APPETITE FOR TRASH

Attacking the plastic flood with enzymes and microbes

22-08-2022 I NICK WIERCKX I AALBORG SUMMER SCHOOL

 A lesson from history: The carboniferous age

1
http://www.geologyin.com/2016/12/10-interesting-facts-about-geological.html http://www.ias4sure.com/wikiias/prelims/geological-time-scale/
 A lesson from history: The carboniferous age

The age of large plants Caused by the invention of lignin

2 https://www.jing.fm/iclip/mJiohR_connections-to-chemistry-concepts-wood-cellulose-lignin/
https://forces.si.edu/atmosphere/04_00_07.html
https://www.kissclipart.com/plant-cell-clipart-plants-the-plant-cell-wall-8dgzb3/
 A lesson from history: The carboniferous age

Microbes lost their appetite for wood Caused by the invention of lignin

For 50 million years!

3 https://www.jing.fm/iclip/mJiohR_connections-to-chemistry-concepts-wood-cellulose-lignin/
https://www.kissclipart.com/plant-cell-clipart-plants-the-plant-cell-wall-8dgzb3/
 Jülich

4
 Jülich

5 https://www.youtube.com/watch?v=kNDjPlZDmKs
 A lesson from history: The carboniferous age

50 million years of accumulated dead wood

6
https://imgur.com/uRyaQEZ https://reneweconomy.com.au
 Is it really?

7
 A lesson from history: The carboniferous age

The “invention” of a new polymer

…led to a golden age of plants

…but caused an incredible imbalance

…that took 50 million years to restore

8
 The invention of new polymers

PET

PE

PUR

10
 Contribute to a golden age of humanity

• Conserve our food

15 → 36 days

• Light weight

200 → 25 grams
• Technical innovations

• Safety innovations

• Energy conservation

11
 Causing an incredible imbalance

greenpeace

335 348
350 322
311
299
Plastic mill t

300 44 kg per person per year! 288

Million tonnes plastic per year

279
270
250 250
245
200 200 reddit.com

150
100 100
50
50
1.5
0
1950 1960 1970 1980 1990 2000 2010 2020 axiomgear.com
Quelle: PlasticsEurope (PEMRG); Consultic;
 The anthropocene age

The invention of new polymers

…contributes to a golden age of humanity

…but is causing an incredible imbalance

…that will take 50 million years to restore?

13
 The challenge: increase plastic recycling

14
www.mckinsey.com/industries/chemicals/our-insights/how-plastics-waste-recycling-could-transform-the-chemical-industry
 Closing cycles in the plastic bioeconomy

15
Ellis, Wierckx et al. (2021) Nature Catalysis 4:539-556 Wierckx et al. (2015) Microb. Biotechnol. 8:900
 Natural polymer degradation

Polymer Monomer

Cellulose Glucose

16
 Non-natural polymer degradation

Polymer Monomers

4,4’-methylenedianiline, 1,4-butanediol, ethylene glycol, adipic acid

polyurethane

17
 Natural plastic degradation

Cutin
Suberin

PET

PUR

18 Wikimedia commons
www.sciencedirect.com/topics/agricultural-and-biological-sciences/cutin www.sayerlack.com/water-based-wood-coatings-complete-guide/
 Natural plastic degradation
Cutinase enzyme degrading PET
untreated treated
Cutin
Suberin top view

PET
side view

87% weight loss

PUR Wei et al., (2017) Microb Biotechnol 10:1308-1322
Zimmermann, Billig (2011) Adv Biochem Eng Biotechnol 125:97-120
Alisch et al (2004) Biocatal. Biotrans. 22:347

19
https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/cutin
 Natural plastic degradation

Ideonella sakaiensis Cutinase enzyme degrading PET

untreated treated

top view

side view

87% weight loss

Wei et al., (2017) Microb Biotechnol 10:1308-1322

Yoshida et al. (2016) Science 351:1196
Zimmermann, Billig (2011) Adv Biochem Eng Biotechnol 125:97-120
Alisch et al (2004) Biocatal. Biotrans. 22:347

20
 Natural plastic degradation

Ideonella sakaiensis PET metabolism

Yoshida et al. (2016) Science 351:1196

21
 Natural plastic degradation
50 yrs
Ideonella sakaiensis

Yoshida et al. (2016) Science 351:1196

22
 Problem not solved!

greenpeace

335 348
350 322
311
299
Plastic mill t

300 44 kg per person per year! 288

Million tonnes plastic per year

279
270
250 250
245
200 200 reddit.com

150
100 100
50
50
1.5
0
1950 1960 1970 1980 1990 2000 2010 2020 axiomgear.com
Quelle: PlasticsEurope (PEMRG); Consultic;
 Closing cycles in the plastic bioeconomy

24
Ellis, Wierckx et al. (2021) Nature Catalysis 4:539-556 Wierckx et al. (2015) Microb. Biotechnol. 8:900
 Isolation of plastic-degrading enzymes

Image by Stephan Thies, IMET @ FZJ

25
Wikimedia commons
 PETase enzymes: the importance of temperature
& thermstable enzymes

Initial PET degradation rate PET degradation over time

Logarithmic
scale!

