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Page 1: Fermentative production of butanol – challenges and solutions

Fermentative production of butanol – challenges and solutions

Peter Dürre Sao Paulo, July 25, 2012

Page 2: Fermentative production of butanol – challenges and solutions

Industrial use of butanol

bulk chemical precursor for production of

- acrylate and methacrylate esters

- glycol esters

- butyl acetate

- butylamines

- amino resins

used for production of adhesives/scalants, alkaloids, antibiotics, camphor, deicing fluid,

dental products, detergents, elastomers, electronics, emulsifiers, eye makeup, fibres

flocculants, flotation aids (e.g. butyl xanthate), hard-surface cleaners, hormones and

vitamins, hydraulic and brake fluids, industrial coatings, lipsticks, nail care products,

odorant standard, paints, paint thinners, perfumes, pesticides, plastics, printing ink,

resins, safety glass, shaving and personal hygiene products, surface coatings, super

absorbents, synthetic fruit flavoring, textiles, as mobile phases in paper and thin-layer

chromatography, as oiladditive, as well as for leather and paperfinishing

Page 3: Fermentative production of butanol – challenges and solutions

Advantages of butanol as a biofuel over ethanol

1. Can be blended in any concentration with gasoline (ethanol only

up to 85 %).

2. No modification of existing car engines required.

3. Has lower vapor pressure and is thus safer to handle.

4. Since it is not hygroscopic, blending is already possible in refinery.

5. Less corrosive. Complete existing infrastructure (tanks, pipelines,

pumps, filling stations, etc.) can be used.

6. Energy content is higher, resulting in a higher mileage/gasoline

blend ratio.

7. Dibutyl ether derivative has the potential for a diesel fuel.

Page 4: Fermentative production of butanol – challenges and solutions

Clostridium acetobutylicum -

biotechnological and political impact

(courtesy by H. Hippe)

-Isolation by Weizmann (between 1912 and 1914) in an research

project aiming at producing synthetic rubber from fermentation

products

-Large-scale fermentation of acetone for ammunition production

during World War I

-Balfour Declaration in 1917

-New production plants in Canada and the U.S.

-Stop of production after armistice in November 1918

-Introduction of prohibition in the U.S. in 1920, as a consequence

shortage of amyl alcohol, a solvent for laquers

-Henry Ford‘s assembly line automobile production required large

amounts of solvents for laquers

-Reopening of plants for butanol production (app. 2/3 of the world

market)

-After 1950, decline of the fermantation due to cheaper crude oil

prices

-Closure of last plants in South Africa (1982) and China (2004)

Page 5: Fermentative production of butanol – challenges and solutions

NCP, South Africa: 12 fermenters, working volume of 90,000 l each ( Jones, in: Clostridia (Bahl, Dürre, eds.))

Page 6: Fermentative production of butanol – challenges and solutions

Carbohydrates

Acid formation

Solvent formation Toxin synthesis

Clostridial form

Forespore

Spore

Spore maturation

Vegetative cells

Page 7: Fermentative production of butanol – challenges and solutions

Metabolism of Clostridium acetobutylicum

Hexose

Acetyl-CoA

Acetoacetyl-CoA

Butyryl-CoA Butyrate

Acetate Ethanol

Acetone

Adc

Butanol

AdhE

CtfA/B

Page 8: Fermentative production of butanol – challenges and solutions

Genes encoding solventogenic enzymes in

C. acetobutylicum

2000 4000 6000

orf5solBorfLadhEctfActfBadc

1000200030004000

adhE2 adcSadcR

500 1000150020002500

bdhB bdhA

megaplasmid

chromosome

Page 9: Fermentative production of butanol – challenges and solutions

Regulation of solvent formation

At the level of transcription:

Induction/repression controlled by known

transcription factors (Spo0A-P, CcpA, CodY)

as well as novel ones (AdcR/AdcS),

Posttranscriptional:

mRNA processing of the sol operon transcript

Posttranslational:

Protein modification of acetoacetate decarboxylase

Page 10: Fermentative production of butanol – challenges and solutions

Distribution of regulator binding sites

Page 11: Fermentative production of butanol – challenges and solutions

Genes encoding solventogenic enzymes are induced

by the master regulator Spo0A~ P

Ravagnani et al., Spo0A directly controls the swithch from acid to solvent production in

solvent-forming clostridia.

Mol. Microbiol. 37, 1172-1185, 2000

Most data stemming from investigation of C. beijerinckii.

