenome Research Applications of Metagenomics in Enzyme Bioprospecting Novel Enzymes discovered through marine metagenomic Approaches Conclusion References

1. Seminar Presentation on
“Synthetic Biology Approaches to identify Enzymes from
Metagenome”
Presented By: Maliha Rashid
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2. Table of Contents
1. Synthetic Biology and Metagenomics
2. Metagenomic Technology
3. Metagenomic Approaches to identify enzymes and biomolecules
4. Function-based metagenomics
5. Synthetic Biology overcome limiting steps in activity-based metagenomic library screening
6. Enzymes discovered by function-based metagenomics
7. Tools for function-based metagenomic analysis
8. Sequenced-based screening of metagenomic libraries
9. High-Throughput Metagenomic Sequencing and Screening Strategies
10. Omic Technologies Integrated with Metagenome Research
11. Applications of Metagenomics in Enzyme Bioprospecting
12. Novel Enzymes discovered through marine metagenomic Approaches
13. Conclusion
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3. Synthetic Biology
• Synthetic biology is the design and
construction of new biological parts,
devices, and systems, and the re-design
of existing, natural biological systems for
useful purposes.
Metagenomics
• Metagenomics is the study of the structure
and function of entire nucleotide
sequences isolated and analyzed from all
the organisms (typically microbes) in a
bulk sample.
• Metagenomics is often used to study a
specific community of microorganisms,
such as those residing on human skin, in
the soil or in a water sample.
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4. Metagenomic Technology
• based on the direct isolation of genomic DNA from environmental samples
• found to be powerful tools for tapping the genetic and metabolic diversity
of complex ecosystems
• used for the identification of novel enzymes encoded by a single gene or a
small-sized operon
• whereas large-sized insert libraries are required for the isolation of large
biosynthetic gene clusters, which encode for complex pathways containing
several genes
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5. Metagenomic
Approaches for
screening
Enzymes and
Biomolecules
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6. Function-Based Metagenomic Screening
Most Common: enzyme activity-based
screens performed in culture plates
• Examples:
1. starch-iodine test for amylase
2. cellulose screening assay for cellulase
As gene sequence information is not
required, functional screening is best for
the identification of novel genes encoding
novel enzymes
Screening Strategies
1. phenotypic detection
2. heterologous
complementation of host
strains
3. induced gene expression
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7. Continued…
Advantages
• prior knowledge of sequence is
not required, and we may get
novel gene sequence without
any similarity to previously
existing sequences
Disadvantages
the chances for failure in gene
expression mainly due to
difficulties in
• promoter recognition
• translational inefficiency
• misfolding of proteins
• defective post-translational
modification of desired proteins
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8. Continued…
• This can be resolved by
1. using vectors capable of accommodating large insert size
2. using vectors with broad host range which allow expression in
multiple hosts i.e., E. coli, Streptomyces lividans and Pseudomonas
putida
3. using rossetta E. coli strains which contain tRNA for rare amino acid
codons
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9. Synthetic Biology overcome limiting steps in activity-based
metagenomic library screening
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10. Enzymes discovered from function-based screening
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Glycosyl
Hydrolases
Novel cellulases Esterases
Amylases
Novel ß-
glucopyranosidase

11. Tools for Function-based metagenomic screening of
Clones
Substrate-Induced Gene
Expression (SIGEX)
Product-Induced Gene
Expression (PIGEX)
Metabolite Regulated
Expression (METREX)
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12. Sequence-Based Metagenomic Screening
• metagenomic clones are screened using an oligonucleotide primer or
probes for the target gene using the colony hybridization technique to
shortlist the clones
• The desired gene may also be amplified by PCR cloned in appropriate
expression vectors.
• This technique leads to discovery of novel sequences.
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13. Continued…
• However, these sequences may share similarity to pre-existing
sequence.
• This technique opens the possibility of finding enzymes with high
activity and efficiency. Example:
• Chitinase
• Hydrogenase
• Phosphatase
• glycerol dehydratase
• hydrazine oxidoreductase
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14. Continued…
• The sequencing strategy depends on the complexity of metagenomic
community
• If the complexity of the microbial community is high, new sequencing
strategy has to be used
Warnecke et al. created metagenomic sequencing data from wood
feeding termites. They generated about 71 million base pairs of
sequence information. They could identify 700 domains of different
glycosyl hydrolases, 45 different carbohydrate active enzymes, etc.
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15. Flow Chart Diagram Explaining the Enzyme Prospecting
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16. High-Throughput Metagenomic Sequencing and
Screening Strategies
Targeted Gene Sequencing
DNA is isolated from
environmental samples using
highly efficient DNA extraction
and purification methods. The
target genes are amplified
using designed primers with
oligonucleotide tags and also
with sequencing adaptors
which sequence the pooled
multiple sample.
Shotgun Metagenome Sequencing
The genomic DNA is
fragmented, end repaired and
ligated with adaptors which
allow amplification of template
and subsequent sequencing
generates large number of short
reads, which can be further
assembled and annotated with
various computational tools and
techniques
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18. Omic Technologies Integrated with Metagenome
Research
• Metatranscriptomics - RNA-based sequence information of microbial
communities in a complex ecosystem
• Metaproteomics - the study of total proteome expressed by the
microorganisms within an ecosystem at a particular period of time
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19. Applications of Metagenomics in Enzyme Bioprospecting
Enzyme Vector/host Environment
Esterase Plasmid library/E. coli Soil from river valley
Thermo-stable esterase Fosmid library/E. coli Mud sediment
Glycosyl hydrolase Λ phage library/E. coli Cow Rumen
Halotolerant tannase Plasmid library/E. coli Soil (cotton field)
Cellulase E. coli Anaerobic beer less
Endo-1,4-endoglucanase YEP356/E. coli Seaweed
Laccase pIndigo BAC5/E. coli Seawater
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20. 20
Applications of Enzymes in various industries

21. Novel Enzymes discovered through marine metagenomic
Approaches
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22. Conclusion
• access to the genetic and metabolic diversity of the microbial communities
• revolutionized industrial production system w.r.t the identification and isolation
of novel biocatalysts
• formulation of various computational tools for the efficient analysis
• link to the genetic diversity and metabolic activities of uncultivable microbes
• elucidate the functional dynamics, activities and production capabilities of
microbial consortia
• opportunity to identify, isolate and develop novel biocatalysts
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23. References
1. Appl Biochem Biotechnol (2017) 183:636–651 DOI 10.1007/s12010-
017-2568-3
2. The Authors (2014) Microbial Biotechnology published by John
Wiley & Sons Ltd and Society for Applied Microbiology, Microbial
Biotechnology, 8, 52–64
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