Reasonably Random Synthetic Biology at Amyris Tim Gardner - - PowerPoint PPT Presentation

Reasonably Random Synthetic Biology at Amyris

Tim Gardner Director, Research Programs & Operations October 27, 2010

2

Overview

► Amyris is an integrated renewable products company producing advanced renewable fuels and chemicals ► Founded in 2003 on principle of social responsibility: use our know-how to address biggest health and environmental challenges ► Public company (IPO September 2010) with R&D, Manufacturing and Distribution facilities in the Emeryville, CA, Campinas, Brazil & Chicago, IL

3

Artemisinin is 95% effective against malaria

The Challenge: Supplying Artemisinin Anti-Malarials

50X increase in production 10X decrease in price Treating malaria would require: 300 to 500 million treatments per year Artemisinin treatments needed: 225 to 400 tons of artemisinin per year This would require: 6,000,000 tons of plant material Malaria causes: 1 to 3 million deaths per year

Total Chemical Synthesis too expensive

Amyris’ fouding product: Artemsinin

4

ispA G6P FDP G3P PEP PYR AcCoA

OAA

MAL

CIT

IPP

TCA Cycle

FPP idi DMAP Glucose

Mevalonate Pathway

Amorphadiene (arteminin precurser)

Artemisa annua

steroids quinones membranes

Non-profit effort to manufacture Artemsinin

5

Strain performance targets reached

10 20 30 40 50

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 Amorphadiene [g/L]

Time (hrs)

Improvement 1 Improvement 2 Improvement 3 Improvement 4 25g/L target

• Sanofi-aventis now ramping production, formulation and product stability testing
• Aim for world-wide distribution in 2012.

6

From drugs to fuels

Phase-contrast micrograph of Amyris engineered microbes producing precursor to Amyris Renewable Diesel

Isoprenoid technology platform capable of making more than 50,000 molecules

• Hydrocarbons, not alcohols or

esters

• Can be used in existing engines

with no performance trade-offs

• Superior environmental profile

– substantially lower greenhouse gas emissions than petroleum – No sulfur – Lower particulates and NOx

• Can be delivered using existing

distribution infrastructure

7

ispA G6P FDP G3P PEP PYR AcCoA

OAA

MAL

CIT

IPP

TCA Cycle

FPP idi DMAP Glucose Farenesene steroids quinones membranes

Mevalonate Pathway

Diesel production

8

Note: Amyris diesel will be used in blends with conventional fuels; values shown for Amyris diesel is for our biomass derived blending component; SME = Soy Methyl Esters

+1 < – 50 Amyris Diesel FAME # 2-D 47 58.1 40-55 Cetane Number Amyris Diesel FAME # 2-D Energy Density 1000 BTU/gal 118 121 115-142 Amyris Diesel FAME # 2-D

0 50 100 150 0 20 40 60

• 75 -50 -25 0
• 9 to-30

Cloud Point (°C) (cold temp operation)

Additional benefits of Amyris renewable diesel compared to #2-Diesel

• 90%+ lower greenhouse gas emissions
• No sulfur & produces lower NOx and particulate emissions
• Registered with the EPA for 20% blends

Amyris Renewable Diesel: a better fuel

9

>$1 Trillion dollar market accessible

fermentation

• Consumer products

– detergents – Cosmetics – fragrances

• Lubricants

– family of base oils – designed to be high performance

• Polymers

– adhesives – oxygen scavenger – toughening agent

• Renewable diesel

– “plug-in” fuels – meets or exceeds stds – substantially lower emissions

• Other applications

– crop protection – many others

Farnesene

By combining biology and chemistry, Biofene becomes a building block of renewable products for a diverse set of applications biology chemistry $48B $337B $809B >$50B

10

Lower cost of production enables access to larger markets

11

Low-cost production drives everything in strain R&D

Short term Medium term Long term Time to value Familiar Unfamiliar Uncertain Level of risk 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Capital cost-saving opportunities

Engineering decisions drive strain performance criteria Multi-parameter strain

• ptimization problem
• yield
• productivity
• reduced media supplements
• temperature
• biocatalyst stability
• GMM certification

Amount of savings

For fuel synthesis we aim to direct >90% of cell resources to the synthesis of byproducts under stringent productivity, temperature, and media conditions 2M ton/yr plant

12

like getting a toddler to eat salad

13

Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2007 2008 2009

Fene production

But we’ve made rapid progress

Fuels strain improvement since program start

Artemisinin base strain (modified for fuel synthesis)

14

Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2007 2008 2009

Fene production

How we got there

+ process development + rational engineering + breeding mutagenesis Artemisinin base strain (modified for fuel synthesis)

15

What can we learn from the mutants?

