BEM Dr Philippe Gabant - June 10, 2022

1. Break out session: Synthetic Biology
1
Dr Philippe Gabant
Co-founder & CSO
pgabant@syngulon.com
Friday June 10, 2022
Disruptive industrial applications of Synthetic Biology

2. Agenda
1. Syngulon presentation
2. The importance of microbes for life on our planet
3. Syngulon’s markets
4. Which genes to fight AMR / to tune microbiota?
5. Historic view point of antimicrobials
6. Syngulon’s technology: industrial applications
7. PARAGEN collection and expansion via Synbio
8. Q&A
2

3. R&D Partners
Scientific Advisory Board
Pr Joseph Martial (Chairman), ULg, Liège (BE)
Pr Bruno André, ULB, Brussels (BE)
Adj-Pr Mike Chandler, University of Georgetown (USA)
Pr Pascal Hols, UCL, Louvain-la-Neuve (BE)
Pr Didier Mazel, Institut Pasteur, Paris (FR)
Pr Laurence Van Melderen, ULB, Charleroi (BE)
Pr Ruddy Wattiez, UMons, Mons (BE)
IN MEMORIAM
Dr Régis Sodoyer, ex-Sanofi Pasteur, Lyon (FR)
Collaboration with:
Universidad Complutense Madrid (UCM)
Dr. Juan Borrero 3
1. Syngulon presentation
Team
Guy Hélin, Co-founder, CEO
Dr. Philippe Gabant, Co-Founder, CSO
Dr. Mohamed El Bakkoury, CTO Yeast
Dr. Luz Perez, R&D Project Manager
Dr. Baptiste Dumont, R&D Project Manager
Félix Jaumaux, PhD Student
Dr. Anandi Martin, Senior Project Manager - Infectious Disease
Loïc Mues, R&D Scientist
Dr. Silvia Soto Diaz, R&D Project Manager
Dr. Jérôme Coppine, R&D Project Manager
Dr. Kenny Petit, R&D project manager
Dr. Jessica El Rayes, R&D project manager
Denis Dereinne, R&D Scientist

4. Our position in the Ecosystem
ACADEMIC PARTNERS
R&D FUNDING
CLUSTERS AND
TRADE
ASSOCIATIONS
CUSTOMERS AND
PROSPECTIVE LICENSEES
R&D PARTNERS 4
Ethanol 2G
Cosmetics
Biopharma
Animal Health
Ethanol 1G

5. 2. The importance of microbes for life on our planet
• Microbes are the chemical biocatalysers of our ecosystem
• Microbes are collaborating and fighting with each other to reach
certain equilibrium to form communities: « microbiota »
• These microbiota have evolved to generate unique chemical
reactions via species synergies
• Human health is related to microbiota dynamic
5

6. 3. Syngulon’s technologies address 3 markets
Technologies
Antimicrobial Resistance
Contamination
Antibiotic-free Selection
Production in a fermentation process; key markets are
biopharma and cosmetics but also enzymes;
1 (small) licensing deal with Enzymicals for diagnostic enzymes
1 signed licensing deal with industrial biotech company
Bacteriocins target the bacteria having a negative effect
on the production process; key markets are ethanol,
cooling towers, feed, animal health;
R&D partnership with TEREOS to improve industrial
fermentation / 1 large development and licensing deal
with Chinese EPPEN for feed probiotics with bacteriocins
Human pharma applications of bacteriocins as alternative
or complement to antibiotics in the context of
AntiMicrobial Resistance AMR (e.g. methicilin-resistant
Staphylococcus aureus - MRSA)
6
NDA

7. AntiMicrobial Resistance
7
UPDATE January 2022:
“On the basis of our predictive statistical models, there were an estimated 4.95 million (3.62–6.57) deaths associated with bacterial AMR in 2019, including 1.27
million (95% UI 0.911–1.71) deaths attributable to bacterial AMR.”
Source: “Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis” based on 471 million individual records or isolates and 7585 study-location-years
The Lancet, Published Online January 20, 2022 https://doi.org/10.1016/S0140-6736(21)02724-0
Source: TACKLING DRUG-RESISTANT INFECTIONS GLOBALLY: FINAL REPORT AND RECOMMENDATIONS, May 2016

8. Problems created by AMR/microbiota
• Better use of our current antibiotic arsenal
• Unmet needs:
– technologies and molecules to tackle AMR
– technologies and molecules to tune microbiota
Syngulon’s mission: Balancing & controlling microbial life on earth
8

9. 4. Which genes to fight AMR / to tune microbiota?
• (R)Explore the world of bacteriocins
- Discovered in 1925 by Belgian scientist:
“André Gratia (1893–1950): forgotten pioneer of research into antimicrobial agents” (*)
- Heterogenous group of antimicrobial peptides produced ribosomally by bacteria
- Used to kill related species to reduce competition for resources and space
- Not toxic
André Gratia
• Apply synthetic biology
9
(*) Wainwright M., J Med Biogr. 2000 Feb;8(1):39-42.

