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Prof. Jason K. Sello Department of Chemistry Brown University j ason_sello@brown
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Transcript of Prof. Jason K. Sello Department of Chemistry Brown University j ason_sello@brown
Towards Sustainable Living: Using Streptomyces Bacteria to Produce
Renewable Energy and Commodity Chemicals from Plant Biomass
Prof. Jason K. SelloDepartment of Chemistry
Brown University
Sources of Renewable Energy
SOLARWIND
BIOMASS
HYDRO GEOTHERMAL
Increasing World Biofuels Production
• 15.9 billion gallons of biofuels were produced domestically in 2010
– 13.2 billion gallons of ethanol
– 2.7 billion gallons of biodiesel
• 138.6 billion gallons of gasoline was consumed in the US during 2010
BP Statistical Review of Energy June 2011. bp.com/statisticalreview
Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Biotechnology for Conversion of Plant Biomass to Biofuels
Plant BiomassFeedstocks
Energy Crops (switch grass) Organic Trash
Forestry WasteAgricultural Residue
Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Biotechnology for Conversion of Plant Biomass to Biofuels
Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Hemicellulose
Cellulose
Lignin
Structural Components of Plant Biomass
Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Hemicellulose
Cellulose
Lignin
Structural Components of Plant Biomass
Using Microorganisms for Biofuel Production
Fermentation of yeast on plant sugars is currently used to produce bioethanol
Engineered bacteria are being developed for the production of biodiesel by fermentation of plant sugars (Steen, Nature, 2010)
Image by Marcin Zemla and Manfred Auer, JBEI. http://newscenter.lbl.gov
Synthetic Biology in Production of Biofuels
Keasling and co-workers have engineered E. coli to convert hemicellulose into biofuels.
Steen. Nature 463, 559-564, 2010.
Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Hemicellulose
Cellulose
Lignin
Structural Components of Plant Biomass
Lignin Component of Plant Biomass
Bugg TD, Ahmad M, Hardiman EM & R Singh. Current Opinion in Biotechnology. 22:394–400, 2011.
• Lignin constitute up to 30% of plant biomass
• Highly stable and heterogeneous polymer consisting of aromatic building blocks
• Lignin interferes with utilization of cellulose for the production of biofuels
• Lignin can be enzymatically depolymerized by some bacteria and fungi
Phanerochaete chrysosporium
P. chryosporium (white rot fungus) can consume lignin.
diark.org
Lignin Depolymerization
Bugg TD, Ahmad M, Hardiman EM & R Singh. Current Opinion in Biotechnology. 22:394–400, 2011.
What is the fate of depolymerized lignin?
Catabolism of Depolymerized Lignin (e.g., Sphingomonas)
Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71(1) 1-15, 2007.
Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71(1) 1-15, 2007.
Catabolism of Depolymerized Lignin (e.g., Sphingomonas)
K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, 2010
Triglycerides
Commodity Chemicals from TCA Cycle
Biodiesel
Alkyl ester
R is methyl, ethyl, or propyl.
Triglyceride(Triacylglycerols)
Methanol Biodiesel(Fatty Acid Methyl Ester) Glycerin
(Glycerol)
Conversion of Triglycerides into Biodiesel01.- 0.5%
Sodium or PotassiumHydroxide
OrSodium Methoxide
80° Celsius
Chemical reaction is a “trans-esterification”.
Bioconversion of Lignin to Biofuels
Lignin Aromatic Compounds
Acetyl-CoA TriacyglycerolsAnd
Fatty AcidsSuccinyl-CoA
An organism that can convert all the components of plant biomass into biofuels would be an efficient “biorefinery”.
Complete Conversion of Lignocellulose to Biofuels
Cellulose
Lignin
Hemicellulose
Aromatic Compounds
Acetyl-CoA TriacyglycerolsAnd
Fatty AcidsSuccinyl-CoA
Prospecting for Plant Biomass Degraders
“An antibiotic is a chemical substance produced by microbes that inhibits the growth of or even destroys other microbes”
Selman Waksman (1888-1973)
Timeline of Antibiotic Discovery
Antibiotics in use as Anti-Bacterial Agents
Antibiotics in use as Anti-Tumor Agents
Antibiotics in Use as Immunosuppresants
Diverse Morphologies and Colors of Streptomyces Species
Image courtesy of T. Kieser
Two Evolutionary Oddities
Streptomycetes Duckbill platypus
Streptomyces: An Unconventional Genus of Bacteria
Multi-cellular
Hyphal morphology and mode of growth like fungi
Complex life cycle
Linear chromosomes and plasmids>8 Mb chromosomes are common
Ubiquitous in terrestrial environments, easily cultured
More than 500 species described
Non-pathogenic relative of Mycobacterium tuberculosis
Prodigious producers of antibiotics
The Majority of Antibiotics are Produced by Streptomycetes
Waksman screened soil samples in search of microorganisms that produce antibiotics.
