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The American University in Cairo
School of Sciences and Engineering

The Biotechnology Graduate Program

Polyketide Syntha...
Background

Polyketide Synthases (PKSs) Family

2
 Secondary metabolism

Background

 Polyketide Synthases (PKSs) Family

 Diverse polyketide natural products
 Natural ...
Carbon acetate units

CoA
carrier
molecules

Background

 Biosynthesis of natural Polyketides

Elongation Cycles
Polyketi...
 Most likely PKSs type III retrieved their
functionality from structurally related
homodimeric fatty acid KASs type III

...
Background

Bacterial PKSs type III

6
Background

 Bacterial PKSs type III
1. Evolutionary Origin
Plant Kingdom

Exclusive

Flavonoids

Chalcone Synthase (CHS)...
Background

 Bacterial PKSs type III
2. Importance
Bacterial PKS III

Plant Kingdom

Overall functional similarity
Only 2...
Organism
PKSs type III

Tetra-hydroxy-naphthalene
3. Examples
synthase (THNS)

Streptomyces
griseus

Significance


Role ...
Background

PKSs and Metagenomics

10
Metagenomic polyketide investigations
Soil Metagenomics

Novel antitumor polyketides

Symbiotic Bacteria in
beetles & mari...
Background

Atlantis II deep brine pool , Red Sea

12
Background

 Atlantis II deep brine pool , Red Sea
1. Formation

2194 m depth
60-km2 wide

13

Adapted from http://krse.k...
2. Characteristics
2000m

2194m

Atlantis
II layers

Interphase layer (INP)

Background

 Atlantis II deep brine pool , R...
3. Hydrothermally generated aromatic compounds

Background

 Atlantis II deep brine pool , Red Sea

Aromatic compounds
60...
Background

 Atlantis II deep brine pool , Red Sea
4. Aromatic degrading bacteria

Stabilized
resonance ring
Possible PKS...
Study Objective

AIM
 Screening and Isolation of possible bacterial PKS type III in
Atlantis II deep brine pool using a m...
Methodology

18
Methodology

1. Brine pool samples collection , DNA extraction &
sequencing:

Sample Collection
3 µm

Serial Filtration

0...
Pfam accessions
PF00195 N terminal domain
PF02797 C terminal domain

Methodology

2. Screening the LCL 454 metagenomic dat...
Phylogenetics analysis
dataset : 85 bacterial, plant, fungi &
amoeba PKSs type III + ATII-ChSyn

Methodology

3. Computati...
Screening the LCL environmental DNA
Amplification

Methodology

4.Isolation and identification of the putative PKS type II...
Results and Discussion

23
Results & Discussion

1. Computational screening of the LCL 454
metagenomic database for PKSs type III:
Screening LCL 454 ...
BLASTx 2.2.28 results
 Best hit : “chalcone & stilbene like synthase domain protein”
Organism: Rhizobium sp. PDO1-076 (Ac...
Results & Discussion

Functional annotation

26
350 a.a.
37.23 KDa

Results & Discussion

2. Functional annotation of predicted “ATIIChSyn” ORF :

27

BPROM
http://web.ex...
Conserved Domains & Features

Results & Discussion

2. Functional annotation of predicted “ATIIChSyn” ORF :

28

http://ww...
Multiple Sequence Alignment
Dimer interface
Residues lining
active site

Results & Discussion

2. Functional annotation of...
Results & Discussion

Phylogenetic analysis

30
Evolutionary Origin
1. First Hypothesis

 PKS type III enzymes was recently acquired to bacteria via horizontal
gene tran...
Plant

Results & Discussion

Plant

cyanobacteria

Plant Symbiotic
bacteria

32
Amoeba

Results & Discussion

Eukaryotes

Amoeba symbiotic
bacteria

33
Results & Discussion

Homology Modelling

34
Template
 The crystal structure Medicago sativa CHS complexed with
malonyl CoA as the template (PDB ID 1CML, Resolution 1...
Structural Superimposition

