Andrew Spicer, Ph.D. Algenuity, UK Surf and turf: transferring triterpenoid plant-metabolic pathways to micro- algal hosts and their growth optimisation www.algenuity.com [email protected]
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Andrew Spicer, Ph.D.
Algenuity, UK
Surf and turf: transferring triterpenoid plant-metabolic pathways to micro-
algal hosts and their growth optimisation
Copyright Algenuity 20162
• Background = Who?
• Opportunity = Why? What?
• Approach/Challenge = Where? How? What? When?
• Summary/Conclusions = So what, so when?
product Services/core R&D
Non-GMO Strain
Discovery & Development
Suite
Strain Engineering Strain Analytics
Bioprospecting (Discover)
Growth Optimisation
(Optimize)
Directed Evolution
(Improve)
Lipids Proteins Secondary
Metabolites
Transcriptomics Metabolomics
Who?
Design
Build
Grow
Test
Learn Discover
Optimize
Improve
Who?
5
What if…..?
Where….?
Why?
A microalgae-based, sustainable route to
very high value plant secondary
metabolites and chemicals not limited by
harvest cycles, seasons or geography
>70% of the new
medicines
developed over
the past 30 years
come from a
natural product.
Copyright Algenuity 2016
“Synthetic biology is the design and
engineering of biologically based
parts, novel devices and systems as
well as the redesign of existing, natural
biological systems.”
A Synthetic Biology RoadMap for the UK, 2012
What?
7
Select compatible strain/chassis:- Biochemical
composition/suitability- Growth modes/rate- Co-products?- Current market value?- Harvest?- Economic model &
target/threshold & predictive tools
- Genome & toolkit?
Assemble toolkit & transformation protocol
DoE growth optimisation vs desired biochem comp
Design/synthesise Cassettes for desired pathway with alternate ORFs/recoding
Acquire knowledge on related pathways
1st transformations & test/yield
DoE application of optimised conditions
2nd transformations & test/yield
DoE application of optimised conditions
3rd transformations & test/yield incl multigene transformations
DoE application of optimised conditions
4th….Final transformations & test/yield incl multigene transformations
DoE application of optimised conditions
Improvement rounds/yield over unit time
Process
How?
Product PoC:
TriForC project FP7 project
• Production platform for bioactive triterpenoid saponins
• EU Consortium
• Particular focus on triterpenoid saponins
• Model PoC/feasibility project for metabolic engineering of
novel plant 2º metabolite pathways into microalgae
8
What?
Plant terpenoid metabolism
Moses et al. 2013
• 4-15 genes need to be expressed to drive full pipeline for production of a final triterpene• Control of expression will be essential…..more tools and validated pipeline needed…• Keep applying industrial scale filter and predictive costs tool at each stage
Basic pathway
10
Why?
First round: Lupeol production in P.
tricornutum from AtLUS
Wild Type
LuS-6
Lupeolacetate
Lupeol
11
What? How?
Lupeol standard
Arabidopsis thaliana lupeol synthase
Lotus japonicuslupeol synthase
Lupeol 2,3 beta Hydroxylupane
2nd Round: Improved product specificity and enzymatic activity through
screen of enzymes from different genetic sources
To go to the next step in the metabolic pathway requires expression of two additional geneswith expression control highly desirable. Also evaluating fully synthetic enzyme variants and
enzyme improvement strategies
What? How?
Copyright Algenuity 2016
fcp CY716A12
fcpT
CPR
fcp P-NAT
fcpT
+
fcpT
fcp LUS
+fcpT
fcp Sh-ble
1)
2)
fcpfcpT
CYP716A12
Betulinic AcidLupeol
• New strains derived – multigene assembly of LjLUS and CYP716A12 for specific
production of betulinic acid = 3rd round
• Inducible control on lupeol synthase to drive reaction to completion = 3rd round
What? How?
0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
LJ18 LJ20 LJ25 AtLuS6
ng
of
lup
eolp
er
10
-7
cells
Quantification of P. tricornutum lines producing lupeol (constitutive)
Reality check time:
~50 mg – 100 mg/L yield at present – vs 15mg+/L from inducible S cerevisiae platform….……but largely relative culture density dependent….how do we improve yield &
economics?
What? How?
16
What? How?
Additional improvements needed
– new tools, new thinking
Tool development 1 - Inducible promoters
0
0,2
0,4
0,6
0,8
1
1,2
Re
lativ
e m
RN
A e
xp
ress
ion
Venus expression
PromA PromB
17
How?
H4:Glut1 P. tricornutum
Mixotrophic
growth
Phototrophic
growth
Heterotrophic
growth
Light intensity
Tool development 2 – Mixotrophic/Heterotrophic options
Copyright Algenuity 2016
• Culture volume – critical - sufficient to sample for GC/MS; transcriptomics; follow quality as well as absolute quantity of biomass
• Light and temp calibration – final QC using obligate photoautotroph – reactor matching = reduced noise;
• Temp range achievable (4°C to 50°C);
Multiparameters possible:• Temp• Lighting Intensity/Photoperiod• Wavelength• pH• Mixing
• Nutrients• Strain variants – genetic modifications
°
Tool development 3 – Design of Experiments (DoE) using Algem
Matthijs et al. in submission
Sampling schedule 12:12 photoperiod
Tool development 4 – Impact of light/photoperiod on sterol precursors
Two sets of Algems, time shifted
Speeding up time….
6:30 hours of sampling
• One set of Algems was shifted by 12 hrs
• “Midnight” occurs at 12:00
Matthijs et al. in submission
qPCR result - conclusion
• Dawn and dusk have identical light conditions but opposite expression patterns
• Algae change their nitrogen assimilation during the photoperiod
• Same approach being used to follow expression of genes for sterol catabolism
and anabolism – coupled with GC-MS etc
N- assimilation genes
Amino acid – degradation genes
0
50
100
150
Light Intensity
Copyright Algenuity 2016
So what, so when?.......
1. First demonstration of production of triterpenoid class compounds in a microalga
2. 5 genes co-expressed in Phaeodactylum tricornutum
3. More refinement and tool building, chassis (strain) development and outside-the-
box thinking needed to improve towards any chance of commercial reality
4. Unique combinations of genetics, cultivation and environmental influences need
to be investigated through multi-parametric approaches
5. Importance in understanding drivers, realistic models and price point thresholds
for very high value chemicals and products made in microalgae
6. GM Microalgal products through SynBio approach – 5-25 year horizon estimated
Copyright Algenuity 2016
Algenuity Team Collaborators
Joanna Szaub-Newton, Ph.D. TriForC FP7 Consortium
Henry Taunt, Ph.D. Olga Sayanova – RRESMichiel Matthijs, Ph.D. Saul Purton – UCLSam Sizer Michele Stanley - SAMSGavin Lowe Chris Howe & Alison Smith – University of CambridgePatrick Hickland Mike Allen - PMLGino Schiano di VisconteDenise PallisterSarah D’adamo, Ph.D.
Mike YatesDaniel Hutton
Mark ZarembaMike JarvisRoshan CheraghvandiJohn Sackett, Ph.D.Denis Spicer
Algenuity Advisory BoardSaul Purton – UCLChris Howe – CambridgeMike Goosey – ex-Shell Global R&D BiofuelsHilary Worton – pharma strategy/marketing
THANK YOU!
www.algenuity.com
Copyright Algenuity 2016
www.algenuity.com
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