How can we identify microorganisms that degrade plant biomass?
Identification of Ligninolytic Streptomyces Strains
S. coelicolor
S. lividans
S. griseus
S. natalensis
S. badius
S. viridosporus
S. setonii
S. avermitilis
S. chattanoogensis
Ligininolytic Streptomyces species can decolorize the aromatic dye, Azure B.
Streptomyces viridosporus
D.L. Crawford, Appl. Environ. Microbiol, 53: 2754-2760, 1987D.L. Crawford, Appl. Environ. Microbiol, 41: 442-448, 1981R L. Crawford, Appl. Environ. Microbiol, 45: 898-904, 1983
S. viridosporus is a bona fide ligninolytic streptomycete. It also is capable of consuming cellulose and hemicellulose.
Metagenomic-based Enzyme Discovery in Lignocellulolytic Microbial Communities
DeAngelis, A. Bioengineering Research, 3, 146-158 (2010)
Biodiversity in Tropical Forest Soil from Puerto RicoR
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f Tax
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DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)
Ric
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s (N
umbe
r of T
axa
DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)
Biodiversity in Tropical Forest Soil from Puerto Rico
Biodiversity in Lignin-Enriched CompostR
ichn
ess
(Num
ber o
f Tax
a
Compost Compost + Alkali Lignin DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)
Ric
hnes
s (N
umbe
r of T
axa
Compost Compost + Alkali Lignin
Biodiversity in Lignin-Enriched Compost
Actinobacteria are Populous Soil Bacteria
Mahidul University- Osaka University
- Large group of terrestrial bacteria with high G+C content genomes (e.g., Streptomyces, Corynebacteria, Nocardia, Actinoplanes, and Mycobacteria). - Many are filamentous like fungi- Play a critical role in the decomposition of organic matter in soil - Important organisms in biotechnology source of enzymes and medicinal antibiotics
Actinobacteria Produce Two-Thirds of the 23,000 Known Antibiotics
Streptomyces derived compounds in red boxes
Sir David A. Hopwood
Streptomyces Bacteria
Overview of Research in the Sello Group
Chemical Synthesis and Drug Discovery
Chemical Ecology
Renewable Energy
Biosynthesis and Metabolomics
trpRS1 v
Antibacterial Drug Resistance
cmlR
Streptomyces Bacteria
Overview of Research in the Sello Group
Chemical Synthesis and Drug DiscoveryOkandeji, JOC, 2008Okandeji, JOC, 2009Socha, BMC, 2010
Okandeji, BMC, 2011Carney, JOC, 2012
Compton, ACS Chem. Biol. 2013Nelson, mBio. 2013Carney, JACS, 2014
Chemical EcologyDavis, Org. Lett., 2009Morin, Org. Lett., 2010
Morin, OBC, 2012
Renewable EnergySocha, Energy & Fuels, 2010
Socha, OBC, 2010Davis, AMB, 2010
Davis, J. Bacteriol., 2012Davis, NAR, 2013
Davis, Genome Ann. 2013
Biosynthesis and Metabolomics
Sello, J. Bacteriol., 2008Badu-Nkansah, FEMS Lett., 2010
Totaro, ChemBioChem, 2012
trpRS1 v
Antibacterial Drug Resistance
Vecchione, J. Bacteriol., 2008Vecchione, AAC, 2009Vecchione, AAC, 2009
Vecchione, J. Bacteriol., 2010
cmlR
Actinobacteria are Potential “Lignocellulose Biorefineries”
• Gram-positive soil-dwelling bacteria
• Degrade all components of plant biomass– Cellulose– Hemicellulose– Lignin
• Naturally accumulate triacylglycerols, the precursors of biodiesel, and make commodity chemicals
• Long history in industrial-scale fermentation for the production of antibiotics
E. Wellington
D.L. Crawford, Appl. Environ. Microbiol, 53: 2754-2760, 1987D.L. Crawford, Appl. Environ. Microbiol, 41: 442-448, 1981R L. Crawford, Appl. Environ. Microbiol, 45: 898-904, 1983
A. setonii and S. viridosporus are bona fide ligninolytic bacteria. They also consume cellulose and hemicellulose.
Plant Biomass-Degrading Actinobacteria
Amycolatopsis setonii Streptomyces viridosporus
The first bacterial lignin peroxidase was isolated from Streptomyces viridosporus
Ramachandran et al. Appl. Environ. Microbiol. 53(12): 2754-2760, 1987.
Lignin Depolymerization
Bugg TD, Ahmad M, Hardiman EM & R Singh. Current Opinion in Biotechnology. 22:394–400, 2011.