Results & Discussion

4.Comparative homology modelling of ATII-ChSyn:

36
Results & Discussion

Enviromental abundance

37
Atlantis II layers

 25 INP
 58 UCL
 134 LCL

Discovery Deep layers

 3 INP only
Aromatic compounds

Results & Discuss...
Results & Discussion

Isolation of ATII-ChSyn

39
Screening LCL of ATII environmental DNA

-35

-10

SD

F_ORF

F_read

R_read

ATG

TGA

256 bp
R_downstream1

Results & Di...
Cloning and Sequencing
E.coli Top 10

1128bp amplicon

p-GEM-T®

Sanger sequencing

Results & Discussion

6.Isolation and ...
Analysis of pET SUMO / ATII-ChSyn expression after IPTG induction
0.1mM IPTG

mM IPTG

0.1

0.2

0.5

1

U

37°C for 1 hou...
pET -28b+
C- terminal histidine tagged
Codon optimized sequence

ATII-ChSyn

6-histidine tag

NcoI

HindIII
38.75 kDa 363 ...
pET -28b+
0.1 mM IPTG
15’

30’

40’

60’

Results & Discussion

7.Expression of ATII-ChSyn

Supernatant

44
Denaturation Conditions

Flow
through

Elution Fractions

Results & Discussion

7. Purification of recombinant “ATII-ChSyn...
Conclusions and perspectives

46
 A Continuous need to discover natural polyketides with
promising pharmaceutical applications
 Exploring extreme environ...
 Optimization is required for the expression of the
recombinant protein

 Enzymatic assays for ATII-ChSyn is required to...
Acknowledgment
Dr. Ahmed Moustafa
Dr. Ari J. Scattone
My co-workers (Aya, Sarah, Nahla and Salma)
Lab mates especially Mah...
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Polyketide Synthase type III Isolated from Uncultured Deep-Sea Proteobacterium from the Red Sea – Functional and Evolutionary Characterization

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Screening and Isolation of possible bacterial PKS type III in Atlantis II deep brine pool using a metagenomic approach
and gaining a deeper insights into the evolutionary origin of PKS type III among Prokaryotes and Eukaryotes

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Polyketide Synthase type III Isolated from Uncultured Deep-Sea Proteobacterium from the Red Sea – Functional and Evolutionary Characterization

