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Polyketide Synthase type III Isolated from Uncultured Deep-Sea Proteobacterium from the Red Sea – Functional and
Evolutionary Characterization
By Hadeel El Bardisy
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The American University in Cairo School of Sciences and Engineering
The Biotechnology Graduate Program
Supervised by
Dr. Ahmed Moustafa
Dr. Ari José S. Ferreira
February 2014
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Polyketide Synthases (PKSs) Family
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Secondary metabolism
Diverse polyketide natural products
Natural polyketides of pharmacological and biological advantages
Classified into type I,II and III
Plausible sequence of decarboxylative condensation reactions
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Polyketide Synthases (PKSs) Family
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Biosynthesis of natural Polyketides B
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Fatty Acid Synthase
Polyketide Synthase
CoA carrier
molecules Carbon acetate units
Elongation Cycles
Cyclization patterns
Full chain reduction
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Polyketide Synthases (PKSs) type III B
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Most likely PKSs type III retrieved their functionality from structurally related homodimeric fatty acid KASs type III
Both enzymes confer an overall homology despite low sequence similarity
Main differences include the extent of catalytic loops at the C-terminal and number of other active residues involved in
biosynthesis
PKS type III
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Bacterial PKSs type III
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Bacterial PKSs type III B
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1. Evolutionary Origin
Plant Kingdom
Flavonoids
Chalcone Synthase (CHS) superfamily
Exclusive
1995 First bacterial PKS III
Evolutionary history ??
Ueda, K., Kim, K. M., Beppu, T., & Horinouchi, S. (1995). Overexpression of a gene cluster encoding a chalcone synthase-like protein confers redbrown pigment production in Streptomyces griseus. The Journal of Antibiotics, 48(7), 638–646.
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Bacterial PKSs type III B
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2. Importance
Plant Kingdom Bacterial PKS III
Overall functional similarity
Only 25 – 50 % identity
More diverse
Microbial polyketides show promising pharmaceutical applications
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Bacterial PKSs type III B
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3. Examples
PKS type III Organism Significance
Tetra-hydroxy-naphthalene synthase (THNS)
Streptomyces griseus
Role in pigmentation Biosynthesis of
Naphthoquinines metabolites (antibacterial, antitumor, antioxidant)
Di-hydroxy-phenyl-glycine synthase (DHPG)
Amycolatopsis Biosynthesis of balhimycin (resistance to MRSA)
Germicidin synthase (Gcs) Streptomyces coelicolor
Germicidin (spore germination)
Streptomyces resorcinol synthase (SrsA)
Streptomyces griseus
Biosynthesis of phenolic lipids in cytoplasmic membrane (resistance β-lactam antibiotics)
PKS 10, PKS 11 and PKS 18 Mycobacterium tuberculosis
Role phenolic lipid cell wall (mycolic acid)
Alkyl resorcinol synthase (ArsB & ArsC)
Azotobacter vinelandii
Biosynthesis of alkyl resorcinol in the cyst wall
phloroglucinol synthase (PhlD)
Pseudomonas flourescens
Leading biocontrol agent against soil borne fungal pathogens
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PKSs and Metagenomics
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PKSs and Metagenomics B
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Metagenomic polyketide investigations
Soil Metagenomics Novel antitumor polyketides
Symbiotic Bacteria in beetles & marine sponges “Pedrin” putative antitumor
Most polyketide metagenomic studies investigated PKS type I and II
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Atlantis II deep brine pool , Red Sea
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Atlantis II deep brine pool , Red Sea B
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1. Formation
Adapted from http://krse.kaust.edu.sa/spring-2010/mission.html
2194 m depth 60-km2 wide
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Atlantis II deep brine pool , Red Sea B
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2. Characteristics
Atlantis II layers
Interphase layer (INP)
Upper convective layer (UCL3)
Upper convective layer (UCL2)
Upper convective layer (UCL1)
Lower Convective Layer (LCL)
LCL: 68.2°C, anoxic, high pressure, 25.7% salinity and pH 5.3
Rise in temp. & salinity
50°C
2000m
2194m
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Atlantis II deep brine pool , Red Sea B
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3. Hydrothermally generated aromatic compounds
Aromatic compounds
60°C 150°C
ATII “Suitable Environment ”
Wang & co workers have identified aromatic compounds in Atlantis II deep compared to Discovery deep
Wang, Y., Yang, J., Lee, O. O., Dash, S., Lau, S. C. K., Al-Suwailem, A., … Qian, P.-Y. (2011). Hydrothermally generated aromatic compounds are consumed by bacteria colonizing in Atlantis II Deep of the Red Sea. The ISME Journal, 5(10), 1652–1659.
