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 hyperthermophiles extreme thermophiles Pyrolobus fumarii is a champion (at least, at present time), H 2 /O 2, NO 2-3, S 2 O 2-3 90-113 C (opt = 106 C). Stetter et al, Extremophiles, 1997. psychrophiles 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. 106 C 80 C 70 C moderate thermophiles mesophiles Optimal temperature 40 C 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 => temperature limit is not far away from 113 C Jaenicke, 1998 3
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 Deep-sea hot vents Terrestrial hot springs 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 tolerance to denaturing agents (detergents, solvents) 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: Carbohydrates Cellulose and derivatives Xylan Agarose Chitin Lichenan Laminarin Proteins In situ enrichments cultivation of tubes with insoluble organic substrates in the hot springs Immediately after cultivation: detection of hydrolitic activity detection of dominating microorganisms isolation of dominating microorganisms capable of growing on biopolimers in pure cultures Kublanov et al., AEM, 2009, 75, 286-291 Lipids 10
Thermophilic microorganisms capable of bipolymers degradation Cellulose degradation (incubation for 1.5 days at 70 о С) Beta-keratin (feathers) degradation (incubation for 3 days at 65 о С) A - control Б - Caldicellulosiruptor kronotskyensis (Miroshnichenko et al, 2008, 58:1492-1496 В Dictyoglomus sp. (unpublished) Caldanaerobacter sp. Strain 1523-1 (unpublished) feathers 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 Thermococcus sibiricus Acidilobus aceticus Desulfurococcus fermentans Melioribacter roseus Archaea species 80-85 Bioengineering Center, RAS Archaea species 78 Bioengineering Center, RAS Archaea order 82.5 Bioengineering Center, RAS Archaea species 80 Virginia Bioinformatics Institute + JGI Bacteria phylum 52-55 Bioengineering Center, RAS Published in 2009 Published in 2009 Published in 2010 Under analysis 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, o C 65 85 87) Neutrophile (ph opt 6.5) Obligate anaerobe Casein Albumin Dextran Sucrose 13
Hydrolases-coding genes in genomes of thermophilic microorganisms capable of biopolymers degradation Genome of Desulfurococcus kamchatkensis In collaboration with CB 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 Ravin et al., J. Bacteriol. 2009 191: 2371-2379 Most interesting hydrolases Dkam_0406 Glycoside Hydrolase 53, endo-β-1,4- First GH53, found in extracellular galactanase, EC 3.2.1.89 Archaea Dkam_0582 Alpha-glucosidase, EC 3.2.1.20 or EC 3.2.1.48 The novel GH family introcellular 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 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=85 o C) Substrates: Amorphous cellulose Miroshnichenko et al. Extremophiles, 2001, 5, 85-91 Hyperthermophile (T, o C 40 78 88) Neutrophile (ph opt 7.3) Obligate anaerobe Agarose Dextran Olive oil 15
Hydrolases-coding genes in genomes of thermophilic microorganisms capable of biopolymers degradation Genome of Thermococcus sibiricus In collaboration with CB 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. Tsib_0325 Tsib_0326, Tsib_0327, Tsib_0328 Glycoside Hydrolase 50, betaagarase, EC 3.2.1.81 Putative Glycoside Hydrolase 12, cellulase or novel GH Tsib_1454 Carboxylic ester hydrolase Very distant to other Mardanov et al., AEM, 2009 75: 4580-4588 Most interesting hydrolases 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 16
Thermophilic microorganisms capable of bipolymers degradation New order Acidilobales Acidilobus aceticus Prokofeva et al. IJSEM, 2000, 50, 2001-2008 Acidilobus saccharovorans Prokofeva et al. IJSEM, 2009, 59, 3116-3122 Hyperthermophiles (T, o C 60 85 92) Acidophiles (ph 2.0 3.8 6.0) Obligate anaerobes Substrates: Lichenan Laminarin Sucrose Lactose Arbutin Xylan Starch Beef extract 17
Hydrolases-coding genes in genomes of thermophilic microorganisms capable of biopolymers degradation Genome of Acidilobus saccharovorans In collaboration with CB 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. Asac_0635 Serine protease Very distant to other Asac_0641 Thermopsin-like protease, A5 Very distant to other extracellular Asac_0652 pepstatin-insensitive carboxyl formerly known as A4, extracellular proteinase, G1, well-known in fungi Asac_0737 Serine protease, putative S53 formerly known as A4 probably extracellular Asac_1420 Serine protease, putative S53 formerly known as A4 extracellular Asac_0354 Glycoside Hydrolase 15, putative glucoamylase, EC 3.2.1.3 Mardanov et al., AEM, 2010 76: 5652-5657 Most interesting hydrolases Very distant to other extracellular 18