  1. 1. The American University in Cairo School of Sciences and Engineering The Biotechnology Graduate Program Polyketide Synthase type III Isolated from Uncultured Deep-Sea Proteobacterium from the Red Sea – Functional and Evolutionary Characterization By Hadeel El Bardisy Supervised by Dr. Ahmed Moustafa Dr. Ari José S. Ferreira 1 February 2014
  2. 2. Background Polyketide Synthases (PKSs) Family 2
  3. 3.  Secondary metabolism Background  Polyketide Synthases (PKSs) Family  Diverse polyketide natural products  Natural polyketides of pharmacological and biological advantages  Classified into type I,II and III  Plausible sequence of decarboxylative condensation reactions 3
  4. 4. Carbon acetate units CoA carrier molecules Background  Biosynthesis of natural Polyketides Elongation Cycles Polyketide Synthase Fatty Acid Synthase Cyclization patterns Full chain reduction 4
  5. 5.  Most likely PKSs type III retrieved their functionality from structurally related homodimeric fatty acid KASs type III PKS type III Background  Polyketide Synthases (PKSs) type III Both enzymes confer an overall homology despite low sequence similarity Main differences include the extent of catalytic loops at the Cterminal and number of other active residues involved in biosynthesis 5
  6. 6. Background Bacterial PKSs type III 6
  7. 7. Background  Bacterial PKSs type III 1. Evolutionary Origin Plant Kingdom Exclusive Flavonoids Chalcone Synthase (CHS) superfamily 1995 First bacterial PKS III Evolutionary history ?? 7 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.
  8. 8. Background  Bacterial PKSs type III 2. Importance Bacterial PKS III Plant Kingdom Overall functional similarity Only 25 – 50 % identity More diverse Microbial polyketides show promising pharmaceutical applications 8
  9. 9. Organism PKSs type III Tetra-hydroxy-naphthalene 3. Examples synthase (THNS) Streptomyces griseus Significance  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  Background PKSBacterial  type III Leading biocontrol agent against soil borne fungal pathogens 9
  10. 10. Background PKSs and Metagenomics 10
  11. 11. Metagenomic polyketide investigations Soil Metagenomics Novel antitumor polyketides Symbiotic Bacteria in beetles & marine sponges Background  PKSs and Metagenomics “Pedrin” putative antitumor Most polyketide metagenomic studies investigated PKS type I and II 11
  12. 12. Background Atlantis II deep brine pool , Red Sea 12
  13. 13. Background  Atlantis II deep brine pool , Red Sea 1. Formation 2194 m depth 60-km2 wide 13 Adapted from http://krse.kaust.edu.sa/spring-2010/mission.html
  14. 14. 2. Characteristics 2000m 2194m Atlantis II layers Interphase layer (INP) Background  Atlantis II deep brine pool , Red Sea Upper convective layer (UCL3) Upper convective layer (UCL2) Upper convective layer (UCL1) Lower Convective Layer (LCL) Rise in temp. & salinity LCL: 68.2°C, anoxic, high pressure, 25.7% salinity and pH 5.3 50°C 14
  15. 15. 3. Hydrothermally generated aromatic compounds Background  Atlantis II deep brine pool , Red Sea Aromatic compounds 60°C 150°C ATII “Suitable Environment ”  Wang & co workers have identified aromatic compounds in Atlantis II deep compared to Discovery deep 15 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.
  16. 16. Background  Atlantis II deep brine pool , Red Sea 4. Aromatic degrading bacteria Stabilized resonance ring Possible PKS III substrates Aerobic ring cleavage by oxygenases Anaerobic CoA ligation facilitates ring cleavage 16
  17. 17. Study Objective AIM  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 17
  18. 18. Methodology 18
  19. 19. Methodology 1. Brine pool samples collection , DNA extraction & sequencing: Sample Collection 3 µm Serial Filtration 0.8 µm 0.1 µm DNA Extraction Pyrosequencing 454 Metagenomic database 19
  20. 20. Pfam accessions PF00195 N terminal domain PF02797 C terminal domain Methodology 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 Enviromental abundance of PKS type III (ATII & DD) 20 Pfam accessions 454 metagenomic database (each layer) Reads NCBI BLASTP
  21. 21. Phylogenetics analysis dataset : 85 bacterial, plant, fungi & amoeba PKSs type III + ATII-ChSyn Methodology 3. Computational analysis 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 Structural Superimposition with template MODELLER version 9.12 Discovery Studio® visualizer 3.5 21