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Atlantis II deep brine pool , Red Sea B
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4. Aromatic degrading bacteria
Stabilized resonance ring
Aerobic Anaerobic
ring cleavage by oxygenases
CoA ligation
facilitates ring cleavage
Possible PKS III substrates
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Stu
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Ob
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Screening and Isolation of possible bacterial PKS type III in Atlantis II deep brine pool using a metagenomic approach
Deeper insights into the evolutionary origin of PKS type III among Prokaryotes and Eukaryotes
AIM
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Methodology
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1. Brine pool samples collection , DNA extraction &
sequencing:
Sample Collection
Serial Filtration 3 µm
0.8 µm
0.1 µm DNA Extraction
Pyrosequencing
454 Metagenomic database
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Me
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log
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2. Screening the LCL 454 metagenomic database & Functional annotation of putative ORF:
Hidden Markov Model Search
Against the LCL 454 metagenomic database
Against LCL 454 assembled metagenomic database
ORF1 ORF2 ORF3 ….. ATII-ChSyn ORF67 ORf68 …. 83
Functional annotation
Pfam accessions
PF00195 N terminal domain PF02797 C terminal domain
Pfam accessions
454 metagenomic database
(each layer) Reads NCBI
BLASTP
Enviromental abundance of PKS type III (ATII & DD)
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3. Computational analysis
Phylogenetics analysis
dataset : 85 bacterial, plant, fungi & amoeba PKSs type III + ATII-ChSyn
Alignment by MUSCLE
PhyML version 3.0 program
Interactive Tree Of Life (iTOL) version 2.1 online tool
Comparative homology modelling of ATII-ChSyn
3D Model of ATII-ChSyn
MODELLER version 9.12
Structural Superimposition with template
Discovery Studio® visualizer 3.5
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Me
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log
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4.Isolation and identification of the putative PKS type III “ATII-ChSyn”:
Screening the LCL environmental DNA
Cloning and Sequencing
Amplification
Expression
Champion™ pET SUMO pET -28b+
N- terminal histidine tagged C- terminal histidine tagged Codon optimized sequence
ATII-ChSyn Protein purification
E.coli BL21 (DE3)
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Results and Discussion
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Re
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1. Computational screening of the LCL 454 metagenomic database for PKSs type III:
PF02797 C-terminal Domain
61,132 62, 184
1,053 bp size
Screening LCL 454 metagenomic database
PF00195 N-terminal Domain
81 similar reads 76 similar reads
Assembly
Contig1 1408bp (105 reads )
Screening LCL 454 assembled metagenomic database
Contig 2: 84,461 bp (83 possible ORFs)
ORF1 ORF3 ORF4 ….. ATII-ChSyn ORF67 ORf68 …. 83
HMM search
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Re
sults &
Disc
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1. Computational screening of the LCL 454 metagenomic database for PKSs type III:
BLASTx 2.2.28 results
Best hit : “chalcone & stilbene like synthase domain protein” Organism: Rhizobium sp. PDO1-076 (Accession: WP_009109596.1)
Best biochemically characterized hit: “Chalcone synthase ” Organism: Rhizobium etli CFN42 (RePKS) (Accession: YP_468285.1)
Most hits were from the phylum Proteobacteria, class Alpha-proteobacteria
http://blast.ncbi.nlm.nih.gov/Blast.cgi
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Functional annotation
Re
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Disc
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Re
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2. Functional annotation of predicted “ATII-ChSyn” ORF :
350 a.a.
37.23 KDa
http://web.expasy.org/cgi-bin/translate/dna_aa
BPROM
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Re
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2. Functional annotation of predicted “ATII-ChSyn” ORF :
Conserved Domains & Features
http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
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Re
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2. Functional annotation of predicted “ATII-ChSyn” ORF :
Multiple Sequence Alignment
Catalytic triad CHN
Malonyl CoA binding site
Product binding site (Cyclization pocket)
Plant PKS type III
Bacterial PKS type III
Dimer interface
Residues lining active site
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Phylogenetic analysis
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Re
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3. Phylogenetics Analysis
Evolutionary Origin
1. First Hypothesis
PKS type III enzymes was recently acquired to bacteria via horizontal gene transfer (HGT) events from plants
2. Second Hypothesis
Higher plants acquired PKS type III via HGT events from ancient eubacteria where it was then lost during prokaryotic evolution
1. Austin, M. B., & Noel, J. P. (2003). The chalcone synthase superfamily of type III polyketide synthases. Natural Product Reports, 20(1), 79–110. 2. Moore, B. S., & Hopke, J. N. (2001). Discovery of a new bacterial polyketide biosynthetic pathway. Chembiochem: A European Journal of Chemical Biology, 2(1), 35–38.
