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

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. Background
Polyketide Synthases (PKSs) Family
2

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. 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.  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. Background
Bacterial PKSs type III
6

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. 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. 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. Background
PKSs and Metagenomics
10

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. Background
Atlantis II deep brine pool , Red Sea
12

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. 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. 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. 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. 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. Methodology
18

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. 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. 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. 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. Results and Discussion
23

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. 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. Results & Discussion
Functional annotation
26

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. Conserved Domains & Features
Results & Discussion
2. Functional annotation of predicted “ATIIChSyn” ORF :
28
http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi

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. Results & Discussion
Phylogenetic analysis
30

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. Plant
Results & Discussion
Plant
cyanobacteria
Plant Symbiotic
bacteria
32

33. Amoeba
Results & Discussion
Eukaryotes
Amoeba symbiotic
bacteria
33

34. Results & Discussion
Homology Modelling
34

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. Structural Superimposition
Results & Discussion
4.Comparative homology modelling of ATII-ChSyn:
36

37. Results & Discussion
Enviromental abundance
37

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. Results & Discussion
Isolation of ATII-ChSyn
39

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. 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. 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. 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. pET -28b+
0.1 mM IPTG
15’
30’
40’
60’
Results & Discussion
7.Expression of ATII-ChSyn
Supernatant
44

45. Denaturation Conditions
Flow
through
Elution Fractions
Results & Discussion
7. Purification of recombinant “ATII-ChSyn” from the pET28b+ATIIChSyn construct
45

46. Conclusions and perspectives
46

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.  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. 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