Thermophilic microorganisms
capable of degrading
biopolymers
Ilya V. Kublanov
Laboratory of hyperthermophilic
microbial communities,
Winogradsky Institute of Microbiology,
Russian Academy of Sciences
Summary

Thermophilic microorganisms

Sources of isolation of thermophilic microorganisms

Utilization of thermophilic microorgansisms and their enzymes:
advantages

Thermophilic microorganisms capable of biopolymers
degradation

Hydrolases-coding genes in genomes of thermophilic
microorganisms capable of biopolymers degradation
2
Thermophilic microorganisms
Optimal temperature
106 C
hyperthermophiles
80 C
extreme thermophiles
70 C
moderate thermophiles
40 C
mesophiles
Pyrolobus fumarii is a champion (at least, at present
time), H2/O2, NO32-, S2O32- 90-113 C (opt = 106 C).
psychrophiles
Stetter et al, Extremophiles, 1997.
Methanopyrus kandleri strain 116 is growing up to 122 C
(opt = 105 C) at 40 MPa. Kurr et al, Arch. Microbiol.,1991. Takai et al, PNAS, 2008.
15 C
“Bacteria are able to grow… at any temperature at which there is liquid water…”
Brock, 1967.
BUT: “Above 110 C aminoacids and other metabolites become highly unstable =>
3
temperature limit is not far away from 113 C” Jaenicke, 1998
Thermophilic microorganisms
16S rRNA gene-based tree of life
Woese et al., 1990, edited by Jurgens, 2002.
4
Sources of isolation of
thermophilic microorganisms
Hightemperature
subsurface
biosphere
Shallow-water submarine hot vents
Terrestrial hot springs
Deep-sea hot vents
5
Sources of isolation of
thermophilic microorganisms
Deep-sea
Shallow
Terrestrial
Subsurface
6
Advantages of thermophiles utilization
 Contamination risk is low
 High temperature allows to work with higher substrates
concentrations due to viscosity decrease
 Industrial processes sometimes require high temperatures
 Less studied => many new, undiscovered enzymes
 Produces thermostable enzymes - thermozymes
7
Advantages of thermozymes utilization
 Thermostability
 Higher
solvents)
tolerance
to
denaturing
agents
(detergents,
 High temperature allows to work with higher substrates
concentrations due to viscosity decrease
 Purification of recombinant enzymes is simplified (thermal
treatment)
8
Thermophilic microorganisms in INMI RAS
 Thermophilic and hyperthermophilic bacteria and
archaea (> 350 strains)
 Many represent new taxonomic groups (species,
genera, families, orders, classes), one – novel phylum
 Represent new metabolic groups
 Source of novel thermostable enzymes
9
Thermophilic microorganisms capable of
bipolymers degradation
Polymeric substrates used
by thermophiles from our
culture collection:
„In situ‟ enrichments – cultivation of
tubes with insoluble organic substrates
in the hot springs
Carbohydrates
•Cellulose and derivatives
•Xylan
•Agarose
•Chitin
•Lichenan
•Laminarin
Immediately after cultivation:
•detection of hydrolitic activity
•detection of dominating microorganisms
•isolation of dominating microorganisms capable
of growing on biopolimers in pure cultures
Proteins
Lipids
Kublanov et
al., AEM, 2009,
75, 286-291
10
Thermophilic microorganisms capable of
bipolymers degradation
Beta-keratin (feathers) degradation
Cellulose degradation (incubation
(incubation for 3 days at 65оС)
о
for 1.5 days at 70 С)
feathers
A - control
Б - Caldicellulosiruptor kronotskyensis
(Miroshnichenko et al, 2008, 58:1492-1496
В – Dictyoglomus sp. (unpublished)
Caldanaerobacter sp. Strain
1523-1 (unpublished)
11
Thermophilic microorganisms capable of
bipolymers degradation
Several genomes of our thermophilic and hyperthermophilic