10. 5. Historical context of the discovery of antimicrobials
Félix d'Hérelle
Prof Ernest Chain Prof. Howard Florey
Prof André Gratia
Sir Alexander Fleming Prof Frederick Twort
Antibiotics
Bacteriophages
Bacteriocins
10

11. André Gratia in the USA
A. Gratia with Paul de Kruif at Rockefeller Institute (New-York) in 1920 (Picture from the archives of )
11

12. 1946
Standing: from left to right:
Prof. Selman Waksman (Rutgers University), Nobel Price of Medicine 1952 for the
discovery of streptomycin. Prof Waksman recommended the publication of this picture
with André Gratia in the book of Ph. Rhodes An outline of the history of
medicine (Butterworth, London, 1985)
Prof. Howard Florey (Oxford University), Nobel Price of Medicine 1945 shared with Sir
Alexander Fleming and Prof E.Chain for the discovery of penicillin.
Prof Jacques Trefouël (Institut Pasteur,Paris), discoverer of sulfonamides.
Prof Ernest Chain (Oxford University), Nobel Price of Medicine 1945 shared with Sir
Alexander Fleming and Prof H. Florey for the discovery of penicillin.
Prof André Gratia (University of Liège), discoverer of colicinogenic factors.
Prof E. Chain expressly requested Prof. A. Gratia to be present on his side on this
group picture.
Foreground:
Dr Pierre Frédericq (University of Liège) who showed that colicins are plasmid
encoded. Colicinogenic factors (such as plasmid colE1) were further developed to
become major tools in molecular biology.
Dr Maurice Welsch (University of Liège), developed mass production of antibiotics and
became Rector of the University of Liège.
Source: Belgian society of microbiology: M. Mergeay and G Cornelis tribute to André Gratia 12

13. Bacteriocins Function and Diversity
Collins, F.W.J., O’Connor, P.M., O’Sullivan, O. et al. Bacteriocin Gene-Trait matching across the complete Lactobacillus Pan-genome. Sci Rep 7, 3481 (2017).
Heilbronner, S., Krismer, B., Brötz-Oesterhelt, H. et al. The microbiome-shaping roles of bacteriocins. Nat Rev Microbiol (2021).
Bacteriocins are ecological genetic biocontol elements used by bacteria in nature
13

14. Bacteriocins in food applications
Fermentation of GRAS bacteria can change food taste and protect food against bacterial spoliage
Some bacteria are secreting antimicrobial compounds including peptides
Using fermentation to generate a firewall against problematic microbes
One of those peptides is Nisin that is used in food industry (E234)
14
Nisin (E234, GRAS) illustrates the potential of bacteriocins at industrial scale

15. Bacteriocins potential
15
1925
Production
• Genetic amenability
• Various prey spectrum
• Molecular diversity
• Cyto-friendly
• Stability
• Biological half-life
Pascal Hols, Laura Ledesma-García, Philippe Gabant and Johann Mignolet, Trends Microbiology 1685 No. of Pages 13

16. “Blank” chassis
Constructed by modules (parts)
Behavior code based
Non self replicative
Possible contamination by external
code
“Evolutionary” based chassis
Constructed by modules (parts)
Behavior code based
Self coding and self replicative
Possible contamination by external
code
Similarities with IT exists but fundamental differences
16
6. Syngulon’s technology: industrial applications
Synthetic Biology: “IT versus genes”

17. Heterogeneity in bioreactors
17
Batch 1
Yield: 75%
Batch 2
Yield: 50%
Batch 3
Yield: 90%
A
GOI
B

18. Batch fermentation in 200 L bioreactor
using E. coli BL21 (DE3) production strain containing a
Syngulon’s self-supporting expression plasmid
While producing the protein of interest (in blue) the
production strain also secret bacteriocins (in red) in the
fermentation medium. The population of cells containing
the Syngulon’s patented expression plasmids are therefore
inhibiting the growth of cells that loss the plasmid as well
as sensitive contaminants. Cells that contain the plasmids
also express the bacteriocin’s immunity and are therefore
immune to the bacteriocins
25
15
10
35
40
55
70
100
130
170
Recombinant
protein expression
in E. coli BL21 (DE3)
Inhibition halos due to
bacteriocins present in the
cell culture supernatant
Bacteriocin
Protein of interest
Example of a production in 200 L bioreactor with
Syngulon self-supporting expression plasmids
18
Future development: new microbial chassis ELITHE Project (convention 8527)

19. 19
Press releases

20. 7. The PARAGEN Collection
Physical Collection of Bacteriocin
Genes and Peptides
Relevant publications
> 100 “wild type” bacteriocins
chemically synthesized
~800 bacteriocin genes
To explore the diversity of bacteriocins we have built a collection of synthetic
genes in a standardized format allowing rapid activity measurements of
bacteriocins.
20

21. Int. J. Mol. Sci. 2012, 13(12), 16668-16707;
Review
Class IIa Bacteriocins: Diversity and New Developments
Yanhua Cui 1, Chao Zhang 1, Yunfeng Wang 2,*, John Shi 3, Lanwei Zhang 1,*, Zhongqing Ding 1, Xiaojun
Qu 4 and Hongyu Cui 2
21

22. Perez et al., Front Microbiol, 2018
Collaboration with:
Universidad Complutense Madrid (UCM)
Dr. Juan Borrero
Synbio to expand PARAGEN (1)
22

23. LVATGMAAGVAKTIVNAVSAGMDIATALSLFSGAFTAAGGIMALIKKYAQKKLWKQLIAA
SGAFTAAGGIMALIKKYAQKKLWKQLIAALVATGMAAGVAKTIVNAVSAGMDIATALSLF
Experimental design
Mature sequence of garvicin ML
Use of split inteins
Collaboration with:
Universidad Complutense Madrid (UCM)
Dr. Juan Borrero
Synbio to expand PARAGEN (2)
23

24. Applying Synbio to expand PARAGEN is part of our mission
S F
S F
H
N
O N
H
O
S F
N
H
O
Mature BACTERIOCIN
IC IN SsrA
+
24

25. Q & A
25
Dr Philippe Gabant
Co-founder & CSO
pgabant@syngulon.com
Disruptive industrial applications of Synthetic Biology
Break out session: Synthetic Biology
Guy Hélin, Co-founder & CEO
Philippe Gabant, Co-founder & CSO
Booth 2137