antibiotic-free technologies for production in a fermentation process

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. Syngulon presentation
2. Scientific background
3. The importance of microbes for life on our planet
4. Synthetic biology
5. Heterogeneity + Key issues in the control of microorganisms
6. Regulatory guidance about antibiotics use in bioproduction
7. Bacteriocins
8. Syngulon Technology a peer police system
9. 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. 2. 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)
4

5. 3. 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

6. A Biobased Chemistry
6
• Improvements in energy storage
• An intensification of biotechnological processes

7. Fermentation of microorganisms in industries
7
Microbial communities are the biocatalysts of our planet and bio industries

8. 4. Synthetic Biology
industrialization concept “IT versus genes”
8

9. “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
9
4. Synthetic Biology
industrialization concept “IT versus genes”

10. 5. Heterogeneity in bioreactors
10
Batch 1
Yield: 75%
Batch 2
Yield: 50%
Batch 3
Yield: 90%
A
GOI
B
Confidential

11. 5. 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

12. 6. 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”
12
“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.”

13. 7. 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
13

14. 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
14

15. 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
15

16. 8. A bacteriocin based fermentation peer police

17. 8. 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 and CN
ZL 201480057387.2,
BR1120160035330, IN389267)
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
17

18. 8. Syngulon Technology with immunity only
New selection technology
developed by Syngulon
(EP3035802B1, US patents
9,333,227 / 10,188,114 and
CN ZL 201480057387.2,
BR1120160035330, IN389267
+ new patent application of
Dec 2017) 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
18

19. 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 mg 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
19
8. Syngulon technology: comparisons of overexpression

20. 20
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
Control of contamination and genetic drift: test bench

21. 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
21

22. 22

23. 9. Q & A
23
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