Hydrolases-coding genes in genomes of thermophilic microorganisms capable of biopolymers degradation Genome of Acidilobus saccharovorans Asac_0772 Glycoside Hydrolase 31, putative α-glucosidase, EC Very distant to other introcellular 3.2.1.20 Asac_0825 Glycoside Hydrolase 57, putative alpha-amylase Very distant to other extracellular Asac_1074 Putative Glycoside Hydrolase 13, α-amylase, EC Very distant to other introcellular 3.2.1.1 Asac_1367 Glycoside Hydrolase 38, α-mannosidase, EC Very distant to other introcellular 3.2.1.24 Asac_1378 Glycoside Hydrolase 3, putative β-xylosidase Very distant to other introcellular Asac_1380 Glycoside Hydrolase 12, putative cellulase, EC Very distant to other extracellular 3.2.1.4 Asac_1386 Glycoside Hydrolase 12, putative cellulase, EC Very distant to other introcellular 3.2.1.4 Asac_1415 Glycoside Hydrolase 12, putative cellulase, EC 3.2.1.4 Very distant to other introcellular Mardanov et al., AEM, 2010 76: 5652-5657 Most interesting hydrolases 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, o C 63 82 89) Neutrophile (ph opt 6.0) Obligate anaerobe 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 In collaboration with Contains several GH-coding genes. Despite the ability to grow on cellulose, no cellulases were found so far great challenge for us 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! Podosokorskaya, Kublanov et al., MS in preparation Moderately thermophile (T, o C 35 55 60) Neutrophile (ph opt 7.5) Facultative anaerobe Dissimilatory Fe(III) reduction Substrates: Cellulose Starch Xylan Dextran Glycogen Lichenan Gelatin 23
Hydrolases-coding genes in genomes of thermophilic microorganisms capable of biopolymers degradation Genome of Melioribacter roseus In collaboration with CB 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 90 80 70 60 50 40 30 20 10 0 Mros Chyd Cthe Rmar Dtur Ctha According to CAZy database. # of putative GH genes # of GHs with uncertain GH fam. # of GHs, placed into families 24
Hydrolases-coding genes in genomes of thermophilic microorganisms capable of biopolymers degradation Genome of Melioribacter roseus Most interesting hydrolases Mros505 Glycoside Hydrolase 5, putative cellulase, EC 3.2.1.4 extracellular Mros508 Glycoside Hydrolase 43/32/68 A novel family, probably intracellular Mros512 Glycoside Hydrolase 5, mannanase endo- or exo- Very distant to other extracellular Mros548 Glycoside Hydrolase 43/32/68 A novel family, probably intracellular Mros754 Glycoside Hydrolase 5, putative cellulase, EC 3.2.1.4 extracellular Mros758 Glycoside Hydrolase 9, putative cellulase, EC 3.2.1.4 Very distant to other extracellular Mros971 Glycoside Hydrolase 42, b-agarase, EC 3.2.1.23 Very distant to other extracellular Mros982 Glycoside Hydrolase 30, 2 domains A novel family, probably extracellular Mros969 Glycoside Hydrolase 43 Mros1588 Glycoside Hydrolase 47, a-mannosidase, EC 3.2.1.113 Very distant to other extracellular Mros1596 Glycoside Hydrolase 92, putative mannosidase Very distant to other extracellular Mros2628 Glycoside Hydrolase 5 Very distant to other intracellular 25
Cellulases of Melioribacter roseus 45 kda 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` 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 GH5, GH9, GH12 - endoglucanase (cellulase), exoglucanase, b-mannosidase, licheninase, endoxylanase and other activities GH1 - b-galactosidase, b-glucosidase and other activities GH42, GH50 - b-agarase GH13, GH57 - a-amylase, pullulanase and other activities GH38, GH47, GH92 - different mannosidases A5 thermopsin, an acid endopeptidase S8A - subtilisin-like serine endopeptidase Found in: Thermococcus sibiricus, Acidilobus saccharovorans, Melioribacter roseus, Caldicellulosiruptor kronotskiensis Thermococcus sibiricus, Acidilobus saccharovorans, Desulfurococcus kamchatkensis, Desulfurococcus fermentans, Melioribacter roseus Thermococcus sibiricus, Melioribacter roseus Thermococcus sibiricus, Acidilobus saccharovorans, Desulfurococcus kamchatkensis, Desulfurococcus fermentans, Melioribacter roseus Melioribacter roseus, Acidilobus saccharovorans, Acidilobus saccharovorans 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