Syngulon’s technology expands the capacity for selection of microorganisms. The ability to select individual microbes with a behavior of interest is essential, whether for simple cloning at the bench, or for industry-scale production. Synthetic biology uses the concept of “bioengineering” to improve or modify existing genetic systems to create microbes with desired behaviors, and Syngulon uses this approach to develop its selection technologies. This selection technology is based on bacteriocins, ribosomally-produced peptides naturally made by most bacteria to kill competitive microbial species. These bacteriocins can have a limited or wide target range against other microbial species. This technology offers advantageous over antibiotic selection for several reasons: it avoids the use of antibiotics in the first place, helping to reduce the spread of antibiotic resistant microbes. The technology also increases product yield; as bacteriocins are generally smaller peptides, they do not impose a heavy metabolic burden on the producing cell. They can have a wide target specificity, helping to avoid genetic drift. Finally, our system is 100% plasmid-based (e.g. without chromosomal mutations), making it applicable for use in any E. coli strains.

1. 1
Dr Philippe Gabant
Co-founder & CSO
pgabant@syngulon.com
Guy Hélin
Co-founder & CEO
ghelin@syngulon.com
Selection Technology Using Bacteriocin-Immunity
to Improve Microbial Fermentation

2. Agenda
1. Synthetic biology
2. Heterogeneity
3. Key issues in the control of microorganisms
4. Regulatory guidance about antibiotics use in bioproduction
5. Bacteriocins: What are they? Why use them?
6. A bacteriocin-based fermentation peer police
7. Syngulon Technology self-supporting = with bacteriocins secretion
8. Syngulon Technology with immunity only
9. Comparisons of overexpression
10. Control of contamination and genetic drift: test bench
11. pDNA production with Syngulon technology
12. Syngulon TEAM / SAB / R&D Partners
13. Q&A 2

3. “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
3
1. Synthetic Biology
industrialization concept “IT versus genes”

4. 2. Heterogeneity in bioreactors
4
Batch 1
Yield: 75%
Batch 2
Yield: 50%
Batch 3
Yield: 90%
A
GOI
B
Confidential

5. 3. Key issues in the control of Microorganisms
Self replicative
Gene of interest
GOI
Two technological solutions
1. Induce the suicide of the cheater (self-police)
2. The fermentation population induces a peer police in the system
KEY ISSUES
1. Antibiotic-free selection
2. Yield increase
3. Genetic stability
4. Easy to use: 100% plasmid-based

6. 4. Regulatory guidance about antibiotics use in bioproduction
“As stated in the “Points to consider in the Characterization of Cell Lines
Used to Produce Biologicals,” it is recommended that penicillin and
other beta-lactam antibiotics be avoided during production, due to the
risk of serious hypersensitivity reactions in patients. If antibiotic
selection is used during production, it is preferable not to use selection
markers which confer resistance to antibiotics in significant clinical use,
in order to avoid unnecessary risk of spread of antibiotic resistance
traits to environmental microbes. Also residual antibiotic in the final
product should be quantitated when possible, and potential for allergy
considered”
6
“Concerning environmental impact and
the use of drug resistance traits, consult
the NIH Guidelines for Research Involving
Recombinant DNA Molecules, Section III-
A-1-a (59 FR 34496, amended 61 FR
59732). Non-antibiotic selection systems
can also be used.”

7. 5. Bacteriocins: What are they? Why use them?
- 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
- Small peptides that in many cases do not undergo post-translational
modification to be active = Ideal for gene-based peptide engineering1
- Active against antibiotic-resistant bacteria (VRE, MRSA…)2
- Antimicrobial activity at the nanomolar range scale (more active than
most antibiotics)2
- Naturally produced by probiotic strains
- Nisin (E234) is a bacteriocin widely used in the food industry (GRAS status)
- Availability of broad and narrow spectrum of activity 1Cotter et al., Nat. Rev. Microbiol., 2013
2Mathur et al., Front. Microbiol., 2017
3Oppegård et al., Biochemistry, 2015
Scimat/Science Photo Library
Pediocin PA-1 3
André Gratia
7

