Diversity and Evolution of Secondary Metabolite Genes in the Marine ActinomyceteSalinispora

Nadine Ziemert

Natural Products as Drugs

Ecological Functions

www.rsc.org

Fundamental units with which microbes sense and respond to their environment

Moriah Sandy; Alison Butler; Chem. Rev. 2009, 109, 4580-4595.

Classical drug discovery

fermentationextraction

bioassayschromatographystructure elucidation

Strain isolation

Genome Mining• Biosynthesis• Regulators• Transporters• Resistance

cchem.berkeley.edu

• Huge amount of data • Big gene clusters, can exceed 100 kb• How to assess novelty• How to predict Bioactivity

• Huge amount of data• Bioinformatic analyses possible• Independent of regulation• Full genetic potential• New sources

Nonribosomal Peptide Synthetases and Polyketide Synthases

Schwarzer & Marahiel, Naturw. 2001 Mar;88(3):93-101

Directly connect genes to molecules

Salinispora• Actinomycetes from marine sediments

Jensen et al., Environ Microbiol. 2005,

S. arenicola S. pacifica S. tropica

Bill Fenical, Brad Moore, Paul Jensen

Salinispora Secondary Metabolites

Udwary et al., PNAS 2007, Nett et al. Nat Prod Rep. 2009,

100 Genomes

Diversity and Evolution of Secondary Metabolites1. How much diversity is out there?2. How pathways evolve to yield new

chemical diversity?3. Diversity and distribution of biosynthetic

pathways?4. Can we learn lessons about effective

sampling strategies?

NaPDoS - The Natural Product Domain Seeker

Ziemert et al., PLoS One 2012;7(3):e34064

Salinisporagenomes

Pathways PKS1PKS2PKS3

NRPS1NRPS2NRPS3

(Blin et al., NAR 2013)

Screening for NRPS and PKS genes

(Medema et al., Mol Biol Evol 2013) (Markowitz et al. Nucl. Acids Res 2014)

124 NRPS/PKS Pathways

species analyzed genomes

avg genome size

avg contig number

avg total pathways/genome

# of distinct pathways

S. arenicola 37 5.7 74 17.5 47 S. pacifica 31 5.4 76 14.1 88 S. tropica 7 5.4 90 13.6 19

Ziemert et al, PNAS 2014

Hierarchical Clustering

N. Millan

Species tree

Anna Lechner

&Matthias

Wietz

Pathway swapping

sporolide cluster

cyanosporasidecluster

PKS32 cluster ?

Functional Pathway Swapping

S. pacifica S. arenicolaS. tropica

S. pacifica

S. arenicola

Ziemert and Crüsemann et al., in prep.

Pathway Evolution

Mechanisms

Pathway swapping Pathway duplication

Gene gain and gene loss

Simplified species tree

1

1

genus specific

1

species specific

1

1

34 of 45 pathways (78%) occur at branch tips

Evolve by gene gain and gene loss, duplication and divergence, horizontal gene transfer of whole units and pathway swapping

75 S

alin

ispo

raG

enom

esConstant sampling of pathways

Strong correlations between species phylogeny and pathways

Pathways are mainly located in certain hot spots in the genome

Informs collection strategies: target new phylotypes and new locations

Summary

ThanksBrad MooreBailey BonetMax CrüsemannRobin Teufel

Paul JensenMustafa GulamAnna LechnerNatalie MillanMatthias WietzKate DuncanKrystle Chavarriah

