functional metagenomics of natural products functional...
TRANSCRIPT
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 [email protected]
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
EtA
c
Me
OH
DM
SO
Sup
ern
atan
ts
Gro
wth
re
lati
ve t
o c
on
tro
l
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
ve t
o c
on
tro
l
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
ativ
e ab
un
dan
ce [
cou
nts
]
D
Ctr. Clone 2
Lo
g2
rela
tive
ab
un
dance
of
ma
sse
s u
niq
ue
to
clo
ne
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.