Harris et al., Northern, morphological, and fermentation analysis of spo0A inactivation

and overexpression in Clostridium acetobutylicum ATCC824.

J. Bacteriol. 184, 3586-3597, 2002

Data stemming from investigation of C. acetobutylicum.

Problem solved, next question! Or?

Page 12: Fermentative production of butanol – challenges and solutions

acetone production after growth on MES medium

0

5

10

15

20

25

30

0 100 200 300 400

acet

on

e c

on

cen

trat

ion

[m

M]

time [h]

WT

DadcS 1

Dspo0A

wt

adcS73s::intron

spo0A462s::intron

0

10

20

30

40

50

60

70

80

90

0 100 200 300 400

bu

tan

ol c

on

cen

trat

ion

[m

M]

time [h]

butanol production after growth on MES medium

Growth of C. acetobutylicum WT and mutants on glucose

Page 13: Fermentative production of butanol – challenges and solutions

0

10

20

30

40

50

60

70

0 100 200 300 400

bu

tan

ol c

on

cen

trat

ion

[m

M]

time [h]

0

0,5

1

1,5

2

2,5

3

3,5

4

0 100 200 300 400

acet

on

e c

on

cen

trat

ion

[m

M]

time [h]

WT 1

DadcS 1

Dspo0A 2

wt

adcS73s::intro

n spo0A462s::intron

3.5

2.5

1.5

0.5

Growth of C. acetobutylicum WT and mutants on glucose

plus glycerol

acetone production after growth on MES medium butanol production after growth on MES medium

Page 14: Fermentative production of butanol – challenges and solutions

Regulator binding sites upstream of the sol promoter

Page 15: Fermentative production of butanol – challenges and solutions

Effect of solB overexpression on solventogenesis

Page 16: Fermentative production of butanol – challenges and solutions

200 nt –

Northern blot experiments

for verifying solB presence under

physiological conditions

Page 17: Fermentative production of butanol – challenges and solutions

Transcriptome of the sol operon region

solventogenic phase

acidogenic phase

Page 18: Fermentative production of butanol – challenges and solutions

A

T

A

A

T

A

A

A

G

T

C

T

T

C

A

G

A

T

G

T

T

T

A

A

T

T

C

C T A G

Primer extension experiments

for identification of solB transcription

start point

Page 19: Fermentative production of butanol – challenges and solutions

1 TAAGATATAG CTTCTTTTAT GTAGTATTAT TTCAGAAGTC TACAAATTAA GTTTATATTT

61 AGACCCTGGG GTGTAACTAT AGTATTTAAT ATTGGTACTA TTAATTAGGG TTATATATAC

121 TAGAACTTAT CATGGTAAAC ATAAATATAA ACTCAATTCT ATTTATGCTC CTATAAAATT

181 TTATAATATA GGAAAACTGC TAAATGTAAA TTATACGTTT ACATTTAGCA GTTTATTTT

Length 195 b

Promoter -35- AAGATA (consensus TTGACA)

-10- TATTAT (consensus TATAAT)

Starting point nt 39

Terminator stem loop structure

(rho-independent terminator)

Integration site nt 149

-35 -10 149 39

Features of solB

Page 20: Fermentative production of butanol – challenges and solutions

Secondary structure of solB transcript

Page 21: Fermentative production of butanol – challenges and solutions

Putative mechanism of action of solB transcript

Page 22: Fermentative production of butanol – challenges and solutions

Approaches to improve biological butanol

production

1. Cloning of genes encoding enzymes required for butanol

formation into new host (e. g. E. coli)

This will include all enzymes that convert acetyl-

CoA into butanol (thiolase, 3-hydroxybutyryl-CoA

dehydrogenase, crotonase, butyryl-CoA dehydro-

genase, butyraldehyde dehydrogenase, butanol

dehydrogenase). Important: ETF proteins!

Potential problems:

- expression of enzymes from an anaerobe

- solvent tolerance

Page 23: Fermentative production of butanol – challenges and solutions

2. Tailor-made strain of Clostridium acetobutylicum,

producing only butanol, H2, and CO2

This will be possible by targeted knock-outs of the lactate

dehydrogenase, 2-acetolactate synthase, acetoacetate

decarboxylase, phosphotransacetylase, and phosphotrans-

butyrylase genes, preventing lactate, acetoin, acetone,

acetate, and butyrate formation.

In addition, deregulatory mutations are required to prevent

development of metabolic bottlenecks.

Page 24: Fermentative production of butanol – challenges and solutions

Fermentation substrates

Sugar and starch are excellent substrates for Clostridium

acetobutylicum.

