Efficient synthesis of specialty carbohydrates
through industrial biotechnology
Prof. Wim Soetaert
May 2014 © Inbiose NV –
Bio 11th Annual World Congress on Industrial Biotechnology Inbiose at a glance
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Spin‐off company from InBio, Centre of Industrial Biotechnology and Biocatalysis, Ghent University
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Core competence in the development and production of specialty carbohydrates through industrial biotechnology •
Proprietary production platform based on fermentative synthesis of specialty carbohydrates (cell factory)
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Access to pilot and production facilities •
Experienced team with industrial and academic background
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Carbohydrates are everywhere
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Carbohydrates are everywhere
H OH
H
O
HO
H
OH
HO
H
H
O
HO
H
OH
OH
HO
H
glucose
From the simplest form
HO
O
H OH
HO
H
O
H
H
H
HO
H
HO
H
H
O
NH
H
OH
NH
O
O
O
H
O
HO
H
OH
OH
O
H
H
O
H
OH
HO
H
OH
H
H
O
O
H
H
HO
H
H
OH
H
Specialty carbohydrate
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To very complex structures
What are specialty carbohydrates?
• Very complex carbohydrates with unconventional structures
• Rare in nature or difficult to impossible to produce
• Price range: 10 €/kg – 1.000 €/kg (reference sugar: 0.5 €/kg)
• Quantities: 1 kg – 10.000 tpa
• High‐end applications in pharmaceuticals, nutraceuticals, cosmetics, chemicals, plant protection,...
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What are specialty carbohydrates?
Monosaccharides or oligosaccharides consisting of:
– L‐Fucose
Human Milk Oligosaccharides
– Sialic acid
– Galactose
– Glucosamine
– Glucose
– etc.
chitosans
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Applications of specialty carbohydrates
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Human Milk Oligosaccharides
Infant formula
Prebiotics
Functional foods
Cosmetics
Specialty carbohydrate production
Extraction
Natural source required, availability problems
High price volatility due to natural variability
Complex extraction procedures
Chemical synthesis
Very complex synthesis and costly substrates
Low yields
Use of toxic chemicals and high waste generation
Enzymatic
synthesis
Simple and cheap Only for simple one‐step conversions
Equilibrium can be unfavourable
Microbial synthesis
Very complex carbohydrates produced in one step
Cheap and readily available substrates
Environmentally sustainable
Inbiose production platform
Engineered production strain
Microbes
at work
fermentation
Down‐stream processing
03‐07‐2012
Specialty carbohydrate
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Engineered production strain
1. The cell needs sugar (disaccharide) to grow
2. The cell converts sugar intracellularly into a saccharide and an activated saccharide
3. A biosynthetic pathway from the activated saccharide to the specialty carbohydrate
is metabolically engineered into the cell
saccharide
sugar
activated saccharide
specialty carbohydrate
Cell
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Engineered production strain
4. Growth pulls the equilibrium towards accumulation of the activated carbohydrate
5. Gene knock‐outs split the microbial metabolism in half (metabolic wall)
6. The activated carbohydrate is converted into the specialty carbohydrate
7. The specialty carbohydrate accumulates and is excreted by the cell
saccharide
Biomass (growth)
sugar
activated saccharide
specialty carbohydrate
Cell
Specialty
carbohydrate
Synthesis pathway engineering
Sucrose
F6P
Gln‐6‐P
Gln‐1‐P
GlcNAc‐1‐P
UDP‐GlcNAc
carbohydrate
Pathway of 7 reactions  requires 7 enzymes coded by 7 genes
Invertase
fructokinase
acetyltransferase
uridyltransferase
aminotransferase
glycosyltransferase
phosphomutase
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Pathway assembly
Cloned genes with tunable elements such as promoters and ribosome binding sites
Assemble in an artificial chromosome
Confidential
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Pathway assembly
Transfer into the base strain
Specialty carbohydrate Sucrose 15
Selection of best strain from the library
carbohydrate carbohydrate carbohydrate
carbohydrate carbohydrate
carbohydrate carbohydrate carbohydrate 16
Integrated technology development
Metabolic engineering of the production organism
bug
Development of the fermentation process
Process
Development of the product recovery process
Product
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Integrated technology development
Bug
• Biochemical pathway design
• Expression of suitable genes in the production host
• Strain selection for optimal producer organism
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Integrated technology development
Process
• Development of the fermentation process
• Optimisation of yield and production rate
• Scale‐up of the fermentation process
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Scale‐up of the fermentation process
Confidential
13‐06‐2012
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Integrated technology development
Product
• Development of down‐stream processing of fermentation broth
• Product recovery and purification
• Product conditioning
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Our technology in a nutshell
Sucrose
Cell factory
Specialty carbohydrate
Integrated technology development
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HMO 3: Strain & process improvement
60
54 g/L
50
40
Final titer 30
(g/L)
20
16 g/L
10
0
5,7 g/L
23 mg/L
182 mg/L
2,3 g/L
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Inbiose product portfolio
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Inbiose value proposition
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Generic and scaleable technology platform for producing specialty carbohydrates through industrial biotechnology
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Inbiose can produce any complex carbohydrate for which a biotechnological pathway can be designed
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Inbiose can quickly develop new processes through its base strains
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Inbiose process starts from cheap substrates and delivers
high yields and productivities
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Inbiose has production facilities to produce up to 1.000 tpa of specialty carbohydrates
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Inbiose is looking for partners
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Partners interested in specific specialty carbohydrates (targets) for which Inbiose can develop efficient and scaleable production technology
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Partners with experience in regulatory approval of our portfolio of specialty carbohydrates
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Partners with large scale production facilities (> 1.000 tpa)
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Partners interested in licencing in our technology
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Partners that developed new applications of specialty carbohydrates
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www.inbiose.com
Business development:
[email protected]
May 2014 © Inbiose NV –
Bio 11th Annual World Congress on Industrial Biotechnology