8. 6. A bacteriocin based fermentation peer police

9. 7. Syngulon Technology self-supporting
= with bacteriocins secretion
Immunity
Target
Gene of interest
Bacteriocin
Selection technology
developed by Syngulon
(EP3035802B1, US patents
9,333,227 / 10,188,114 /
11,427,800, CN ZL
201480057387.2 / ZL
201910882176.7, IN38926,
BR1120160035330/BR1220210
154171) to control microbes
providing:
1. Bacteriocin selection of
expressing clones
2. Yield increase
3. Control of genetic drift
4. 100% plasmid-based
(available in most E coli
strains)
Plasmid
Bacteriocin gene
Immunity gene
9

10. 8. Syngulon Technology with immunity only
New selection technology
developed by Syngulon
(EP3035802B1, US patents
9,333,227 / 10,188,114 /
11,427,800 and CN ZL
201480057387.2 / ZL
201910882176.7, IN389267,
BR1120160035330/BR12202101
54171 + US Patent 11,932,672)
to control microbes providing:
1. Bacteriocin selection of
expressing clones
2. Yield increase
3. Control of genetic drift
4. 100% plasmid-based
(available in most E
coli strains)
Plasmid
Closed and controlled batch fermentor
Immunity
Target
Gene of interest
Bacteriocin
Immunity gene
10

11. Protein X in E. coli RV308 at 37°C with KanR (pKan-Plac) and with 2 immunities against
microcins C7 and ColV (pBACT-Plac)
70
55
40
35
25
100
0 15’ 30’ 60’ 120’ 0’ 15’ 30’ 60’ 120’
pKan-Plac-X pBACT6.0-Plac-X
Induction time
X
(~40 kDa)
5 g of total extract were analysed by SDS-PAGE
pKan-Plac-X
pBACT6.0-Plac-X
0.67 0.95 1.12 1.48 2.30
0.50 0.60 0.62 0.74 0.95
T=0 15’ 30’ 60’ 120’
OD600nm at different time of induction
11
9. Syngulon technology: comparisons of overexpression

12. 12
Confidential
BL21 (DE3)
BL21 (DE3) / pAutoColV-X
1 2 3 4 5
0% 10% 30% 50% 100%
BL21 (DE3) / pET28b-X BL21 (DE3)
6 7 8 9
0% 10% 30% 50%
1 2 3 4 5 6 7 8 9
After 1 hour of growth, recombinant protein production
is induced with IPTG for 2 hours followed by bacterial cell
analysis (presence of plasmid) and protein SDS-PAGE
analysis (coomassie + western blot)
M
kDa
70
55
40
35
25
15
100
130
170
1 2 3 4 5 M 6 7 8 9 5
pAutoColV-X pET28b-X
Protein X
(~ 42 kDa)
Protein X
(~ 42 kDa)
Coomassie
SDS-PAGE
Western blot:
anti-X
70
55
40
35
25
15
100
130
170
T = 0
T = 3h
Potential contaminants or cells having lost the
plasmid are killed by the bacteriocin (ColV)
produced by the pAutoColV plasmid
Potential contaminants or cells having lost
the plasmid can take over the bacterial
population harbouring the plasmid
High production of the recombinant protein (X) with the self-supporting plasmid
(pAutoColV) compared to the standard plasmid (pET28b) with antibiotic resistance.
With the self-supporting system, contamination and genetic drift of the bacterial
population are better controlled during the recombinant protein production
10. Control of contamination and genetic drift: test bench

13. 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
13

14. 14

15. Construction of pAAV-ImV vector*
PCR fragment
*Vector without ampicillin resistance gene and with only immunity gene of ColV. The selection is
achieved by the addition of ColV bacteriocin in the medium.
Replace the AmpR gene in the
pAAV-RC2 (AmpR) plasmid by the
immunity gene of ColV
M: Molecular weight (bp)
R: restriction by EcoRV/NsiI (validated: 4,7 – 1,4 kb)
M R
400
200
600
800
1000
1500
2000
2500
3000
10000
4000
6000
Validation by restriction of
the pAAV-ImV vector.
15
11. pDNA production with Syngulon technology
Nat Rev Drug Discov. 2019 May ; 18(5): 358–378

