Towards Sustainable Living: Using Streptomyces Bacteria to Produce

Renewable Energy and Commodity Chemicals from Plant Biomass

Prof. Jason K. SelloDepartment of Chemistry

Brown University

jason_sello@brown.edu

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

LigninAromatic

CompoundsAcetyl-CoA Triacyglycerols

AndFatty 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

ichn

ess

(Num

ber

of T

axa

DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)

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

Biodiversity in Lignin-Enriched CompostR

ichn

ess

(Num

ber

of T

axa

Compost Compost + Alkali Lignin DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)

Ric

hnes

s (N

umbe

r of

Tax

a

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 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

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 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

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