structure and rearrangements of a modular pks
TRANSCRIPT
Some Metagenomics and…..The
Structure and Rearrangements of a
Modular PKS
David H. Sherman
University of Michigan, Life Sciences Institute, Dept. of Medicinal Chemistry
Intriguing Biosynthetic Mechanisms
(R. Berlinck, R. Sarpong, R. Williams)
Notoamide A
(+)-versicolamide B
O
HNH
O
NNH
OO
O
HHN
O
NNH
OO
(-)-versicolamide B
O
HNH
O
NNH
O
O(-)-notoamide B
O
HHN
O
NNH
O
O
(+)-notoamide B
(+)-stephacidin A
O
H
NNH
O
HN O
O
H
NNH
O
HNO
(-)-stephacidin A
Marine Aspergil lus sp. Terrestrial Aspergi l lusversicolor
R S
S
1. Bicyclo [2.2.2] diazaoctane core
2. Indoxyl spiro-center
3. Enantiomeric assembly of the
fungal isoprenylated [2.2.2]
diazaoctane alkaloids
Finefield et al. Angew. Chem. Intl. Ed. 2012
Finefield. et al. J. Nat. Prod.. 2012
• Searching for new drug leads from marine microbes
• Culture previously unidentified bacteria and fungi from a variety of marine sources
• These organisms are grown in liquid culture
• Extracts are screened for bioactivity
• Extracts with interesting activity are purified to isolate the bioactive metabolite
Collection
Primary
Sources
Secondary
Isolation
Fermentation
Extraction
Bioassays & high-throughput screening
3
Traditional Discovery
Collection
High-throughput
sequencing
Bioinformatic assembly, chemical
probe synthesis, biochemical
validation
Heterologous
expression
Extraction
High-throughput structure elucidation
4
Emerging NP Discovery Model
• Explore new drug leads from unculturable marine microbial symbionts
• Assemble genomes and express biosynthetic systems from a variety of marine sources
• High throughput screening for bioactivity
• Pursue priority molecules for molecular probe development and drug discovery
ET-743: Elucidating the origin of a chemotherapeutic natural product
Marine Invertebrates are Rich Sources of Bioactive Compounds
• Potent natural products are thought to serve as a chemical defense for sessile invertebrates
• Many of these compounds are thought to be produced by bacterial symbionts
• Many symbionts remain incapable of being cultured in the laboratory.
• Culture-independent methods are needed
Ecteinascidia turbinata and ET-743• Mangrove tunicate
• Producer of chemotherapeutic ET-743 (Yondelis®)
• The drug is currently produced in a lengthy semi-synthetic process
• Thought to be produced by an uncultivable bacterial symbiont
Moss et. al. (2003) Marine Biology 143(1):99-110Pérez-Matos A, Rosado W, and Govind N (2007) Antonie van Leeuwenhoek 92(2):155-64
Ecteinascidia turbinata and ET-743
• Mangrove tunicate
• Producer of chemotherapeutic ET-743 (Yondelis®)
• The drug is currently produced in a lengthy semi-synthetic process
• Thought to be produced by an uncultivable bacterial symbiont
3. Saframycin Mx1 in Myxococcus xanthus4. Safracin in Pseudomonas fluorescens
1. ET-743 in E. frumentensis2. Saframycin A in S. lavendulae
Endoecteinascidia frumentensis: Producer of ET-743?
