surf and turf: transferring triterpenoid plant-metabolic...

Andrew Spicer, Ph.D.

Algenuity, UK

Surf and turf: transferring triterpenoid plant-metabolic pathways to micro-

algal hosts and their growth optimisation

[email protected]

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.


“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


3rd transformations & test/yield incl multigene transformations


4th….Final transformations & test/yield incl multigene transformations


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?


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


• 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


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


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


www.algenuity.com

surf and turf: transferring triterpenoid plant-metabolic...

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