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

Evolutionary Characterization

By Hadeel El Bardisy

1

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

Ba

ck

gro

un

d

2

Polyketide Synthases (PKSs) Family

Ba

ck

gro

un

d

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

3

Polyketide Synthases (PKSs) Family

4

Biosynthesis of natural Polyketides B

ack

gro

un

d

Fatty Acid Synthase

Polyketide Synthase

CoA carrier

molecules Carbon acetate units

Elongation Cycles

Cyclization patterns

Full chain reduction

5

Polyketide Synthases (PKSs) type III B

ack

gro

un

d

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

Ba

ck

gro

un

d

6

Bacterial PKSs type III

7

Bacterial PKSs type III B

ack

gro

un

d

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.

8

Bacterial PKSs type III B

ack

gro

un

d

2. Importance

Plant Kingdom Bacterial PKS III

Overall functional similarity

Only 25 – 50 % identity

More diverse

Microbial polyketides show promising pharmaceutical applications

9

Bacterial PKSs type III B

ack

gro

un

d

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

Ba

ck

gro

un

d

10

PKSs and Metagenomics

11

PKSs and Metagenomics B

ack

gro

un

d

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

Ba

ck

gro

un

d

12

Atlantis II deep brine pool , Red Sea

13

Atlantis II deep brine pool , Red Sea B

ack

gro

un

d

1. Formation

Adapted from http://krse.kaust.edu.sa/spring-2010/mission.html

2194 m depth 60-km2 wide

14

Atlantis II deep brine pool , Red Sea B

ack

gro

un

d

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

15

Atlantis II deep brine pool , Red Sea B

ack

gro

un

d

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.

16

Atlantis II deep brine pool , Red Sea B

ack

gro

un

d

4. Aromatic degrading bacteria

Stabilized resonance ring

Aerobic Anaerobic

ring cleavage by oxygenases

CoA ligation

facilitates ring cleavage

Possible PKS III substrates

17

Stu

dy

Ob

jectiv

e

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

18

Methodology

19

Me

tho

do

log

y

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

20

Me

tho

do

log

y

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)

21

Me

tho

do

log

y

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

22

Me

tho

do

log

y

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)

23

Results and Discussion

24

Re

sults &

Disc

ussio

n

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

25

Re

sults &

Disc

ussio

n

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

26

Functional annotation

Re

sults &

Disc

ussio

n

27

Re

sults &

Disc

ussio

n

2. Functional annotation of predicted “ATII-ChSyn” ORF :

350 a.a.

37.23 KDa

http://web.expasy.org/cgi-bin/translate/dna_aa

BPROM

28

Re

sults &

Disc

ussio

n

2. Functional annotation of predicted “ATII-ChSyn” ORF :

Conserved Domains & Features

http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi

29

Re

sults &

Disc

ussio

n

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

30

Phylogenetic analysis

Re

sults &

Disc

ussio

n

31

Re

sults &

Disc

ussio

n

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.

32

Re

sults &

Disc

ussio

n

Plant Symbiotic bacteria

Plant

cyanobacteria

Plant

33

Re

sults &

Disc

ussio

n

Eukaryotes

Amoeba symbiotic bacteria

Amoeba

34

Homology Modelling

Re

sults &

Disc

ussio

n

35

Re

sults &

Disc

ussio

n

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

36

Re

sults &

Disc

ussio

n

4.Comparative homology modelling of ATII-ChSyn:

Structural Superimposition

37

Enviromental abundance

Re

sults &

Disc

ussio

n

38

Re

sults &

Disc

ussio

n

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

39

Isolation of ATII-ChSyn

Re

sults &

Disc

ussio

n

40

Re

sults &

Disc

ussio

n

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

41

Re

sults &

Disc

ussio

n

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

42

Re

sults &

Disc

ussio

n

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

43

Re

sults &

Disc

ussio

n

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

44

Re

sults &

Disc

ussio

n

7.Expression of ATII-ChSyn

0.1 mM IPTG

15’ 30’ 40’ 60’

pET -28b+

Supernatant

45

Re

sults &

Disc

ussio

n

7. Purification of recombinant “ATII-ChSyn” from the pET28b+-ATIIChSyn construct

Denaturation Conditions

Flow through

Elution Fractions

46

Conclusions and perspectives

47

Co

nclu

sion

s an

d P

ersp

ectiv

es

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

48

Co

nclu

sion

s an

d P

ersp

ectiv

es

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

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