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Re
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Plant Symbiotic bacteria
Plant
cyanobacteria
Plant
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Re
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Disc
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Eukaryotes
Amoeba symbiotic bacteria
Amoeba
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Homology Modelling
Re
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Disc
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Re
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4.Comparative homology modelling of ATII-ChSyn:
The crystal structure Medicago sativa CHS complexed with malonyl CoA as the template (PDB ID 1CML, Resolution 1.69Ả)
BLASTP: similarity 43% , identity 24%, length coverage 98%
Template
Antiparallel β-sheets
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Re
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4.Comparative homology modelling of ATII-ChSyn:
Structural Superimposition
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Enviromental abundance
Re
sults &
Disc
ussio
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Re
sults &
Disc
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5. Environmental representation of bacterial PKS type III in brine pools:
Atlantis II layers Discovery Deep layers
25 INP 58 UCL 134 LCL
3 INP only
Aromatic compounds
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Isolation of ATII-ChSyn
Re
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Disc
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Re
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6.Isolation and identification of the putative PKS type III enzyme from ATII brine pool:
Screening LCL of ATII environmental DNA
-35 -10 SD
256 bp
F_read R_read F_ORF
R_downstream1
ATG TGA
1128
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Re
sults &
Disc
ussio
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6.Isolation and identification of the putative PKS type III enzyme from ATII brine pool:
Cloning and Sequencing
1128bp amplicon p-GEM-T® Sanger sequencing
E.coli Top 10
Champion™ pET SUMO
6-histidine tag SUMO ATII-ChSyn N- terminal histidine tagged
51kDa 473 amino acid
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Re
sults &
Disc
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7. Expression of ATII-ChSyn
Analysis of pET SUMO / ATII-ChSyn expression after IPTG induction
37°C for 1 hour
0.1 0.2 0.5 1 U
mM IPTG 0.1mM IPTG
S D
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Re
sults &
Disc
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7.Expression of ATII-ChSyn
ATII-ChSyn 6-histidine tag
C- terminal histidine tagged Codon optimized sequence
38.75 kDa 363 amino acid
NcoI HindIII
pET -28b+
U 1hr 2hr 3hr 4hr 5hr
0.1 mM IPTG
0.1 mM IPTG
S D S D
uninduced induced
37°C for 1 hour
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Re
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Disc
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7.Expression of ATII-ChSyn
0.1 mM IPTG
15’ 30’ 40’ 60’
pET -28b+
Supernatant
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Re
sults &
Disc
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7. Purification of recombinant “ATII-ChSyn” from the pET28b+-ATIIChSyn construct
Denaturation Conditions
Flow through
Elution Fractions
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Conclusions and perspectives
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Co
nclu
sion
s an
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ersp
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Conclusions
A Continuous need to discover natural polyketides with promising pharmaceutical applications
Exploring extreme environments as a source of natural polyketides is feasible by metagenomics
A putative PKS type III (ATII-ChSyn) was identified, amplified and sequenced from the LCL of ATII brine pool, Red Sea
Preliminary homology modelling probed an overall conserved fold of ATII-ChSyn structure and predicted a possible interaction of catalytic triad with malonyl-CoA substrate
Evolutionary analysis of bacterial PKS type III propose the possible involvement of amoeba symbiotic bacterium in HGT events from prokaryotes to eukaryotes
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Co
nclu
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s an
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ersp
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Future perspectives
Optimization is required for the expression of the
recombinant protein
Enzymatic assays for ATII-ChSyn is required to characterize its catalytic machinery in terms of substrate specifity, functional capabilities and product identification
Parallel efforts should be exploited to evaluate the enzyme pH, salinity and thermostable characteristics
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Acknowledgment
Dr. Ahmed Moustafa Dr. Ari J. Scattone My co-workers (Aya, Sarah, Nahla and Salma) Lab mates especially Maheera, Bothaina Amgad Ouf Mariam & Yasmeen KAUST Spring 2010 expedition members Ehab Moussa & Mohammed Saad Biotechnology graduate program Professors Dr. Asma Amleh My Dear Biotech Club members