Eric AllenSheila PodellJames Wang

Pieter DorresteinBill Fenical

S. pacifica, CNQ-768 GuamS. pacifica, CNS-103 PalauS. pacifica, CNR-894 PalauS. pacifica, CNR-114 GuamS. pacifica, CNT-003 FijiS. pacifica, CNT-001 FijiS. pacifica, CNT-131 FijiS. pacifica, CNT-603 FijiS. pacifica A, CNT-855 HawaiiS. pacifica, CNY-330 Sea of CortezS. pacifica, CNY-331 Sea of CortezS. pacifica, DSM-45544 FijiS. pacifica D, CNT-796 HawaiiS. pacifica D, CNT-851 HawaiiS. pacifica D, CNT-084 FijiS. pacifica D, CNT-609 FijiS. pacifica A, CNS-055 PalauS. pacifica A, DSM-45548 PalauS. pacifica B, DSM-45549 FijiS. pacifica C, CNT-124 FijiS. pacifica C, CNT-584 FijiS. pacifica C, CNT-854 HawaiiS. pacifica F, CNT-029 FijiS. pacifica C, DSM-45547 FijiS. pacifica C, DSM-45546 FijiS. pacifica C, CNS-860 FijiS. pacifica C, DSM-45543 FijiS. pacifica C, CNS-996 FijiS. pacifica C, CNT-045 FijiS. pacifica E, CNR-942 PalauS. pacifica E, CNT-569 FijiS. tropica, CNB-536 BahamasS. tropica, CNH-898 BahamasS. tropica, CNS-416 BahamasS. tropica, CNB-440 BahamasS. tropica, CNB-476 BahamasS. tropica, CNR-699 BahamasS. tropica, CNS-197 BahamasS. arenicola, CNB-458 BahamasS. arenicola, CNB-527 BahamasS. arenicola A, CNH-962 Sea of CortezS. arenicola, CNH-646 BahamasS. arenicola, CNH-905 BahamasS. arenicola, CNH-877 BahamasS. arenicola, CNY-011 BahamasS. arenicola B, CNH-941 Sea of CortezS. arenicola B, CNP-193 Sea of CortezS. arenicola B, CNH-964 Sea of CortezS. arenicola B, CNP-105 Sea of CortezS. arenicola, CNS-991 FijiS. arenicola, CNY-280 FijiS. arenicola, CNT-857 HawaiiS. arenicola, CNT-859 HawaiiS. arenicola, CNT-850 HawaiiS. arenicola, CNT-798 HawaiiS. arenicola, CNT-800 HawaiiS. arenicola, CNT-849 HawaiiS. arenicola, CNX-482 PalmyraS. arenicola, CNX-508 PalmyraS. arenicola, CNX-814 PalmyraS. arenicola, CNX-481 PalmyraS. arenicola, CNX-891 PalmyraS. arenicola, CNS-051 PalauS. arenicola, CNY-237 FijiS. arenicola, CNY-231 FijiS. arenicola, CNY-234 FijiS. arenicola, CNS-205 PalauS. arenicola, CNR-107 GuamS. arenicola, CNR-921 PalauS. arenicola, CNY-260 FijiS. arenicola, CNQ-748 GuamS. arenicola, CNT-005 FijiS. arenicola, CNS-673 FijiS. arenicola, CNS-744 FijiS. arenicola, CNY-256 Fiji

Des

Des

Des

Island 13

NRPS16

NRPS16

Island 16

NRPS16

© 2010 Universität Tübingen

Functional Pathway Swapping• Desferrioxamine is the more

ancient pathway• NRPS16 is acquired via horizontal

gene transfer • Acquisition of NRPS16 leads to

the deletion of the des pathway• Shows enormous plasticity of

secondary metabolite gene clusters

Questions: What drives evolution of siderophores in marine actinomycetes?Does NRPS16/ Salinichelin provide a better adaptation to the environment?

Cyanosporaside Evolution

???

Lane et al., 2013

Pathway Distribution

• More than half of the pathways were found in only 1 or 2 strains

• These pathways were recently acquired

• Indicates a high rate of “pathway sampling”

Number of strain in which pathways occur

1 (singletons)

2 (doubletons)

>3

Pattern-based Genome Mining

Duncan et al. 2015, Chemistry & Biology, Volume 22, Issue 4, 2015, 460 - 471

Heterologous Expression

Bonet et al. 2015, J Nat Prod. 2015 Mar 27;78(3):539-42

Genome miningSelect strain/culture

De-replicate

Bioinformatics

Extract DNAMe

O OH

OH

MeO

Me

Me

MeO

MePredict novelty

Culture

IdentifySequence

Bioassay

Connecting Genes to Molecules

Kersten, Ziemert et al., 2013 PNAS

Peptido- and Glycogenomics Pattern based Genome Mining

Heterologous Expression

Duncan, Ziemert et al. 2015, Chemistry & Biology

Bonet, Ziemert et al. 2015, J Nat Prod.