Illumina paired-end sequencing performed by Prognosys, Sequence assembly & analysis by Amyris

16

Mutant family tree and performance gains

M N O A B C D E I H G L F P K J

Post child

29 18 8 16

3.1 20 20 11 13

• 2

7 18 high medium

low

No change

Causal mutations found in

• Post-translational regulation
• Cofactor synthesis

Most genes we’d never

• considered. None “on

pathway” One we had tried rationally but not with the right mutation

improvement

Mutant Strain X

X

17

What about rational engineering?

Are electronics and machines the right paradigm? Synthetic Biology: the dream of plug and play biology

18

The neutral chassis hypothesis

Add a little synthetic biology

19

Biology is designed by natural selection It works, but it’s not always pretty

20

Too many parts kinda complexity

21

Promoter strength varies depending on its insertion site

Gene Expression

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 A B C D E

Promoter Genome locus

22

How much does diversity influence pathway production?

Sporulate (haploidize)

4 diverse hybrid haploids. Select for production

Farnesene Pathway

23

Impact of diversity on Mevalonate production

Reference strain

Fold increase in mevalonate titer over reference

24

Frequencies for top and bottom Mevalonate pools

25

A practical approach

Enzyme kinetics Doable but hard Context-effects & post- transcriptional regulation Shooting in the dark Stoichiometry & mRNA expression Routine

We always start here

1 Rational Semi-rational Random Random

Pathway PoC Pathway Optimization

We have targeted activities here when bottlenecks become clear

2 3

This is where most of the strain improvement “action” is.

26

• 1. Rational: Modeling / Isotopomers

Input

• Public models
• Yeastcyc
• Amyris knowledge
• Experimental data

Output

• Balanced models
• Simulatable models
• Matlab, Excel, GAMS format

Yeast metabolic database

27 Fermentation Downstream Processing (DSP) Scale-Up

• 2. & 3. Industrialize strain improvement

Analytics Knowledge Management Screening Rational Strain Design Random Mutagenesis Strain Engineering

Capacity: Screen >70,000 strains / week Test >40 2L fermentations / week

28

Continuous process improvement is critical

0.0% 1.0% 2.0% 3.0% 4.0% 5.0% 6.0% 7.0% 8.0% 5 10 15 20 25 30 Yield of parent strain

Relative improvement for a constant absolute yield gain CV required to detect winning mutant

Assuming constant absolute yield gain per improved mutant strain.

• S/N will drop as yield increases.
• So too must CV.

29

The value of process control

Original strain screening assay New assay

Relative production by shake plate assay Relative production by shake plate assay Yield in 2L tanks Yield in 2L tanks

The reward:

Overall screening process CV <4% Enables detection of 4% improvements w/ 5% FN and 5% FPs through 2 tiered screen

30

HT screening pipeline

Process improvement is easier said than done

31

Diagnosing sources of variation

32

Y4921 Count

Better decisions via informatics integration

LIMS systems is identifying and eliminating sources of error

Systematic drops in median plate titer traced to worn posts in one plate shaker

Strain score

33

0.94

0.26

0.00

Multivariate optimization – picking winners

Informatics integration is critical to good decisions (get data out of silos) winner Stress resistance

34

Strain hit from Screening (HTS Proj.) Calculate Yield (PV Proj.) Tank Testing (PV Proj.) Plate, pick, assay (HTS proj.) MAD DB Plate re-testing (MAD Proj.) PV DB HTS DB Data Warehouse

Calculator App

Filter, calculate, store, visualize (K2Y proj.)

Informatics integration enables assessment of stress resistance

Stress resistance metric

35

Let the data guide

Use empirical data mining to guide library construction, screening conditions, process dev.

36

Conclusions

• Rational engineering gets the ball rolling
• Industrialization enables rapid strain optimization

– Harnessing nature’s way of “thinking”: randomness and diversity – We are doing in 4 years what used to take 12

• Continuous process improvement is critical to the

success of an industrial platform

– Informatics is fundamental – Data mining & omics is fundamental

Thanks to the >200 folks in R&D contributing to our success