1. ET-743 in E. frumentensis2. Saframycin A in S. lavendulae
3. Saframycin Mx1 in Myxococcus xanthus4. Safracin in Pseudomonas fluorescens
Rath CM et al. (2011) ACS Chem Biol
ET-743 Putative Biosynthetic Pathway
Rath CM et al. (2011) ACS Chem Biol
Metagenomic Sequencing Efforts
Tunicate
Metagenomic Sequencing Efforts
Tunicate
PacBio sequencing at UM core (1 kB library)
MDA single cell sequencing
Illumina and PacBio (10 kB) combined sequencing at JGI
X X X
Metagenomic Sequencing Efforts
Tunicate
PacBio sequencing at UM core (1 kB library)
MDA single cell sequencing X X Illumina Metagenomic Sequencing at JGI
Overview of the Metagenome
15233 15306 19872 21664
Total Assembled Sequences
427549 466685 493145 465849
Total Bases 808986041 839356773 847549657 837783164
Protein Coding Genes 1588700 1658335 1683129 1648896
Largest Contig (bp) 97417 391789 163783 171962
Smallest Contig (bp) 200 200 200 200
SM COGS 1102 1160 1200 1160
Sequenced Tunicate Samples
Expanding the ET-743 Biosynthetic Gene Cluster
35 kb contig
428 kb contig
Name kb kd Blast ID Function
EtuA1 1.9 76.636 NRPS module C-A-T
EtuA2 1.3 167.313 NRPS module C(PS)-A-T-RE
EtuA3 5.4 210.893 NRPS dimodule FA-T-C-A-T
EtuD1 0.8 29.929 TatD Mg2+ depedent cytoplasmic DNase
EtuD2 0.8 36.693 DNA polymerase III delta prime subunit
EtuD3 0.7 25.232 DNA polymerase I 5'-3' exonuclease domain
EtuF1 1.3 43.122 Acetyl-CoA carboxylase biotin carboxylase subunit
EtuF2 0.5 17.098 Acetyl-CoA carboxylase biotin carboxylase subunit
EtuF3 1.8 87.021 Penicillin acylase
EtuH 0.4 18.42 Catechol hydroxylase
EtuM1 1.1 41.556 SAM dependent methyltransferase
EtuM2 0.7 25.554 SAM dependent O-methyltransferase
EtuN1 1.4 56.151 Asp/Glu-tRNA amidotransferase subunit B
EtuN2 1.4 43.367 Asp/Glu-tRNA amidotransferase subunit A
EtuN3 0.2 11.963 Asp/Glu-tRNA amidotransferase subunit C
EtuO 1.5 57.587 FAD dependent monoxygenase
EtuP1 2 76.412 Pyruvate dehydrogenase E1 component
EtuP2 1 42.531 Pyruvate dehydrogenase E2 component
EtuR1 0.9 32.435 Bacterial symbiont gene for protein found in host
EtuR2 0.3 13.328 Transcriptional regulator MerR family
EtuR3 0.4 15.575 DNA K suppressor protein
EtuT 0.8 35.189 Drug metabolite transporter superfamily protein
EtuU1 1.4 53.601 EtuP peptidase U62 modulator of DNA gyrase
EtuU2 0.5 20.221 Shikimate kinase I
EtuU3 0.3 14.111 Hypothetical protein
Gene Product Classification Designation
Pyruvate Dehydrogenase EtuP3
Methyltransferases
EtuM3, EtuM4, EtuM5,
EtuM6, EtuM7
Fatty acid biosynthesis EtuF4
Putative P450 EtuO2
Acytltransferase TBD
Drug Transport TBD
Phosphopantotheoylcysteine synthetase TBD
Origin of glycolic acid (EtuP1/P2/gene 287)
EtuP1 obtains α,β-dihydroxyethyl-Thiamine diphosphate (ThDP) from
a ketose phosphate (xylulose, fructose, or
sedoheptulose suggested)
Expanding the ET-743 Biosynthetic Gene Cluster
35 kb contig
428 kb contig
What we’re missing:1. Acetylation after EtuO 2. Thioether ring formation after EtuO 3. N-methylation 4. Transamination? 5. Methylene dioxybridge formation 6. Incorporation of tyrosine derivative
Have a candidate for acetylation Several methyltransferase candidates
Still digging for transamination candidate
EtuO2 is a candidate for methylene dioxybridge formation EtuA2 repeat Pictet-Spengler?
From Clusters to Genomes
• Do we have the complete genome?
