Technology presentation focusing on bacteriosins applications for the development of new genetic control of microbial strains in industrial fermentation with examples using metagenomic analysis and the production of bacteriocins.

1. 1
Bacteriocins applications for the
development of new genetic control of
microbial strains in industrial fermentation
Dr Philippe Gabant,
Founder-CSO
pgabant@syngulon.com

2. Structure of the talk
• Syngulon
• Synthetic Biology: “IT versus genes”
• A need for a genetic firewall
• Conclusions
• Questions
2

3. A Biobased Chemistry
3
• Improvements in energy storage
• An intensification of biotechnological processes

4. Applications of microorganisms in
industries
4
“ it may be possible to create high-value substances such as biofuels by
modifying simple organisms such as E. coli bacteria.” May 2013, “Next-
generation genomics” in Disruptive technologies: Advances that will
transform life, business, and the global economy (McKinsey Global
Institute)

5. BiocatalysisRecombinant products
KEY ISSUES
Gene containment1.
Yield increase2.
Contamination prevention3.
Genetic drift4.
Genosphere
Inside
Outside
KEY ISSUES
1. Antibiotic-free selection of expressing clones
2. Yield increase
3. Genetic stability
4. Easy to use: 100% plasmid-based
5
Sterile fermentor Gene of
interest
Different industries different technological needs

6. Synthetic Biology: An approach with
many industrial applications
6

7. 7
Synthetic Biology: industrialization
concept “IT versus genes”

8. 8
Synthetic Biology: Industrialization
concept “IT versus Genes”
“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 replicative
Possible contamination by external code
Similarities with IT exists (both code based industries) but fundamental differences exist

9. 9
Industrial biotech environment
Genosphere
Inside
Outside
Genetic code of interest

10. 10
The Problem:
How to control industrial
Microorganisms?
KEY ISSUES
1. Gene containment
2. Yield increase by industrial Genetic Bio-control
3. Contamination prevention against microbial invaders
4. Genetic drift Self replicative

11. Industrial and large-scale applications need
to design a Genetic Firewall
11
Outside
Inside
Do we have the building blocks of this firewall in Nature?

12. 12
Bacteriocins : the origins
Gratia, A., 1925 Sur un remarquable exemple d’antagonisme entre
deux souches de colibacille. C. R. Soc. Biol. 93: 1040–1042.
2000 by the Genetics Society of America
Perspectives
Anecdotal, Historical and Critical Commentaries on Genetics
Edited by James F. Crow and William F. Dove
Andre ́ Gratia: A Forerunner in Microbial and Viral Genetics
Jean-Pierre Gratia
Microbial Genetics and Ecology Unit, Brussels University School of Medicine, B-1070 Brussels,
Belgium
Prof André Gratia 1893-1950

13. 13
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

14. Microcin B17 used in E. coli BL21
14
Bacteriocins:

15. Taking control of the bacteriocin loci
15

16. Syngulon Technology
for industrial biotech
Key issues
• Genetic containment
• Genetic security / competitors
• Selection of recombinants presenting
the designed behaviour
• Alternative to antibiotic used to fight
environmental contaminants)
• Control of contaminants (bacterial
invaders, divergent micro-refineries,
escaping host)
• Genetic drift
• Partners control
Non-issues (because impossible) : physical
containment, sterile
(Syngulon US Patent 9,333,227)
Immunity
Target
Genetic code of interest
Bacteriocin (molecule
naturaly secreted to kill
or control other
bacteria)
Need : selection not only focused
on the host of interest (suicidal
approach) but also allowing the
production microorganisms
(micro-refineries) to control its
growing environment and limit
the spreading of micro-refineries
outside the production plan
Approach : bacterial bacteriocins
loci are adapted for a “muti-
dimensional” selection
controlling the hosts and
environment during production
Applications : genetic vigilance of
the production plant allowing
continuous production
Open / semi-open continuous production – could be multicellular
16
Plasmid

17. | 17

18. PARAGEN
Collecting samples
and
Metagenomic analysis

19. Metagenomic Data

20. How to produce bacteriocins?

21. Pediococcus Pentosaceus
(HELA: CWBI-B29)
Pediococcus Pentosaceus
(PROVITAL: CWBI-B73)
- PedA gene
Application of bacteriocin for yeast
protection

22. In vitro Synthesis
- +

23. Chemical Synthesis
Chemical synthesis
ColV EntL50A Ent35 PedA1

24. Take Home Message
24
Selection technologies are essential for efficient•
microbial strain construction and fermentation
New industrial challenges induce the need of new•
selection technologies
Syngulon• propose an unique and innovative genetic
firewall to boost fermentation based on bacteriocin
genetics circuits

25. 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)
SYNGULON Team
Guy Hélin, Co-founder, CEO
Dr Johann Mignolet, R&D Project Manager
Dr Mohamed El Bakkoury, CTO Yeast
Marylène Maistriau, R&D Project Manager
Dr Philippe Gabant, Co-Founder, CSO
Bertrand Delahaye, R&D Scientist
Christine Ganshof, Lab assistant
Thomas Gosset, Ir, R&D Engineer
ACKNOWLEDGEMENTS
25