hydrolytic microorganisms were sequences and annotated
Organism
Domen
Novel… Topt °C
Were sequenced
status
Desulfurococcus
kamchatkensis
Archaea
species
80-85
Bioengineering Center,
RAS
Published in
2009
Thermococcus
sibiricus
Archaea
species
78
Bioengineering Center,
RAS
Published in
2009
Acidilobus
aceticus
Archaea
order
82.5
Bioengineering Center,
RAS
Published in
2010
Desulfurococcus
fermentans
Archaea
species
80
Virginia Bioinformatics Under
Institute + JGI
analysis
“Melioribacter
roseus”
Bacteria phylum
52-55
Bioengineering Center,
RAS
Under
analysis
12
Thermophilic microorganisms capable of
bipolymers degradation
Desulfurococcus kamchatkensis
First archaeum,
growing on keratin!
Substrates:
•Alpha-keratin
•Gelatin
Kublanov et al., IJSEM,
2009, 59, 1743-1747
•Hyperthermophile (T, oC 65 – 85 – 87)
•Neutrophile (pHopt 6.5)
•Obligate anaerobe
•Casein
•Albumin
•Dextran
•Sucrose
13
Hydrolases-coding genes in genomes of
thermophilic microorganisms capable of
biopolymers degradation
In collaboration with CB
Genome of Desulfurococcus kamchatkensis
Contains a number of peptidases-coding genes, including those hydrolyzing alphakeratin
Contains genes of alpha-GHs which are in accordance with substrate specificity of
D. kamchatkensis
Most interesting hydrolases
Dkam_0406
extracellular
Dkam_0582
Glycoside Hydrolase 53, endo-β-1,4First GH53, found in
galactanase, EC 3.2.1.89
Archaea
Alpha-glucosidase, EC 3.2.1.20 or EC 3.2.1.48 The novel GH family
Dkam_0433
Trypsin-like serine protease S1
Trypsin-like
intracellular
Dkam_1142
Cystein aminopeptidase, C15
The nearest - bacterial
ND
Dkam_1274
Subtilisin-like serine endopeptidase, S8A
Very distant to other
extracellular
Ravin et al., J. Bacteriol. 2009 191: 2371-2379
introcellular
14
Thermophilic microorganisms capable of
bipolymers degradation
Thermococcus sibiricus
Isolated from high-temperature oil
reservoir in Western Siberia Russia,
from the depth 2350 m (T=85oC)
Substrates:
•Amorphous cellulose
Miroshnichenko et al.
Extremophiles, 2001, 5, 85-91
•Hyperthermophile (T,
•Neutrophile (pHopt 7.3)
•Obligate anaerobe
oC
40 – 78 – 88)
•Agarose
•Dextran
•Olive oil
15
Hydrolases-coding genes in genomes of
thermophilic microorganisms capable of
biopolymers degradation
In collaboration with CB
Genome of Thermococcus sibiricus
Contains 15 genes encoding esterases (and lipases in particular), among them 4
extracellular. No beta-oxidation enzymes. T. sibiricus was found to be able to grow
on olive oil and glycerol, but not on fatty acids.
Contains saccharolytic gene island - a region with many genes of extracellular and
intracellular GHs and transporters. Acquired by lateral gene transfer, presumably,
from extremally thermophilic bacteria of phylum Thermotogae.
Most interesting hydrolases
Tsib_0325
Tsib_0326,
Tsib_0327,
Tsib_0328
Tsib_1454
Glycoside Hydrolase 50, betaagarase, EC 3.2.1.81
Putative Glycoside Hydrolase
12, cellulase or novel GH
The first GH50, found in Archaea. In general, the family
consist of 58 proteins, non of them was characterized
Extremely distant form other GH12. CAZy does not put them
into GH12
Carboxylic ester hydrolase
Very distant to other
Mardanov et al., AEM, 2009 75: 4580-4588
16
Thermophilic microorganisms capable of
bipolymers degradation
New order Acidilobales
Substrates:
•Lichenan
•Laminarin
•Sucrose
•Lactose
•Arbutin
Acidilobus aceticus
Prokofeva et al. IJSEM,
2000, 50, 2001-2008
Acidilobus saccharovorans
Prokofeva et al. IJSEM,
2009, 59, 3116-3122