Problems:

1. Limitation of arable land, biofuels from biomass will only

represent a fraction of the total fuel required.

However, this will have a substantial effect on greenhouse

gas emissions.

2. Ethical problem of competition between nutrition and

biofuels.

Possible solutions: Conversion of lignocellulose to sugars or

transfer of butanol production feature to syngas-using bacteria

Page 25: Fermentative production of butanol – challenges and solutions

Commercial Cellulosic Butanol Production

• Cheaper feedstocks & more efficient

fermentation technology required to

improve economics.

• Laihe Rockley Bio-Chemicals resolved to

restart operation of their biobutanol plant

with biomass 150K tonne/year plant in NE China (corn belt)

Largest biobutanol plant in the world

Developed hydrolysis technology for generating

sugars from corn residues

Transitioning (with help grom GBL) from corn

starch to corn residues (bagasse, stover, and

shells).

Green Biologics Ltd., Abingdon, UK:

Industrial technology leader in butanol fermentation

Page 26: Fermentative production of butanol – challenges and solutions

Characteristics:

Gram-positive

obligatly anaerobic

motile

rod (0.6-1 x 2-3 μm)

few endospores

Products:

acetate, ethanol

Doubling times:

fructose (tD = 2,5 h)

synthesis gas (tD = 6-8 h)

1 μm

Clostridium ljungdahlii

Drake et al., 2006. In: The Prokaryotes, 3rd ed., vol. 2, 354-420

Page 27: Fermentative production of butanol – challenges and solutions

CO dehydrogenase/

Acetyl-CoA synthase

Formyl-THF yynthetase

Formate dehydrogenase

Acetyl-CoA

2 e- CO2

CO

Methyl branch Carbonyl branch

Formate Tetrahydrofolate (THF)

Formyl-THF+

ATP

ADP + Pi

Methenyl-THF cylohydrolase

Methenyl-THF

H+

H2O

Methylene-THF dehydrogenase

2 e-

Methylene-THF Methylene-THF reductase

2 e-

Methyl-THF

Methyl-C-FeS-P

C-FeS-P

C-FeS-P

Methyltransferase

[CO] CO

CO dehydrogenase/Acetyl-CoA synthase

HSCoA

Catabolism Anabolism

CO dehydrogenase 2 e-

CO2

2 e-

H2O

(Corrinoid-iron/

sulfur protein)

Wood-Ljungdahl pathway in

C. ljungdahlii

Page 28: Fermentative production of butanol – challenges and solutions

Development of C. ljungdahlii into a novel microbial

production platform from syngas

Genome sequencing and annotation

Suitable shuttle plasmids with antibiotic resistance cassettes (pIMP1,

erythromycin or clarithromycin, thiamphenicol)

Reliable DNA transfer procedure (electroporation, conjugation)

Expression of heterologous genes from other bacteria (clostridia)

Page 29: Fermentative production of butanol – challenges and solutions

butyraldehyde dehydrogenase (AdhE)

butanol dehydrogenase (BdhA)

butyryl-CoA dehydrogenase (Bcd)

crotonase (Crt)

3-hydroxybutyryl-CoA dehydrogenase (Hbd)

thiolase (ThlA)

3-hydroxybutyryl-CoA

crotonyl-CoA

acetyl-CoA acetate acetyl-P acetylaldehyde ethanol

butyryl-CoA

butyraldehyde

butanol

acetoacetyl-CoA

synthesis gas

Butanol synthesis with C. ljungdahlii

Page 30: Fermentative production of butanol – challenges and solutions

pIMP1 pSOBPptb

Butanol production by recombinant C. ljungdahlii

Page 31: Fermentative production of butanol – challenges and solutions

Gene expression in recombinant C. ljungdahlii

Page 32: Fermentative production of butanol – challenges and solutions

Further optimization by "metabolic engineering"

Inactivating genes responsible for butanol degradation

Prohibiting formation of side products by targeted knock-outs

Enhancing butanol formation by increasing plasmid copy number and using stronger promoters

Page 33: Fermentative production of butanol – challenges and solutions

Application vision

Page 34: Fermentative production of butanol – challenges and solutions

Former and current coworkers

in the field presented

Sonja Linder

Niklas Nold

Bettina Schiel-Bengelsdorf

Thiemo Standfest

Kai Thormann

Simone (Lederle) Thum

Brigitte Zickner

Tobias Zimmermann

Financial Support

BMBF-GenoMik/GenoMik-Plus

BMBF-SysMO


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