1. Zaaien,
oogsten
en eten
AgriFoodTop
Symposium
7 juni 2017, Wageningen

2. Green chemicals via microbes: Nootkatone
Wat is er aan de hand?
Green chemicals via microbes: Nootkatone
Natural grapefruit flavor
Isoprenoid, derived from valencene
Very scarce, high impact flavor molecule
Excellent insect and tick repellent

3. Wat is er aan de hand?
Citrus greening disease: a threat to Citrus-derived products
Increased use of pesticides, lower harvests, increasing prices

4. Alternative: Microbial fermentation
Wat is er aan de hand?
Alternative: Microbial fermentation
Constant quality & availability
Not depending on harvest
Scaleability
Cost effective supply
Biobased with low carbon footprint
No pesticides
Fermentation
Distillation
Glucose
Rhodobacter
with terpene synthase

5. } Wat is er aan de hand? Plug-in terpene synthases
Produce specific terpene or terpene mixture
Access to > 1,000 terpene products
High carbon flux directed to terpene precursors
Sugar }
R. sphaeroides MEP pathway
FPP
Heterologous mevalonate pathway genes
Terpene synthase
Terpene product

6. Wat hebben we gedaan?
2014: P450 enzymes for conversion to nootkatone: Making P450 work in Rhodobacter was more difficult than expected !
=> More focus on increasing valencene output & proof of plug-in terpene synthase concept
Valencene
Nootkatone
P450

7. Increase Valencene output:
Wat hebben we gedaan?
Increase Valencene output:
Variants of Valencene synthase were tested in vitro & in vivo (E.coli & Rhodobacter)
=> Some variants with improved performance

8. Improved fermentation conditions
Wat hebben we gedaan?
Improved fermentation conditions
Chemostat study to determine the optimal growth rate for production
Robust, scaleable fermentation protocol => reliable 50,000-fold scaling

9. Wat hebben we gedaan?
Other terpenes: plugin synthases
=> Rhodobacter platform allows to produce a wide range of different terpenes

10. Wat levert het op?
Follow-up projects:
TKI-AF “Harness bacterial platform for mono- and diterpene production”,
2016: ZON-MW hotel project on transcriptomics
NWO groen: Wageningen University vakgroep BioProceskunde & Isobionics & WPR – Improving Rhodobacter metabolism
STW: Wageningen University vakgroep Plant physiology : Terpene synthases

11. Wat levert het op?
Increased valencene productivity
Contribution to stable, scaleable Rhodobacter bioprocess
Platform proven as suitable for many C15- terpene products (plug-in)

12. Wie deden er mee? MicCell (until 03/2016) Thamara Hesselink
Bert Schipper
Aalt-Jan van Dijk
Adele van Houwelingen
Dirk Bosch
Jules Beekwilder
Wouter Wisselink
Niels Willems
Elena Melillo
Frank Fluitman
Robert Trokowski
MicCell (until 03/2016)