• What could the genome tell us about this organism
– Direct bacterial link to ET-743 production
– Endosymbiosis
– Cultivation
– Host and symbiont evolution
ESOM puts bacterial DNA into distinct bins
Sunit Jain
ESOM puts bacterial DNA into distinct bins
Sunit Jain
An Ideal ESOM Map
Sunit Jain
Bin 1
Bin 2More
Bins
Sunit Jain
ET
CyanoMore Bins
Sunit Jain
Combining Sequenced Samples
Combining Sequenced Samples
Combining Sequenced Samples
General Genome Comparison
Organism Type GenomeSize
GC Content Coding % Pseudogenes
Endoecteinascidia frumentensisSuspected Endosymbiont 1.263 23.29 90.76 22 (in progress)
Coxiella burnetiiIntracellular Pathogen 2.033 42.60 87.81 83
Francisella novicidaIntracellular Pathogen 1.9 32.24 89.88 --
Rickettsiella grylliIntracellular Pathogen 1.561 37.93 89.63 --
Fangia hongkongensisFree living 2.693 37.94 91.42 --
Buchnera aphidicolaEndosymbiont 0.64 26.29 86.53 --
Wigglesworthia glossinidia Endosymbiont 0.72 25.22 87.95 --
General Genome Comparison
Organism Type GenomeSize
Coding % GC Content Pseudogenes
Endoecteinascidia frumentensisSuspected Endosymbiont 1.263 90.76 23.29 22 (in progress)
Coxiella burnetiiIntracellular Pathogen 2.033 87.81 42.60 83
Francisella novicidaIntracellular Pathogen 1.9 89.88 32.24 --
Rickettsiella grylliIntracellular Pathogen 1.561 89.63 37.93 --
Fangia hongkongensisFree living 2.693 91.42 37.94 --
Buchnera aphidicolaEndosymbiont 0.64 86.53 26.29 --
Wigglesworthia glossinidia Endosymbiont 0.72 87.95 25.22 --
Organism Type GenomeSize
Coding % GC Content Pseudogenes
Mycobacterium leprae TN
Intracellular Pathogen(Actinobacteria)
3.268 74.52 57.8 --
Rickettsia prowazekii Breinl
Intracellular Pathogen(Alphaproteobacteria)
1.109 76.76 29.01 --
Candidatus Endolissoclinum faulkneriL5
Endosymbiont(Alphaproteobacteria)
1.51 57% ~35% --
Summary
• Expanded the ~35 kB ET-743 biosynthetic gene cluster to a > 400 kB scaffold that contains additional (dispersed) biosynthetic genes.
• Sequence assembly resulted in a single bin representing majority of genome for ET-743 producing organism.
• Genome is completely annotated and analysis is underway.
• Previous studies combined with new genomic evidence suggest E. frumentensis is a novel endosymbiont undergoing genome reduction with its tunicate host.
• We have preliminary insights into possible reasons the organism has resisted cultivation.
Sequencing Pathway annotation DNA fragments for assembly of entire pathwayA.PCR from metagenomeB.Synthetic double stranded DNA
Invitrogen GeneArt® Strings™:; 1000bp at $149
IDT gBlocks™; 500bp at $99
Gibson Assembly
plasmid
DNA fragments
Bacterial artificial chromosome (BAC)
Plasmid assembly A
Plasmid assembly BAssembled pathway Plasmid assembly C
Sample
Metagenomicextraction
Heterologousexpression
Streptomyces venezuelae ATCC 15439
The Methymycin and Pikromycin Biosynthetic
Gene Cluster
0 10 20 30 40 50 60 (kb)
pLZ51
pLZ62
pLZ71
pLZ82
pME43
pLZ81
pLZ78
pLZ4
pLZ56
pikAI pikAII pikAIII pikAIVpikR1pikR2
desVIII desVII desVI desV desIV desIII desII desIdesR
A B CR
pikAV pikC pikD
D
Xue et al., PNAS, 1998; Gene, 2000
pikC pikR des (pikB)
I II III IVR1
R2
V C DIIIIIIIVVVIVIIVIII R
pikDpikA
The Macrolide Pathways of
Streptomyces venezuelae
O
O
O
O OHO
NMe2
Me
O
O
O O
O OHO
NMe2
Me O
O
O O
O OHO
NMe2
Me
OH
O
O
O
O OHO
NMe2
Me
R1
R2
O
O
O O
OH
O
O
O
OH
narbonolide
1 malonyl-CoA +5 methylmalonyl-CoA
pikA
pikB (des)pikA
pikB (des)
YC-17
narbomycin
pikC
pikC
10-deoxymethynolide