Genomics Approaches in Bioenergy Technology
In collaboration with the Joint Genome Institute (JGI), the genomes of A. setonii and S. viridosporus has been sequenced.
http://www.jgi.doe.gov/education/bioenergy/
2012
2013
A. setonii S. viridosporus S. coelicolor A3(2)
A. mediterranei U32
Genome Size 8,442,518 8,292,505 9,054,847 10,236,715
% GC 71.9 72.5 72.0 71.3
Total Genes 8,328 7,648 8,325 9,292
Protein Coding Genes
8,264 7,553 8,210 9,228
Proteins with Predicted Functions
6,446 5,653 5,226 6,431
Predicted Secreted Enzymes
1,750 1,618 1,949 3,019
Global Genome Comparisons of Four Actinomycetes
Data are from JGI (DOE JOINT GENOME INSTITUTE)https://img.jgi.doe.gov
Numbers of Genes in Certain COG Functional Categories
A. setonii S. viridosporus
Description Gene # % of Genome Gene # % of Genome
Amino Acid Transport and Metabolism
539 8.4 452 8.5
Carbohydrate Transport and Metabolism
587 9.2 503 9.4
Coenzyme Transport and Metabolism
303 4.7 238 4.5
Energy Production and Conversion
584 9.1 340 6.4
Lipid Metabolism 448 6.9 310 5.82
Secondary Metabolism 397 6.2 288 5.4
Signal Transduction 1018 15.86 689 12.93
Posttranslational Modification, Protein turnover, chaperones
149 2.32 169 3.17
Number of Genes with (or without) a homolog in:
Comparison Organism
A. setonii S. viridosporus S. coelicolor A3(2)
A. mediterranei U32
Comparisons for Unique Genes
A. setonii - (3,730) (2,300) (3,545)
S. viridosprous (3,522) - (3,441) (1,719)
Comparisons for Common genes
A. setonii - 4,534 5,964 4,719
S. viridosporus 4,030 1,618 1,949 3,019
Global Genome Comparisons of Four Actinomycetes
Number of genes without a homolog in the organism being compared are indicated in parenthesis.
A. setonii S. viridosporus
Pfam Description Gene # Gene #
Glyco_hydro 36 71
Carbohydrate Binding Module
1 18
Polysacc_deac 5 9
a-amylase 9 15
Pectate Lyase 0 3
Total # 51 116
Predicted Carbohydrate Degrading Genes in A. setonii and S. viridosporus
A. setonii S. viridosporus
Pfam Description Gene # Gene #
An_Peroxidase 2 1
Catalase 1 4
CMD* 5 6
Cu-oxidase 3 2
Dyp_perox 3 1
GSHPx 1 1
Mn_catalase 2 1
peroxidase 1 1
Total # 18 17
Predicted Lignin Degrading Genes in A. setonii and S. viridosporus
Both species have a comparable number of genes encoding enzymes with potential activity against lignin.
*(CMD) Carboxymuconolactone decarboxylase
Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71(1) 1-15, 2007.
Pathways for Catabolism of Depolymerized Lignin in Sphingomonas
Homologs of Sphingomonas Lignin Catabolism Pathway Genes in Amycolatopsis setonii
Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71(1) 1-15, 2007.
PCA 3,4- cleavage pathway
Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71, 1-15 (2007)
PCA 3,4- cleavage pathway
Homologs of Sphingomonas Lignin Catabolism Pathway Genes in Streptomyces viridosporus
Complete Conversion of Lignocellulose to Biofuels
Cellulose
Lignin
Hemicellulose
Aromatic Compounds
Acetyl-CoA TriacyglycerolsAnd
Fatty AcidsSuccinyl-CoA
Streptomyces viridosporus as a Model for Catabolism of Lignin-Derived Aromatic Compounds
Catabolism of a Lignin-Derived Aromatic Compound via the β-Ketoadipate Pathway in S. viridosporus
pcaLpcaB
pcaGpcaH
pcaFpcaJ
pcaIregulator
pcaL β-ketoadipate enol-lactone hydrolase/decarboxylase
pcaB β-carboxymuconate cycloisomerasepcaG protocatechuate 3,4 dioxygenase, α-subunitpcaH protocatechuate 3,4 dioxygenase, β-subunitpcaF β-ketoadipyl CoA thiolasepcaJ β-ketoadipate succinyl-CoA transferase, β-subunitpcaI β-ketoadipate succinyl-CoA transferase, α-subunit
K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, 2010
Triglycerides
Commodity Chemicals from TCA Cycle
Lignin Derived Aromatics to Commodity Chemicals
MMAMutase
MMAEpimerase
Succinyl-CoA (S)-methyl Malonyl CoA
(R)-methyl Malonyl CoA
DEBS
Tet
Tetracycline
Malonyl CoAAcetyl-CoA
ACC Carboxylase
Complete Conversion of Lignocellulose to Biofuels
Cellulose
Lignin
Hemicellulose
Aromatic Compounds
Acetyl-CoA TriacyglycerolsAnd
Fatty AcidsSuccinyl-CoA