ABSTRACT117 PKS-positive Contigs

Detected by anti-SMASH 21,2

Representative Structure

Predictions

Functional Metagenomics of Natural Products

ACKNOWLEDGEMENTSThis work was supported by NIH and NSF grants

Abstract

16S rRNA Genes in 925 BACs

16S rRNA containing contigs from 925 BACs were identified by

BLAST search of the soil metagenomic library using E. coli 16S rRNA

sequence as the query. 17 contigs were identified, corresponding to 11

unique clones. Each clone was then BLASTed against NCBI, and top

hits were identified. Note that clone 35L8 (bold) also contains a Type I

PKS gene cluster.

Clone

Contig

length Accession Top hit

%

Identity21B7 106,238 CP007268 Halorhodospira halochloris str. A 84.46

21K7 155,294 FJ625356 Uncultured bacterium clone HF_NC_17 87.96

24N15 21,183 JQ371560 Uncultured bacterium clone FL3Aa9_7480 92.33

24O4 22,253 JX120409 Uncultured bacterium clone UA_39 95.12

31F6 12,075 CP007207 Flavobacterium psychrophilum FPG3 92.27

33E21 29,532 CP007035 Niabella soli DSM 19437 88.34

35L8 81,829 CP002826 Oligotropha carboxidovorans OM5 82.23

46F13 28,292 JX120409 Uncultured bacterium clone UA_39 95.12

46N9 30,069 AF507711 Uncultured soil bacterium clone S098 96.16

48M7 64,008 KC172332 Uncultured soil bacterium clone DM8-64 97.44

50F24 85,939 GQ396906 Uncultured bacterium clone AK4AB1_09G 97.06

Functional Metagenomics for Natural ProductsHigh Diversity, Low Rediscovery Rate of Small Molecule Pathways

in Large Insert Soil Metagenomic Libraries

Varigen Biosciences, 505 S. Rosa Rd., Madison WI (608)444-9518 info@varigenbio.com

A new technology to unleash the full potential of uncultivated microbial

natural products has been developed. Large-insert soil metagenomic

libraries > 100 Kb were constructed and the contents of 19,200

individual BAC inserts have been determined using a NGS sequence

pooling strategy and bioinformatics to identify novel natural product-

encoding pathways from previously undescribed metagenomic diversity.

The cloned pathways are very divergent from known pathways, with the

GC content varying from 41 to 76% and the amino acid identity of the

KS domains ranging from 32 to 83% to the best matching BLAST hit.

Screening the entire library in the original E. coli cloning host revealed

28 BACs with anti-MRSA activity and 14 compounds. 593 BACs were

identified by NGS to contain novel PKS and/or NRPS pathways not

seen before. Expression of PKS pathway-containing clones in an E.

coli strain engineered for polyketide metabolite support resulted in

multiple cases of heterologous expression as determined by anti-

infective screening and structural analysis. These results indicate a high

degree of unique sequence space has been recovered on large-insert

metagenomic clones. Multiple types of small molecule pathways have

been discovered and shuttled to a novel E. coli strain for functional

expression of active compounds. These libraries are ideal for anti-

microbial, anti-fungal, anti-viral, anti-cancer or enzyme discovery

research.

Three Libraries for Natural Product Pathways & Compounds1) Soil Diversity 1: A BAC library with 19,200 clones from agricultural

soil has been extensively characterized and validated to contain

~110,000 kb inserts with hundreds of full length PKS pathways. Every

clone has been sequenced.

2) Soil Diversity 2: A BAC library with 103,680 clones from a native

prairie soil has been partially characterized and validated to contain

~100,000 kb inserts.

3) A PKS/NRPS enriched library from the agricultural soil that has

been shuttled to an engineered E. coli strain (295 clones) that provides

metabolic support for small molecule production. Every clone has been

sequenced.