  22. 22. Screening the LCL environmental DNA Amplification Methodology 4.Isolation and identification of the putative PKS type III “ATII-ChSyn”: Cloning and Sequencing Expression pET -28b+ Champion™ pET SUMO E.coli BL21 (DE3) N- terminal histidine tagged C- terminal histidine tagged Codon optimized sequence 22 ATII-ChSyn Protein purification
  23. 23. Results and Discussion 23
  24. 24. Results & Discussion 1. Computational screening of the LCL 454 metagenomic database for PKSs type III: Screening LCL 454 metagenomic database PF00195 N-terminal Domain PF02797 C-terminal Domain HMM search 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) 1,053 bp size ORF1 ORF3 ORF4 ….. 61,132 ATII-ChSyn ORF67 62, 184 ORf68 …. 83 24
  25. 25. 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) Results & Discussion 1. Computational screening of the LCL 454 metagenomic database for PKSs type III:  Most hits were from the phylum Proteobacteria, class Alphaproteobacteria 25 http://blast.ncbi.nlm.nih.gov/Blast.cgi
  26. 26. Results & Discussion Functional annotation 26
  27. 27. 350 a.a. 37.23 KDa Results & Discussion 2. Functional annotation of predicted “ATIIChSyn” ORF : 27 BPROM http://web.expasy.org/cgi-bin/translate/dna_aa
  28. 28. Conserved Domains & Features Results & Discussion 2. Functional annotation of predicted “ATIIChSyn” ORF : 28 http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
  29. 29. Multiple Sequence Alignment Dimer interface Residues lining active site Results & Discussion 2. Functional annotation of predicted “ATIIChSyn” ORF : Plant PKS type III Bacterial PKS type III Catalytic triad CHN Malonyl CoA binding site Product binding site (Cyclization pocket) 29
  30. 30. Results & Discussion Phylogenetic analysis 30
  31. 31. Evolutionary Origin 1. First Hypothesis  PKS type III enzymes was recently acquired to bacteria via horizontal gene transfer (HGT) events from plants Results & Discussion 3. Phylogenetics Analysis 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. 31
  32. 32. Plant Results & Discussion Plant cyanobacteria Plant Symbiotic bacteria 32
  33. 33. Amoeba Results & Discussion Eukaryotes Amoeba symbiotic bacteria 33
  34. 34. Results & Discussion Homology Modelling 34
  35. 35. Template  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% Results & Discussion 4.Comparative homology modelling of ATII-ChSyn: Antiparallel β-sheets 35
  36. 36. Structural Superimposition Results & Discussion 4.Comparative homology modelling of ATII-ChSyn: 36
  37. 37. Results & Discussion Enviromental abundance 37
  38. 38. Atlantis II layers  25 INP  58 UCL  134 LCL Discovery Deep layers  3 INP only Aromatic compounds Results & Discussion 5. Environmental representation of bacterial PKS type III in brine pools: 38
  39. 39. Results & Discussion Isolation of ATII-ChSyn 39
  40. 40. Screening LCL of ATII environmental DNA -35 -10 SD F_ORF F_read R_read ATG TGA 256 bp R_downstream1 Results & Discussion 6.Isolation and identification of the putative PKS type III enzyme from ATII brine pool: 1128 40
  41. 41. Cloning and Sequencing E.coli Top 10 1128bp amplicon p-GEM-T® Sanger sequencing Results & Discussion 6.Isolation and identification of the putative PKS type III enzyme from ATII brine pool: Champion™ pET SUMO N- terminal histidine tagged 6-histidine tag SUMO ATII-ChSyn 51kDa 473 amino acid 41
  42. 42. Analysis of pET SUMO / ATII-ChSyn expression after IPTG induction 0.1mM IPTG mM IPTG 0.1 0.2 0.5 1 U 37°C for 1 hour S D Results & Discussion 7. Expression of ATII-ChSyn 42
  43. 43. pET -28b+ C- terminal histidine tagged Codon optimized sequence ATII-ChSyn 6-histidine tag NcoI HindIII 38.75 kDa 363 amino acid 0.1 mM IPTG U 1hr 2hr 3hr 4hr 5hr Results & Discussion 7.Expression of ATII-ChSyn 0.1 mM IPTG induced uninduced S D S D 43 37°C for 1 hour
  44. 44. pET -28b+ 0.1 mM IPTG 15’ 30’ 40’ 60’ Results & Discussion 7.Expression of ATII-ChSyn Supernatant 44
  45. 45. Denaturation Conditions Flow through Elution Fractions Results & Discussion 7. Purification of recombinant “ATII-ChSyn” from the pET28b+ATIIChSyn construct 45
  46. 46. Conclusions and perspectives 46
  47. 47.  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 Conclusions and Perspectives Conclusions 47
  48. 48.  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 Conclusions and Perspectives Future perspectives 48
  49. 49. 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 49

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