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Towards Sustainable Living: Using Streptomyces Bacteria to Produce
Renewable Energy and Commodity Chemicals from Plant Biomass
Prof. Jason K. SelloDepartment of Chemistry
Brown University
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Sources of Renewable Energy
SOLARWIND
BIOMASS
HYDRO GEOTHERMAL
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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
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Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Biotechnology for Conversion of Plant Biomass to Biofuels
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Plant BiomassFeedstocks
Energy Crops (switch grass) Organic Trash
Forestry WasteAgricultural Residue
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Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Biotechnology for Conversion of Plant Biomass to Biofuels
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Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Hemicellulose
Cellulose
Lignin
Structural Components of Plant Biomass
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Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Hemicellulose
Cellulose
Lignin
Structural Components of Plant Biomass
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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
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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.
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Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Hemicellulose
Cellulose
Lignin
Structural Components of Plant Biomass
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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
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Phanerochaete chrysosporium
P. chryosporium (white rot fungus) can consume lignin.
diark.org
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Lignin Depolymerization
Bugg TD, Ahmad M, Hardiman EM & R Singh. Current Opinion in Biotechnology. 22:394–400, 2011.
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What is the fate of depolymerized lignin?
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Catabolism of Depolymerized Lignin (e.g., Sphingomonas)
Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71(1) 1-15, 2007.
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Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71(1) 1-15, 2007.
Catabolism of Depolymerized Lignin (e.g., Sphingomonas)
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K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, 2010
Triglycerides
Commodity Chemicals from TCA Cycle
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Biodiesel
Alkyl ester
R is methyl, ethyl, or propyl.
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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”.
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Bioconversion of Lignin to Biofuels
LigninAromatic
CompoundsAcetyl-CoA Triacyglycerols
AndFatty AcidsSuccinyl-CoA
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An organism that can convert all the components of plant biomass into biofuels would be an efficient “biorefinery”.
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Complete Conversion of Lignocellulose to Biofuels
Cellulose
Lignin
Hemicellulose
Aromatic Compounds
Acetyl-CoA TriacyglycerolsAnd
Fatty AcidsSuccinyl-CoA
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Prospecting for Plant Biomass Degraders
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“An antibiotic is a chemical substance produced by microbes that inhibits the growth of or even destroys other microbes”
Selman Waksman (1888-1973)
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Timeline of Antibiotic Discovery
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Antibiotics in use as Anti-Bacterial Agents
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Antibiotics in use as Anti-Tumor Agents
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Antibiotics in Use as Immunosuppresants
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Diverse Morphologies and Colors of Streptomyces Species
Image courtesy of T. Kieser
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Two Evolutionary Oddities
Streptomycetes Duckbill platypus
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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
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The Majority of Antibiotics are Produced by Streptomycetes
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Waksman screened soil samples in search of microorganisms that produce antibiotics.
How can we identify microorganisms that degrade plant biomass?
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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.
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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.
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Metagenomic-based Enzyme Discovery in Lignocellulolytic Microbial Communities
DeAngelis, A. Bioengineering Research, 3, 146-158 (2010)
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Biodiversity in Tropical Forest Soil from Puerto RicoR
ichn
ess
(Num
ber
of T
axa
DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)
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Ric
hnes
s (N
umbe
r of
Tax
a
DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)
Biodiversity in Tropical Forest Soil from Puerto Rico
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Biodiversity in Lignin-Enriched CompostR
ichn
ess
(Num
ber
of T
axa
Compost Compost + Alkali Lignin DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)
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Ric
hnes
s (N
umbe
r of
Tax
a
Compost Compost + Alkali Lignin
Biodiversity in Lignin-Enriched Compost
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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
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Actinobacteria Produce Two-Thirds of the 23,000 Known Antibiotics
Streptomyces derived compounds in red boxes
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Sir David A. Hopwood
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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
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Streptomyces Bacteria
Overview of Research in the Sello Group
Chemical Synthesis and Drug Discovery
Okandeji, JOC, 2008Okandeji, JOC, 2009Socha, BMC, 2010
Okandeji, BMC, 2011Carney, JOC, 2012
Compton, ACS Chem. Biol. 2013Nelson, mBio. 2013Carney, JACS, 2014
Chemical Ecology
Davis, Org. Lett., 2009Morin, Org. Lett., 2010
Morin, OBC, 2012
Renewable Energy
Socha, Energy & Fuels, 2010Socha, 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
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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
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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
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The first bacterial lignin peroxidase was isolated from Streptomyces viridosporus
Ramachandran et al. Appl. Environ. Microbiol. 53(12): 2754-2760, 1987.
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Lignin Depolymerization
Bugg TD, Ahmad M, Hardiman EM & R Singh. Current Opinion in Biotechnology. 22:394–400, 2011.
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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/
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2012
2013
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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
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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
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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.
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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
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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
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Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71(1) 1-15, 2007.
Pathways for Catabolism of Depolymerized Lignin in Sphingomonas
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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
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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
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Complete Conversion of Lignocellulose to Biofuels
Cellulose
Lignin
Hemicellulose
Aromatic Compounds
Acetyl-CoA TriacyglycerolsAnd
Fatty AcidsSuccinyl-CoA
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Streptomyces viridosporus as a Model for Catabolism of Lignin-Derived Aromatic Compounds
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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 cycloisomerase
pcaG protocatechuate 3,4 dioxygenase, α-subunit
pcaH protocatechuate 3,4 dioxygenase, β-subunit
pcaF β-ketoadipyl CoA thiolase
pcaJ β-ketoadipate succinyl-CoA transferase, β-subunit
pcaI β-ketoadipate succinyl-CoA transferase, α-subunit
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K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, 2010
Triglycerides
Commodity Chemicals from TCA Cycle
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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
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Complete Conversion of Lignocellulose to Biofuels
Cellulose
Lignin
Hemicellulose
Aromatic Compounds
Acetyl-CoA TriacyglycerolsAnd
Fatty AcidsSuccinyl-CoA