40°C 65°C

26
Tournier et al. Nature 580, 216–219 (2020). Wikimedia commons
 PETase enzymes: the importance of temperature
& thermstable enzymes

Initial PET degradation rate Differential Scanning Calorimetry

• Increased chain mobility

• More accessible to enzyme
40°C 65°C • Tg lowered by water absorption (→ 70°C)

27
Tournier et al. Nature 580, 216–219 (2020). Wikimedia commons
 PETase enzymes: the importance of PET crystallinity

Differential Scanning Calorimetry

→ Only amorphous PET is degraded

→ “real-world substrates are 30-70% crystalline
28
Wikimedia commons
 PETase enzymes: the importance of PET crystallinity

PET degradation vs. crystallinity

→ Only amorphous PET is degraded

→ “real-world substrates are 30-70% crystalline
29
Lu et al. Nature 604, 662–671 (2022). Wikimedia commons
 PETase enzymes: the importance of PET crystallinity

PET degradation vs. crystallinity

Water bottle after

pretreatment around
2% crystallinity

→ Only amorphous PET is degraded

→ “real-world substrates are 30-70% crystalline
→ Pretreatment needed
30
Lu et al. Nature 604, 662–671 (2022). Wikimedia commons
 Plastic degrading enzymes

Cleave “bio-similar” bonds Cleave low Tg plastics

• PBS (-32 °C)
• Ester • PBAT (-30 °C)
• PLA (50-60 °C)
→PET, PLA, PBAT, PBS • PET (65-80 °C)
• Nylon (45-60 °C)
• Amide
→ BUT: not crystalline
• →Nylon, polyurethanes

(some vinyl (C-C) and ether (C-O-C) cleaving enzymes)

31
 Engineering of plastic-degrading enzymes

Image by Stephan Thies, IMET @ FZJ

32
Wikimedia commons
 PETase directed evolution

33
Lu et al. Nature 604, 662–671 (2022). Wikimedia commons
 PETase industrial applications

34
www.carbios.com
 Bio-depolymerization of plastics

• Find enzymes that break down plastic

• Improve them by laboratory evolution

• Mixed plastic → “plastic hydrolysates”

35
 Closing cycles in the plastic bioeconomy

36
Ellis, Wierckx et al. (2021) Nature Catalysis 4:539-556 Wierckx et al. (2015) Microb. Biotechnol. 8:900
 Biological funneling of plastic hydrolysates

37
Ellis, Wierckx et al. (2021) Nature Catalysis 4:539-556 Wierckx et al. (2015) Microb. Biotechnol. 8:900
 Biological funneling of plastic hydrolysates
Plastic biotech Classic biotech

38
 Adipic acid metabolism in P. putida

39
 α,ω-dicarboxylate metabolism in P. putida

Metabolic engineering Laboratory Genome Reverse

evolution sequencing engineering

adipic acid

dcaAKIJP from Acinetobacter baylyi ADP1

Parke et al. (2001) Appl. Environ. Microbiol. 67: 4817

40
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 Adipic acid metabolism
Metabolic engineering

Acinetobacter baylyi
Gram-negative γ-proteobacteria

41
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 Adipic acid metabolism
Laboratory evolution

adipic acid
glucose,
4-hydroxybenzoate

42
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 Adipic acid metabolism
Genome sequencing

Plasmid vs genomic expression

plasmid

genomic

Growth PCN

43
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 Adipic acid metabolism
Genome sequencing

E80* in A12.1

44
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 Adipic acid metabolism
Reverse engineering

5 KT2440

4 A12.1
OD600 equivalent

0
0 10 20 30 40 50 60 70
time (h)

45
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 Adipic acid metabolism
Reverse engineering

5 KT2440

4 A12.1
OD600 equivalent

3
KT2440

KT2440
2

KT2440
1

0
0 10 20 30 40 50 60 70
time (h)

46
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 Adipic acid metabolism

5 KT2440

4 A12.1
OD600 equivalent

3
KT2440

KT2440
2

KT2440
1
KT2440
0
0 10 20 30 40 50 60 70
time (h)

47
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 Adipic acid metabolism
Reverse engineering

Combination of genomically integrated dcaAKIJP cluster and the double mutant lead to evolved growth

5 KT2440

4 A12.1
OD600 equivalent

3
KT2440

KT2440
2

KT2440
1
KT2440
0
0 10 20 30 40 50 60 70 KT2440
time (h)
KT2440

48
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 PHA production from adipic acid

Up to 25% of CDW is PHA Monomer chain length varies

monomeric fraction (%)

0.8 100
CDW
C12
PHA (g/L)
0.6 C10

CDW (g L-1)
C8
0.4
50 C6
0.2

0.0
The handbook of polyhydroxyalkanoates

ge

rA
1p

g
14
s
.

(2020) ed: M. Koller .1

12

p

:P
12
A

X:

-p g ge

::P A
pa ::P 12 p
g
A

g
14

P YX A 2.1
Y

YX s r
14
aa
:P

aF 14 .1
aa p
1
-p
X:

A
aF
Y
aa

pa
-p

P
aF

40
pa

4
P

T2

0 aa
K
40

44 -p
4

T2 F
T2

K paa
K

P
440
T2
49

K
Ackermann et al. (2021) Metabolic Engineering 67:29-40
 Biological funneling of plastic hydrolysates
Plastic biotech

Feed plastic hydrolysate to microbes

• Combine metabolic pathways
• Laboratory evolution
• Genetic engineering
• Produce value-added chemicals

50
 Bio-upcycling of plastic waste

• Circular thinking
• Incentivize waste management
• Zero pollution
• Engineering enzymes and microbes
• Laboratory evolution
• Enzyme engineering
• Metabolic engineering
• Inspired by nature
• Moonlighting enzymes
• Connecting pathways

51
 And pick up after yourself!

52
Interested in a paid MSc or PhD thesis in Germany?
-fungal biotechnology
-aromatics production → n.wierckx@fz-juelich.de
-solvent-tolerance
-plastics upcycling