•Hyperthermophiles (T, oC 60 – 85 – 92)
•Acidophiles (pH 2.0 – 3.8 – 6.0)
•Obligate anaerobes
•Xylan
•Starch
•Beef extract
17
Hydrolases-coding genes in genomes of
thermophilic microorganisms capable of
biopolymers degradation
In collaboration with CB
Genome of Acidilobus saccharovorans
Contains endopeptidase-coding genes, including rear acid endopeptidase –
thermopsin. Contains many GH-coding genes. Due to distant phylogenetic position
and acidophilic origin of A. saccharovorans its GHs might possess novel features,
like acid tolerance.
Most interesting hydrolases
Asac_0635
Asac_0641
Asac_0652
Very distant to other
Very distant to other
formerly known as A4,
well-known in fungi
formerly known as A4
extracellular
extracellular
Asac_0737
Serine protease
Thermopsin-like protease, A5
“pepstatin-insensitive carboxyl
proteinase”, G1,
Serine protease, putative S53
Asac_1420
Serine protease, putative S53
formerly known as A4
extracellular
Asac_0354
Glycoside Hydrolase 15, putative Very distant to other
glucoamylase, EC 3.2.1.3
Mardanov et al., AEM, 2010 76: 5652-5657
probably extracellular
extracellular
18
Hydrolases-coding genes in genomes of
thermophilic microorganisms capable of
biopolymers degradation
Genome of Acidilobus saccharovorans
Most interesting hydrolases
Asac_0772
Asac_0825
Asac_1074
Asac_1367
Asac_1378
Asac_1380
Asac_1386
Asac_1415
Glycoside Hydrolase 31, putative α-glucosidase, EC
3.2.1.20
Glycoside Hydrolase 57, putative alpha-amylase
Putative Glycoside Hydrolase 13, α-amylase, EC
3.2.1.1
Glycoside Hydrolase 38, α-mannosidase, EC
3.2.1.24
Glycoside Hydrolase 3, putative β-xylosidase
Glycoside Hydrolase 12, putative cellulase, EC
3.2.1.4
Glycoside Hydrolase 12, putative cellulase, EC
3.2.1.4
Glycoside Hydrolase 12, putative cellulase, EC
3.2.1.4
Mardanov et al., AEM, 2010 76: 5652-5657
Very distant to other
introcellular
Very distant to other
Very distant to other
extracellular
introcellular
Very distant to other
introcellular
Very distant to other
Very distant to other
introcellular
extracellular
Very distant to other
introcellular
Very distant to other
introcellular
19
Thermophilic microorganisms capable of
bipolymers degradation
Desulfurococcus fermentans
Substrates:
•Cellulose
•Agarose
•Arbutin
•Sucrose
•Starch
Perevalova et al., IJSEM, 2005, 55, 995-999
•Hyperthermophile (T,
•Neutrophile (pHopt 6.0)
•Obligate anaerobe
oC
63 – 82 – 89)
•Alpha-keratin
•Casein hydrolysate
20
Hydrolases-coding genes in genomes of
thermophilic microorganisms capable of
biopolymers degradation
Desulfurococcus fermentans
starch
First archaeum growing
on cellulose!
cellulose
control
Genome of Desulfurococcus fermentans
Contains several GH-coding genes. Despite the
ability to grow on cellulose, no cellulases were
found so far – great challenge for us
In collaboration with
21
Thermophilic microorganisms in INMI RAS
“Melioribacter roseus” – represents a novel phylum
Novel phylum
16S rRNA gene-based tree of life
22
Thermophilic microorganisms capable of
bipolymers degradation
“Melioribacter roseus”
New Phylum!
Substrates:
•Cellulose
•Starch
•Xylan
Podosokorskaya, Kublanov et al., MS in
preparation
•Dextran
•Glycogen
•Moderately thermophile (T, oC 35 – 55 – 60)
•Neutrophile (pHopt 7.5)
•Facultative anaerobe
•Dissimilatory Fe(III) reduction
•Lichenan
•Gelatin
23
Hydrolases-coding genes in genomes of
thermophilic microorganisms capable of
biopolymers degradation
In collaboration with CB
Genome of “Melioribacter roseus”
Contains about 90 GH-coding genes, that`s comparable to the most active