pikromycin
methymycin (R1=OH, R2=H)neomethymycin (R1=H, R2=OH)
1 malonyl-CoA +6 methylmalonyl-CoA
novamethymycin (R1=OH, R2=OH)
Xue et al., Chem. Biol. 1998; Wilson et al., J. Bact. 2001
Pikromycin Pathway Studies
• Pathway characterization
• Sugar pathway engineering and
glycosylation
• Chain elongation, channeling and release
• Docking domain interactions
• Polyketide termination and cyclization
• Hydroxylation/C-H functionalization
• Structural analysis during complete catalytic
cycle (Cryo Electron Microscopy)
module 0 module 1 module 2 module 3 module 4 module 5 module 6
PikAI PikAII PikAIII PikAIV
KSQ
AT
ACP KSAT KR
ACP KS
ATMKR
ACP
DHKS
AT KR*
ACP KS
ATKR
ACP
ER
DHKS
AT KR
ACPKS
AT
ACP TE
Starter unit Extender units
(2S)-Methylmalonyl-CoA (2S)-Methylmalonyl-CoA Malonyl-CoA 1 X 5 X 1 X
10-Deoxymethynolide
(10-DML)
Narbonolide (NBL)
467-481 883-915
1361-14031479-1492
1519-1543dimerizationhelices
dockinghelix
dockinghelix
1-38
1544-1562
AT5 KR5
ACP5
KS5
Xue et al., PNAS, 1998; Gene, 2000; Dutta, Whicher et al., 2014
Mechanism of Modular Type I Polyketide Synthases (PKS)
KS
S
O
Me
AT ACP
CoA-S
O
Me
AT
CoA-S
O
OH
O
Me
KRACP
SH S
O
Me
KS
SH
KS ATKR
ACP
S
O
Me
OH
Me
NADPH
S
O
Me
OH
Me
KS etc.
AT
KRACP
S
O
CO2H
Me -CO2
KS
SH
ATKR
ACP
S
O
Me
O
Me
LOAD MODULE 1
1
Propionyl CoA
Methylmalonyl CoA
MODULE 1
2 3
MODULE 1
MODULE 1
4
MODULE 2
1) The AT of the loading module loads the KS of module 1 with propionyl CoA
5
2) The ACP is loaded with methyl malonyl CoA by the AT of module 1
3) Decarboxylation and attack on the KS-bound propionate gives the extended -ketothioester
4) The KR reduces the -ketothioester
5) No more reductive modules are present, so the chain is transferred to module 2
Chemoenzymatic Synthesis
Hansen et al. JACS 135(30):11232-11238 (2013)
This methodology delivered both macrolide antibiotic classes in 13 steps
(longest linear sequence) from commercially available (R)-Roche ester in >10%
overall yields.
Structure and Molecular Dynamics of a Complete Modular PKS
Electron Cryo-Microscopy (Cyro EM)
Freeze Sample
Imaging
Pick
Particles/proce
ss images
Image Stack
Initial modelRefined model
Refine Reconstruction
Yiorgo Skiniotis, Somnath
Dutta
Employed Cryo EM and different combinations
of substrates to trap PikAIII in each state
State 1
Resting state
holo-PikAIII
State 2
MM-PikAIII
State 5
β-hydroxyhexaketide-
PikAIII
State 4
β-Ketohexaketide-
PikAIII
State 3a
Upstream ACP
fusion
State 3b
Pentaketide
Holo-PikAIII
Cryo Electron Microscopy
Dutta et al., 2014; Whicher et al., 2014
Structure and Molecular Dynamics of a Complete Modular PKS
Chemoenzymatic Synthesis
Hansen et al. JACS 135(30):11232-11238 (2013)
This methodology delivered both macrolide antibiotic classes in 13 steps
(longest linear sequence) from commercially available (R)-Roche ester in >10%
overall yields.
Current substrate panel
PhS
O O OH
conserve C1-7 vary C8-11
PhS
O O OH
PhS
O O OH
PhS
O O OH
PhS
O O OH
PhS
O O OH
PhS
O O OH
PhS
O O OH
PhS
O O OH
PhS
O O OH
PhS
O O NH2
PhS
O O NHMe
stereoisomers truncations amino
First analogs with PikAIII-TE
66%
<5%
32%
14%
23%
ND
PikAIII-TE
Enzymatic reaction conditions: 1 mM pentaketide, 20 mM MM-NAC, 0.1 mM NADP+, 2.5 mM G6P, 0.5 unit/mL G6PDH, and 1 μM purified PikAIII-TE. 4hrs RT
Pentaketide Epimer AnalysisO
O
O
OH
S
O O OH
O
O
O
OH
S
O O OH
O
O
O
OH
S
O O OH
O
O
O
OH
S
O O OH
PikAIII-TE
66%
5%
8%
13%
Question: How do we improve PKS catalysis with unnatural substrates?