LibraryBAC

Clones

Average

Insert Size

BAC

Vector

Complete

Insert

Sequence

Anti-

Microbial

Activity

Known

Structures

BigDNA

Soil Diversity 119,200 110 Kb

pSMART

BACSYes 33 Clones 9

BigDNA

Soil Diversity 2103,680 100 Kb pBAC-SBO No NA NA

PKS Express

Extract96+ 100 Kb

pSMART

BACSYes Yes NA

Natural Product Type # PathwaysType I PKS 78

Type I PKS-NRPS 55

Type II PKS 10

Type III PKS 117

Type IV PKS 1

NRPS 603

Bacteriocin-NRPS 3

Arylpolyene 75

Bacteriocin 210

Homo-serine-lactone 23

Indole 13

Ladderane 17

Lantipeptide 49

Lassopeptide 46

Microviridin 3

Phosphonate 10

Resorcinol 19

Siderophore 4

Terpene 320

Other KS 17

other 237

Total 1910

Novel non-PKS Anti-MRSA Compounds from Metagenomic Clones in E. coli Cloning Host

Very active against Vibrio anguillarum (MIC, 0.03 mg/mL)

Fdhila F et al. J Nat Prod 2003, 66, 1299-1301.

Diketopiperazines from Metagenomic Clones

MIC (mg/mL)

E. coli

ATTC 25922

S. aureus

ATCC 25923

B. subtilis

ATCC 66923

P. aeruginosa

ATCC 27853

Cyclo-prolinyl-tyrosine 27.0 80.2 73.7 68.7

Tetracycline 64.0 256.0 128.0 12.5

Streptomycin 50.0 130.0 120.0 61.0

Reported antimicrobial activities in literature

Sunaryanto et a. Micr Biology 2011, 5:81-87.

NGS of the

Soil Diversity 1

metagenomic

library

reveals

hundreds of

BGCs

encoding

natural

products

28 Anti-MRSA & 3 Anti-P. aeruginosa Clones

E. coli BTRA strain engineered for polyketide expression

Pigmented and fluorescent polyketide products

-0.200

0.000

0.200

0.400

0.600

0.800

1.000

Initial 16h 40h 64h

Op

tica

l Den

sity

at

60

0 n

m

Time

E. coli BTRA (P7H3) extract activity against Cryptococcus neoformans

KS Domains from

Type I PKS Pathways

Novel clade

KS domains discovered by PCR

KS domains discovered by NGS

Novel clade

Novel clade

Novel clade

Novel clade

Dendogram of KS domains derived from Type I PKS

pathways encoding known polyketide products along

with metagenomic clones identified via PCR (red) or

NGS screening (blue)

020406080

100120140

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clone 2

clone 3

clone 12

clone 46

clone 53

clone 64

clone 65

clone 730

20

40

60

80

100

120

140

Gro

wth

re

lati

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UACC-62

SF-295

A498

COLO205

SW-620

DU-145

A B

Identifying novel compounds with LC-MS in engineered E. coli BTRA strain

Eight candidate clones

Potential candidates

masses

A) Extracts of the PKS express library were tested for anticancer activity

against six cell lines. B) Eight candidates were re-cultivated, and four

different extraction methods were tested. C) Methanol extracts were

analyzed with LC-DAD-MS, and molecules that were unique to clone 2

were identified by MS-profiling. D) The extract was re-analyzed with

MSMS, and a molecular network with library search against compounds

with similar MSMS pattern was generated. Further analysis remains,

however one potential candidate mass was identified, and the MS pattern

analysis indicated structural similarities to a cyclic peptide. The candidate

from MS2 was in coherence with the MS1 profiling results.

C WELL2

Time [min]

Rel

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e ab

un

dan

ce [

cou

nts

]

D

Ctr. Clone 2

Lo

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rela

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ab

un

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2

Expression of natural products by metagenomic clone P7H3 in E. coli incubated for 48 hr,

during which the induction agents L-arabinose and isopropyl β-D-1-thiogalactopyranoside

were added at the time point of 24 hr: a) empty vector with one-fold concentration of

induction agents; b) the clone with 1-fold concentrations of induction agents expressing

compounds 1 and 3; and c) the clone with 2-fold concentrations of induction agents

expressing compounds1−6. The LC-MS data showed that these compounds are small

molecules with MW <500. The similar UV absorption patterns indicated that they belong to

the same chemotype.