polysaccharide-degrading microorganisms. Also, possess many GTs (glycosyl
transferases), CE (carbohydrate esterases) and PL (polysaccharide lyases).
100
# of putative GH
genes
90
80
70
# of GHs with
uncertain GH fam.
60
50
40
30
# of GHs, placed into
families
20
10
0
Mros
Chyd
Cthe
According to CAZy database.
Rmar
Dtur
Ctha
24
Hydrolases-coding genes in genomes of
thermophilic microorganisms capable of
biopolymers degradation
Genome of “Melioribacter roseus”
Most interesting hydrolases
Mros505
Mros508
Mros512
Mros548
Mros754
Mros758
Mros971
Mros982
Mros969
Mros1588
Glycoside Hydrolase 5, putative cellulase, EC 3.2.1.4
Glycoside Hydrolase 43/32/68
Glycoside Hydrolase 5, mannanase endo- or exoGlycoside Hydrolase 43/32/68
Glycoside Hydrolase 5, putative cellulase, EC 3.2.1.4
Glycoside Hydrolase 9, putative cellulase, EC 3.2.1.4
Glycoside Hydrolase 42, b-agarase, EC 3.2.1.23
Glycoside Hydrolase 30, 2 domains
Glycoside Hydrolase 43
Glycoside Hydrolase 47, a-mannosidase, EC
3.2.1.113
Mros1596 Glycoside Hydrolase 92, putative mannosidase
Mros2628 Glycoside Hydrolase 5
extracellular
A novel family, probably intracellular
Very distant to other
extracellular
A novel family, probably intracellular
extracellular
Very distant to other
extracellular
Very distant to other
extracellular
A novel family, probably extracellular
Very distant to other
extracellular
Very distant to other
Very distant to other
extracellular
intracellular
25
Cellulases of “Melioribacter roseus”
1
2
3
4
5
6
Zymogram. Substrate CMC.
Incubation: 77°C, pH 7.7, 3 h.
1. Markers
2. “aerobic” cells
3. “anaerobic” cells
4. “aerobic” cells. Preincubation at 96°C, 5`
5. “aerobic” cells. Preincubation at 96°C, 10`
45 kDa
6. “aerobic” cells. Preincubation at 96°C, 30`
M. Roseus is growing optimally at 55 C
Its cellulases (presumably GH5) are stable at 96 C
26
Novel hydrolases, found in genomes of
our microorganisms
Enzyme class
Found in:
GH5, GH9, GH12 - endoglucanase (cellulase), Thermococcus sibiricus, Acidilobus saccharovorans,
exoglucanase, b-mannosidase, licheninase,
“Melioribacter roseus”,
endoxylanase and other activities
Caldicellulosiruptor kronotskiensis
GH1 - b-galactosidase, b-glucosidase and other Thermococcus sibiricus, Acidilobus saccharovorans,
activities
Desulfurococcus kamchatkensis, Desulfurococcus
fermentans, “Melioribacter roseus”
GH42, GH50 - b-agarase
Thermococcus sibiricus, “Melioribacter roseus”
GH13, GH57 - a-amylase, pullulanase and
other activities
Thermococcus sibiricus, Acidilobus saccharovorans,
Desulfurococcus kamchatkensis, Desulfurococcus
fermentans, “Melioribacter roseus”
GH38, GH47, GH92 - different mannosidases
“Melioribacter roseus”, Acidilobus saccharovorans,
A5 – thermopsin, an acid endopeptidase
Acidilobus saccharovorans
S8A - subtilisin-like serine endopeptidase
Desulfurococcus kamchatkensis and other
And many others!
27
Possible applications:

Wastes utilization

Biomass conversion

Biofuels

Food bioprocessing

Detergents
28
Thank you!
Laboratory of hyperthermophilic microbial communities, INMI RAS
Laboratory:
E.A. Bonch-Osmolovskaya
A.V. Lebedinskiy, T.G. Sokolova
A.I. Slobodkin, N.A. Chernyh
M.I. Prokofeva, G.B. Slobodkina
I.V. Kublanov, O.A. Podosokorskaya
S.N. Gavrilov, D. Kozhevnikova
M.Yu. Merkel, A.A. Perevalova
Funding:
•Russian Academy of Sciences
•Russian Foundation of Basic Research
•Russian Federation Ministry of
Education and Science
•FP7-KBBE “Hotzyme” project –
systematic screening for novel
hydrolases from hot environments (just started)
29