KS-KetoSynthaseAT-Acyl TransferaseKR-KetoReductaseACP-Acyl Carrier ProteinTE-ThioEsterase
Thioesterase Enzyme Mechanism
• Pik TE catalysis is facilitated via Ser148-His268-Asp176 triad
His
ACP
SH
TE
Ser
AspO
O
NN H
O
O
O
Me
Me
OH
Me
Me
O
Me
OH
His
ACP
SH
TE
Ser
AspO
O
N
NH
O
O
OO
O
Me
Me
HMe
Me
HO
Me Me
O
O
Me
O
Me
MeMe
OHMe
O
His
ACP
O
O
Me
Me
OH
Me
Me
O
Me
OH
S
TE
Ser
AspO
O
N
N HO
H
His
ACP
SH
TE
Ser
AspO
O
NN H
O
O
O
Me
Me
OH
Me
Me
O
Me
OH
His
ACP
SH
TE
Ser
AspO
O
N
NH
O
O
OO
O
Me
Me
HMe
Me
HO
Me Me
O
O
Me
O
Me
MeMe
OHMe
O
His
ACP
O
O
Me
Me
OH
Me
Me
O
Me
OH
S
TE
Ser
AspO
O
N
N HO
H
HisHis
ACPACP
O
O
MeMe
MeMe
OHOH
MeMe
MeMe
O
MeMe
OHOH
S
TETE
SerSer
AspAspO
O
N
N HO
H
Pikromycin TE with Diphenylphosphonate
Pentaketide
Akey et al., Nature Chem. Biol. 2006
Insights into Macrolactonization
“creating the curl”
His268
Thr77
Gln183
Ser148
Ala217 Ala221
Hydrophilic Barrier
Anchor Point
Akey et al., Nature Chem. Biol. 2006
New Biosynthetic Challenges
O
OOO
O O
O O
O
O
H
O
OH
HH
H
HH
H
H
HH
H
H
HH
H
HH
O
O
O
O O
O
O
O O
O
O
O O
H
OH
OH
H
H
H
H
H
H
H
H
H
H
HH
H
H
H
H
H
H
H H
H
OH
OH
OH
HO
O
O
O
O
OO
OO
O
OO O
O O
O
O
O
O
O
OHOH
O
OO
O
O
O
O
OHO
S
O
O
O
O
O
O
OH
OHOS
OH
OH
OH OOH
O
H
HO OH
H
HO
HH
H HOH
H H HOH
HOH
O OHO
H H H H H
HO
H
H
OH
OHHHH
OH
OH OHH
H
HOH
H
OHH
HO
H
H
H
H
H
H
HO
H
H
H
H
OH
H
H
HH
OHH
H
H
H H HHOH
5
6
7
HO
O O
O
O
O
O
O
O
OH
HO
OH
8
Acknowledgements Sherman LaboratoryMichael-Marie SchofieldDrishti KaulDr. Fengan Yu Dr. Joe ChemlerPam ShultzShamilya Williams
Former Sherman Lab membersDr. Chris RathDr. Tyler NuscaDr. George Chlipala
Joint Genomics Institute Tijana Glavina del Rio (Illumina/PacBio)Susannah Tringe (Illumina/PacBio)Tanja Woyke (Single Cell)
Center for Chemical Genomics Martha J. LarsenTom McQuade
Collaborating LabsDr. Greg Dick & LabDr. Phillip Hanna & LabDr. Robert Williams & Lab (CSU)Dr. Xiaoxia (Nina) Lin & Lab
Tunicate CollectionsErich Bartels at Mote Marine Labs
Support
ICBG – Costa RicaNSF Graduate Research Fellowship ProgramCellular Biotechnology Training Program Rackham Graduate Student Research GrantsRackham International Research Award
Acknowledgements
University of Michigan
• Doug Hansen
• Dr. Joe Chemler
• Dr. Alison Narayan
• Dr. Courtney Aldrich
• Dr. Brian Beck
• Dr. Somnath Dutta
• Jon Whicher
• Wendy Hale
• NIH grant GM078553
• Hans W. Vahlteich Professorship
• U-M College of Pharmacy
Prof. Yiorgo Skiniotis Prof. Janet Smith
Prof. Kicki Hakansson