16. Construction of pAAV-MccV vector*
R1 R2
PCR fragment
ColV (MccV) operon
*Vector without ampicillin resistance gene and without addition of other compounds (i.e. antibiotics) in
the culture medium. The selection is achieved by the expression of the ColV bacteriocin and the
associated immunity protein from the ColV operon
Validation by restriction of
the pAAV-MccV vector
M: Molecular weight (bp)
R1: restriction by EcoRI / SpeI (validated: 4,893 - 3 - 1,535 - 0,667 - 0,218 kb)
R2: restriction by BamHI / PmeI (validated: 6 - 3,5 - 0,8 kb)
M R1
400
200
600
800
1000
1500
2000
2500
3000
10000
8000
4000
6000
R2
5000
16
11. pDNA production with Syngulon technology
Replace the AmpR gene in the
pAAV-RC2 (AmpR) plasmid by
the ColV bacteriocin operon.
Nat Rev Drug Discov. 2019 May ; 18(5): 358–378

17. 17
11. pDNA production with Syngulon technology
Figure 3. Competition experiments. (A) Competition Experiment Without Selection. The upper section is a schematic representation of the competition experiments set
up with cell counts at the time of flask inoculation (Time 0) in CFU/mL ± SD. Flask 1 only contains the ampicillin resistant E. coli STABLE (AAV-RC2) strain (blue);
Flask 2 was inoculated with 99% of the strain STABLE (AAV-RC2) (blue) and the 1% of the plasmid-free strain STABLE (red). The competition was made without
adding ampicillin. The lower section of the panel shows the average pDNA extraction obtained from each culture (pDNA µg/mL of Culture) after 20 hours of growth.
60% of pDNA production drift was observed due to the population evolution resulting from the competition by 1% of empty bacteria. (B) Competition Experiment With
Peer Selection. The upper section of the panel represents the experimental set up with cell counts at the time of flask inoculation (Time 0) in CFU/mL ± SD. Flask 1,
contains only the STABLE (AAV-MccV) strain (green); Flask 2 contains 50% of the strain STABLE (AAV-MccV) (green) and 50% of the plasmid-free STABLE strain
(red). The lower section of the panel shows the average pDNA extraction obtained from each culture (pDNA µg/mL of Culture) after 20 hours of growth. No pDNA
production drift was observed from the competition experiment. Showing that the peer selection based of ColV produced by individual’s bacteria containing the pDNA
(AAV-MccV) was able to outcompete pDNA empty STABLE population during fermentation. The experiments were performed in triplicate with duplicate samples.
From El Bakkoury et al, 2024
Control of contamination and
genetic drift with pAAV-MccV vector

18. 18
11. pDNA production with Syngulon technology
Supercoiled plasmid DNA analysis
All three plasmid preparations successfully achieved 95% of the supercoiled form, being only the remaining 5% OC forms.
These results demonstrate that efficient and high quality pDNA production is possible with stable and antibiotic-free plasmids
like pAAV-MccV and pAAV-ImV
pAAV-RC2 (AmpR) pAAV-MccV pAAV-ImV
Superhelical Density
Percentage of supercoiled DNA
pAAV-RC2 pAAV-MccV pAAV-ImV
pAAV-RC2
pAAV-MccV
pAAV-ImV

19. 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)
Juan Borrero, PhD – Senior Scientist
Team / SAB / R&D Partners
Team
Guy Hélin, Co-founder, CEO
Philippe Gabant, PhD - Co-Founder, CSO
Mohamed El Bakkoury, PhD - CTO Yeast
Luz Perez, PhD - R&D Project Manager
Félix Jaumaux, PhD - R&D Project Manager
Loïc Mues, Ir - R&D Scientist
Kenny Petit, PhD - R&D project manager
Denis Dereinne, PhD student - R&D Scientist
Alex Quintero, PhD - R&D Project Manager
19

20. 13. Q & A
20
Selection Technology Using Bacteriocin-Immunity
to Improve Microbial Fermentation
Dr Philippe Gabant
Co-founder & CSO
pgabant@syngulon.com
Guy Hélin
Co-founder & CEO
ghelin@syngulon.com

21. Scientific background
Genetic selection (definition)
• A selection is an approach allowing only a sub-population of interest to survive
• The result is an enrichment of only individuals presenting the behavior of interest
(i.e. production of a certain compound)
Development of selections
1. In the 1990 selection for bacteria (clones) having recombinant plasmids (ULB patents):
R&D kits, exclusive license Invitrogen
2. In the 2000 selection for biopharma expression system without antibiotics (ULB
patents): licensing via Delphi Genetics (today part of Catalent)
3. From 2013, NEW selection technology for recombinants products or plasmidic DNA
(Syngulon patents - Dr. Gabant, inventor)
21